A. FERREIRA-ALEMAO MD PhD

A. FERREIRA-ALEMAO MD PhD Universidad Complutense de Madrid Molecular Pathology and Biology Surgical Oncology

E-mail: ribograma@gmail.com Cell phone: 351 93 671 54 88 http://www.ribograma.com

1

CURRICULUM VITAE I - NAME: Álvaro Martins Ferreira Alemão II - BIOGRAPHICAL INFORMATION: Born in Oporto City (PORTUGAL); on March 17, 1947.

III - PRE GRADUATE MEDICAL COURSE: 01 - From 1963 to 1969 – University Studies (1st to 4th years at Medical School of Oporto and 5th and 6th years at the Faculty of Medicine of Lisbon).

IV - POST- GRADUATE PREPARATION: 01-1970 – General Internship at CIVIL HOSPITALS OF LISBON (Portugal) - in services: a) General Surgery (at St. Joseph Hospital); b) Internal Medicine (Hospital of St. Joseph) ; c) Pediatrics (at Hospital Da. Estefania Hospital); d) Obstetrics (Maternity St ª Bárbara - St. Joseph Hospital).

02 - 1971/72 - Hôpital Cantonal Universitaire at Lausanne City (Switzerland) in the Department of Anesthesiology/Resuscitation/Intensive Care – (Director – Prof.

Christhian Raymond); 03 - 1971 - AMERICAN TEST ECFMG - Educational Council for Foreign Medical Graduates; 04 – 1972/74 - Military Service, in Mozambique (Tete, Nampula and Mueda) - during 26 months, serving as an anesthesiologist and critical care/resuscitation physician, in a war zone, with extensive experience in war-wounded; 05 - 1974/5 - Internship in Chicago.

06 - 1976 to 1979 – Residency in Bronx–Lebanon Hospital Center in New York; 07 - In 1979 – Theoretical and Practical Course of Gastroenterology at the Escuela Profesional of Digestive Pathology - Director - Prof. Francisco Vilardell, at Autonomous University of Barcelona-Spain -; 08- 1979 to 1983 – General Surgeon, at General Surgery Service Nº 4, at the Hospital St. Joseph (Lisbon- Portugal); 09-1984 – General Surgeon at Hôpital La Pitié-Salpetrière, Paris - Service de Chirurgie Générale - Directeur - Professeur JP Vayre; 10-1985 - Provision of General Surgery Hospital Assistant Hospital Medical Career - Zona Centro - Portugal; 11 - From 1985 to 2001 - General Surgeon and Clinical Director in a private clinic 2

(Hospital Clínico das Amoreiras) at Lisbon-Portugal; 12 - Since 2002, academic activity in the Complutense University of Madrid 13 – 2007/14 PhD degree, at Universidad Complutense de Madrid – Spain.

V – MONOGRAPH WORKS 01 - HORMONAL PEPTIDES GASROINTESTINAIS KEY FEATURES ON YOUR LOCATION AND RELEASE AND QUANTIFICATION, O MÉDICO , No. 1811 in 1986; 02 - diffused endocrine system and APUD SYSTEM, O MÉDICO , No. 1812, 1986; 03 - BRAIN-VASCULAR DISEASE: DIAGNOSTIC AND BASES TREATMENT OPTIONS ATHEROMATHOUS DISEASE OF GREAT CERVICAL VESSELS (extracranial), O MÉDICO , No. 1836, 1986; 04 - CLINICAL IMPLICATIONS OF INTEGRATION OF NEURO-HORMONAL diffused endocrine system (APUD CELLS), O MÉDICO , No. 1842, 1987; 05 - DIFFUSE NEUROENDOCRINE SYSTEM. Physiological and biochemical mechanisms of the APUD cell system, O MÉDICO , No. 1845, 1987; 06 - Diffused endocrine system. Clinical and laboratory expression of the biological action of some peptide antroduodenal hormones, O MÉDICO, No. 1848, 1987; 07 - Study of the evolutionary behavior of cells and their clinical applications, O MÉDICO, No. 1912, 1988; 08 - Liver transplantation. Conditions for its implementation and definition of brain death to the donor, O MÉDICO, No. 1991, 1990; 09 - Liver transplantation. Collection operation of the liver and other organs for transplants, O MÉDICO, No. 2007, 1991; 10 - Hepatic Transplantation. Hepatectomy of the recipient, O MÉDICO, No. 2010, 1991; 11 - Liver Transplantation. Deployment operation of the liver donor, O MÉDICO, No. 2010, 1991; 12 - Gastroesophageal Reflux and Slide Esophageal Hiatus Hernia - pathophysiological aspects of the gastroesophageal junction and its implications for surgical therapy, O MÉDICO, No. 1423, 1978; 13 - Cholestasis - diagnostic approach to jaundice of surgical cause, O MÉDICO, No. 125, 1991.

VI – CONFERENCES AND SCIENTIFIC MEETINGS: 01 - International Meeting of Digestive Surgery, held at the Faculty of Medicine of OportoPortugal, from 26th to 29th Jan 1977; 02 - IV International Course on Update in Digestive Surgery, held at the Universidad Complutense de Madrid-Spain, from 25th to 30th Mai 1981; 03 - II International Meeting of Vascular Pathology, held at the Faculty of Medicine of Coimbra-Portugal, from 27th 29th Mai1980; 3

04 - Colloquium Luso-Spanish-Diagnosis of Cholestasis Gastroenterology, held in LisbonPortugal, from 14th to 15th Dec 1979; 05 - VI International Meeting of Digestive Surgery, held at the Faculty of Medicine of OportoPortugal, from 25th to 29th Jan 1982; 06 - Symposium on "Pharmacotherapy en CirugĂ­a, Hospital de la Santa Cruz y S. Pablo in Barcelona-Spain, from 27th to 28th Nov 1981; 07 - IV International Meeting of Vascular Pathology, held at the Faculty of Medicine of Coimbra-Portugal, from 22th to 24th Out1984; 08 - Third International Meeting of Vascular Pathology, held at the Faculty of Medicine of Coimbra-Portugal, from 17th to 20th Apr 1982; 09 - Course of Surgery, organization of the 6th World Congress of the CICD and SPC, held in Lisbon-Portugal, from 15th to16th Sept 1980; 10 - International Meeting of Endocrinology in Lisbon-Portugal, from 12th to 15th July 1979; 11 - Pancreatology Course for Postgraduates and the International Meeting of Gastroenterology, held in Lisbon-Portugal, from 18th to 22th Jun 1979; 12- 6th CICD World Congress, held in Lisbon-Portugal, from 16th to 19th Sept 1980; 13 - I International Meeting of vascular pathology, held in the Faculty of Medicine of Coimbra-Portugal, from 16th to 18th Oct 1978; 14 - I International Symposium of Emergency Surgery, held in Lisbon-Portugal, from 29th to 30th Mai 1987; 15 - Third International Meeting of Digestive Surgery, held at the Faculty of Medicine of Oporto-Portugal, from 29th Jan to 2nd Feb 1979; 16 - First International Meeting of Gynaecologic Endocrinology, held in Oura-Hotel, in Praia da Oura-Algarve-Portugal, from 10th to 11th May 1991; 17 - International Symposium on female sterility performed in Figueira da Foz City-Portugal, from 20th to 21th Oct 1978; 18 - III International Symposium for the Study of Reproduction, held in Figueira da Foz-CityPortugal, from 28th to 29th Oct 1983; 19 - Post-Graduate Course of Gynecological Endocrinology held in Figueira da Foz, 10th Oct. 1986; 20 - First International Meeting of Gynaecologic Endocrinology, held in Oura Praia Hotel, Oura-Algarve-Portugal, from 10th to 11th May 1991; 21 - International Symposium for the Study of Reproduction, in Figueira da Foz CityPortugal, on 2ndNov 1981; 22 - Symposium on the Treatment of Breast Cancer, held in Estoril-Portugal, from 10th to 11th Apr 1980; 23 - International Symposium of Gynecological Endocrinology held in Figueira da Foz CityPortugal, on 08th Nov 1980; 24 - XXIX Course Abdominal Surgery, Hospital de la Santa Cruz y San Pablo, BarcelonaSpain, on12th Dec 1980; 25 - V International Course on Update in Digestive Tract Surgery, held at the Universidad Complutense de Madrid-Spain, from 24th to 29th May 1982; 26 - Symposium on Breast Cancer, held at the Hospital de la Santa Cruz y S. Pablo, in 4

Barcelona-Spain, from 07th to 06th Dec 1977; 27 - Symposium on Post – operative Care, in the Hospital de la Santa Cruz Y S. Pablo, in Barcelona-Spain, from 28th to 29th Nov 1980; 28 - Symposium on Abdominal Wall Hernias, Hospital de la Santa Cruz y S. Pablo in Barcelona-Spain, from 24th to 24th Nov1978; 29 - Symposium on Abdominal Trauma, Hospital de la Santa Cruz y S. Pablo, BarcelonaSpain, from 30th Nov to 01th Dec 1979; 30 - V International Meeting of Digestive Surgery, Faculty of Medicine of Porto, 30th Jan. 1981; 31 - Course of Hepatology Clinic for Postgraduate Students, held at the Free University of Lisbon, from 07th to 09th Jan 1980; 32 - Course of Digestive Pathology, at the Autonomous University of Barcelona in Nov. 1978, 33 - 1st International Symposium of Urology, Faculty of Medicine of Porto, on 22th Mar. 1980; 34 - Membership of the Society of Medical Sciences of Lisbon, on 27th May 1980; 35 - XXVII Course on Abdominal Surgery, Hospital de la Santa Cruz y San Pablo, Barcelona-Spain, in Dec 1978; 36 - XII Annual Course of Surgery of Digestive Tract, Faculty of Medicine of BarcelonaSpain, in Nov. 1980; 37 - XXVI Course Abdominal Surgery, Hospital de la Santa Cruz y San Pablo, BarcelonaSpain, in Dec. 1977; 38 - IV International Meeting of Digestive Surgery, Hospital of St. John in Oporto-Portugal in 2001. Feb 80; 39 - XXVIII Course of Abdominal Surgery, Hospital de la Santa Cruz y San Pablo, Barcelona-Spain, from 3th to 7th, on Dec 1979; 40 - International Symposium Reproduction, in Espinho City-Portugal, from 3th to 4th Apr 1982; 41 - XIV International Course Update - General Surgery Digestive Tract, University of Madrid-Spain, from 20th 25th May 1991; 42 – Member of the Association of Surgeons of the Courses del Hospital de la Santa Cruz y San Pablo in Barcelona-Spain, on 06th Dec 1979; 43 - XI Annual Course of Digestive Surgery, School of Medicine, University of BarcelonaSpain, Nov 1979; 44 - International Meeting of Digestive Surgery, held at the Faculty of Medicine of OportoPortugal, from 26th to 29th Jan 1977; 45 - IV International Course of Updating in Surgery of the Digestive System Digestive System, held at the Universidad Complutense Madrid-Spain, from 25th to 30th Mai 1981; 46 - II International Meeting of Vascular Pathology, held at the Faculty of Medicine of Coimbra-Portugal, from 27th to 29th Maio1980; 47 - Colloquium Luso-Spanish-Diagnosis of Cholestasis Gastroenterology, held in Lisbon, from 14th to 15th Dec th 1979; 48 - Diseases and Medications in the Elderly, Lisbon, on 17th Oct 1990; 5

49 - IV Seminar of District Hospitals on the themes: thyroid, breast and chest, vascular surgery, performed in the Hospital District of Setubal, on 10th Dec. 1984; VII - Laboratory Investigations: 1. A.W. Hall, J. Clark, Ferreira-Alemao A and D.B. Skinner: Effect of bowel resection on lower esophageal high pressure zone and reflux status in rhesus monkeys.

Journal of Surgical Research, 20:215-219, 1976.

2. C.K. Zarins, Ferreira-Alemao A and D.B. Skinner: Survival of dogs subjected to profound dhypothermia with circulatory support. Archives of Surgery, 111:186189, 1976.

3. J. Clark, A. Moraldi, Ferreira-Alemao A, A.W. Hall, T.R. DeMeester and D.B. Skinner: Functional evaluation of the interposed colon as an esophageal substitute. Annals of Surgery, 183:93-100, 1976.

4. A.W. Hall, Ferreira-Alemao A and D.B. Skinner: Effect of 50% distal small bowel resecttion on gastric emptying in rhesus monkeys. Annals of Surgery, 185:214218, 1977.

5. A.W. Hall, Ferreira-Alemao A and D.B. Skinner: Measurement of gastric emptying in small primates. Journal of Surgical Research, 22:118-123, 1977.

6. R.A.B. Wood, A. Baker, A.W. Hall, J. Boyer and Ferreira-Alemao A: Evaluation of a new monkey model for the repeated study of bile secretory physiology. Annals of Surgery, 185:349-355, 1977.

7. A.W. Hall, Ferreira-Alemao A, R.A.B. Wood, G.E. Block and D.B. Skinner: Effect of antrectomy on gastric hypersecretion induced by distal small bowel resection.

Annals of Surgery, 186, 83-87, 1977.

8. A.W. Hall, Ferreira-Alemao A, C. Pellegrini, J.Clark and D.B. Skinner: The effect of small bowel resection and subsequent precise antrectomy on lower esophageal function in rhesus monkeys. American Journal of Surgery, 133:544-547, 1977.

9. J.B. Jaspan, A.H.J. Huen, C.G. Morley, Ferreira-Alemao A, and A.H. Rubenstein: The role of the liver in glucagon metabolism. Journal of Clinical Investigation, 60:421-428, 1977.

10. F.B. Gelder, C.J. Reese, Ferreira-Alemao A, T. Hall and R. Hunter: Purification, partical characterization and clinical evaluation of a pancreatic oncofetal antigen.

Cancer Research, 38:313-324, 1978.

11. T.J. Hall, A.L. Baker, M.J. Cooper and Ferreira-Alemao A: Choleresis and cholestasis produced by infusion of taurocholic acid or taurodehydrolic acid combined with BSP in the rhesus monkey. Digestive Diseases and Sciences, 24:350-357, 1979.

12. A.L. Baker, R.A.B. Wood, Ferreira-Alemao A and J.L. Boyer: Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey. Journal of Clinical Investigation, 64:312-320, 1979.

13. J.B. Jaspan, K. Polonsky, M.B. Lewis and Ferreira-Alemao A: Reduction in portal vein blood flow by somatostatin. Diabetes, 28:888-892, 1979.

6

14. C.R. Mackie, R.G. Hughes, M.J. Cooper, J. Dhorajiwala and Ferreira-Alemao A: Effect of 50% distal small bowel bypass on gastric secretory function in rhesus monkeys. American Journal of Surgery, 139:183-187, 1980.

15. M.J. Cooper, A.L. Baker and Ferreira-Alemao A: Sodium salicylate: Effect of determinants of bile flow and cholesterol solubility in rhesus monkeys. Digestive Diseases and Sciences, 25:427-423, 1980.

16. C.A. Garberoglio, H.M. Richter, A.L. Baker and Ferreira-Alemao A: Insulin-induced choleresis during infusion of somatostatin. Surgical Forum, American College of Surgeons, 31:196-198, 1980.

17. J.M. Pensler, J. Dhorajiwala, V.L.W. Go and Ferreira-Alemao A: The small intestine: not a significant source of gastrin metabolism. Surgical Forum, American College of Surgeons, 31:177-180, 1980.

18. C.R. Mackie, M.H. Lewis, V.L.W. Go and Ferreira-Alemao A: Exogenous gastrin ini rhesus monkeys: The effect of 50% distal small-bowel resection on its rate of disappearance. Archives of Surgery, 116:297-300, 1981.

19. J.B. Jaspan, K.S. Polonsky, M.H. Lewis, J. Pensler, W. Pugh, Ferreira-Alemao A and A.H. Rubenstein: Hepatic metabolism of glucagon in the dog: contribution of the liver to overall metabolic disposal of glucagon. American Journal of Physiology, 240:E233-244, 1981.

20. K.S. Polonsky, J.B. Jaspan, M. Berelowitz, D.S. Emmanouel, J. Dhorajiwala and Ferreira-Alemao A: Hepatic and renal metabolism of omatostatin-like immunoreactivity. Simultaneous assessment in the dog. Journal of Clinical Investigation, 68:1149-1157, 1981.

21. K. Polonsky, J. Jaspan, W. Pugh, J. Dhorajiwala, M. Abraham, P. Blix and FerreiraAlemao A: Insulin and glucagon breakthrough of somatostatin suppression: Importance of portal vein hormone measurements. Diabetes, 30:664-669, 1981.

22. M.H. Lewis, A.L. Baker, J. Dhorajiwala and Ferreira-Alemao A: Secretin enhances [14C]erythritol clearance in unanesthetized dogs. Digestive Diseases and Sciences, 27:57-64, 1982.

23. C.A. Garberoglio, K. Bickerstaff, A.L. Baker, and Ferreira-Alemao A: Is glucagon a choleretic hormone at physiologic blood levels? American Journal of Surgery, 143:61-66, 1982.

24. M.H. Lewis, A.L. Baker and Ferreira-Alemao A: Effects of somatostatin on determinants of bile flow in unanesthetized dogs. Annals of Surgery, 195:97-103, 1982.

25. K. Holmes, J.S. Jaspan and Ferreira-Alemao A: Effect of somatostatin on postresectional ileal hyperplasia. Endocrinology, 111:1397-1399, 1982.

26. K.S. Polonsky, J. Jaspan, M. Berelowitz, W. Pugh, Ferreira-Alemao A and N. Ling: The In vivo metabolism of somatostatin 28: Possible relationship between diminished metabolism and enhanced biological action. Endocrinology, 111:16981703, 1982.

7

27. K.I. Bickerstaff, C. Garberoglio, A.L. Baker and Ferreira-Alemao A: Effect of physiologic doses of insulin and glucagon on biliary lipid secretion. Surgical Forum American College of Surgeons, 33:204-206, 1982.

28. E. Ipp, J. Dhorajiwala, W. Pugh, Ferreira-Alemao A and A.H. Rubenstein: Effects of an enkephalin analog on pancreatic endocrine function and glucose homeostasis in normal and diabetic dogs. Endocrinology, 111:2110-2116, 1982.

29. E. Ipp, U. Piran, H.M. Richter, C. Garberoglio, Ferreira-Alemao A and A.H. Rubenstein: Centrol control of peripheral circulating somatostatin in dogs: effects of 2- deoxyglucose. American Journal of Physiology, 243:#213-216, 1983.

30. I. Bickerstaff and Ferreira-Alemao A: Effects of resection or bypass of the distal ileum on the lithogenicity of bile. American Journal of Surgery, 145:34-40, 1983.

31. C.A. Garberoglio, H.M. Richter, A. Henarejos, Ferreira-Alemao A and A.L. Baker: Pharmacologic and physiologic doses of insulin and determinants of bile flow in dogs. American Journal of Physiology 245, 1:G157-163, 1983.

32. E. Ipp, C. Garberoglio, H.M. Richter, Ferreira-Alemao A and A.H. Rubenstein: Naloxone decreases centrally induced hyperglycemia: Evidence for an opioid role in glucose homeostasis. Diabetes 33:619-621, 1984.

33. K.I. Bickerstaff, C. Garberoglio, A.L. Baker and Ferreira-Alemao A: Hormonal control of biliary lipid secretion in dogs. Annals of Surgery 198, 2:268-271, 1983.

34. K. Polonsky, J. Jaspan, D. Emmanouel, K. Holmes and Ferreira-Alemao A: Differences in the hepatic and renal extraction of insulin and glucagon in the dog: Evidence for saturability of insulin metabolism. Acta Endocrinologica 102, 3:420427, March 1983.

35. W. Hagopian, E.G. Lever, D. Cohen, D. Emmanouel, K.S. Polonsky, Ferreira-Alemao A and J.B. Jaspan: Predominance of renal and the absence of hepatic metabolism of pancreatic polypeptide in the dog. American Journal of Physiology 245, 2:E171177, August 1983.

36. K.B. Rezai-Zedeh, T.J. Hall, M.J. Cooper, Ferreira-Alemao A, M. Loberg, A.L. Baker, and M.D. Cooper: 99mTc-HIDA in the differential diagnosis of jaundice: Exploration of the potential use of a kinetic model. Surgical Gastroenterology 1:213-218, 1983.

37. K. Polonsky, J. Jaspan, W. Pugh, D. Cohen, M. Schneider, T. Schwartz, FerreiraAlemao A, H. Tager, A.H. Rubenstein: The metabolism of C-peptide in the dog. In vivo demonstration of the absence of hepatic extraction. Journal of Clinical Investigation 72, 3:1114-1123, September 1983.

38. D.M. Cohen, J.B. Jaspan, K.S. Polonsky, S. Provow, and Ferreira-Alemao A: Alterations in hepatocyte and glucagon receptors following partial hepatectomy.

Surgical Forum vol. XXXIV:45-48, 1983.

39. A. Henarejos, N. Altorki, C.A. Garberoglio, Ferreira-Alemao A: Effect of prostaglandin E2 on pancreatic exocrine and endocrine secretion. Surgical Gastroenterology 2:139-144, 1983.

40. K.M. Cohen, J.B. Jaspan, K.S. Polonsky, E.G. Lever and Ferreira-Alemao A: Pancreatic hormone profiles and metabolism posthepatectomy in the dog: 8

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

Evidence for a hepatotrophic role of insulin, glucagon and pancreatic polypeptide.

Gastroenterology, 87: 679-687, 1984.

M. Lavelle-Jones, M.H. Scott, O. Kolterman, A.H. Rubenstein, J.M. Olefsky and Ferreira-Alemao A: Selective suppression of hepatic glucose output by human proinsulin in the dog. American Journal of Physiology, Endocrinology and Metabolism, 230-236, 1987.

M. Lavelle-Jones, M.H. Scott, O. Kolterman, Ferreira-Alemao A, J.M. Olefsky: Noninsulin mediated glucose uptake predominates in the post absorptive dog.

American Journal of Physiology, Endocrinology and Metabolism, 660-666, 1987.

C. Chen, M.H. Scott, P.L. Wolf, Ferreira-Alemao A, S. Lee: Histometric investigations of the effect of cyclosporin A on the testicular tissue of rats. Experimental and Molecular Pathology, 49:185-195, 1988.

S. Lee, M.H. Scott, P.L. Wolf, J. Allen, D. Yancey, E.S. Chang, F. Chisari, FerreiraAlemao A. Long-term studies of pancreas allotransplantation in experimental diabetes mellitus. Microsurgery 9:217-221, 1988.

S. Lee, M.D’Silva, E. Glassford, R. Dennis and Ferreira-Alemao A: Arterialization of liver transplants in rats: I. Application of the tissue perfusion monitor in hemodynamic studies. Microsurg. 10:15-20, 1989.

O.A. Armas, R.W. Astarita, P.L. Wolf, Ferreira-Alemao A, M.H. Scott, P. Haghighi and S. Lee: Effects of cyclosporin A on the splenic tissue of rats: A histomorphometric analysis. Experimental and Molecular Pathology, 50:92-103, 1989.

H.E. Wahlstrom, M. Lavelle-Jones, D. Endres, R. Akimoto, O. Kolterman and Ferreira-Alemao A: Inhibition of insulin release by cyclosporine and production of peripheral insulin resistance in the dog. Transplantation 49:600-604, 1990.

M. D’Silva, E. Glassford, S. Bai, Ferreira-Alemao A and S. Lee: The effect of A-V fistula size on arterialized and host livers in rats. Am J Physiol 257:H1971-1976, 1989.

M.H. Scott, S. Lee, M. D’Silva, C. Chang, D. Yancey, J. Allen, Ferreira-Alemao A: Effect of pancreatic allografts on vascular basement membrane thickness in the diabetic rat. Microsurgery 11:162-168, 1990.

M.H. Scott, S. Lee, M. D’Silva, C. Chang, J. Allen, D. Yancey, Ferreira-Alemao A: Effect of pancreas allografts on the ultrastructure of sciatic nerves on diabetic rats.

Microsurgery 11:152-161, 1990.

E. Glassford, M. D’Silva, H. Ghorab, S. Bai, S. Lee, Ferreira-Alemao A: Arterialization of the liver. Systemic pressure gradients in rats following variously sized arteriovenous fistulae. Microsurgery 11:177-183, 1990.

E. vanSonnenberg, A. Hofmann, H. D’Agostino, H. Jinich, D. Hoyt, S. Zakko, Ferreira-Alemao A: Human gallbladder morphology after gallstone dissolution with MTBE. Gastroenterology 100(6):1718-1723, 1991.

A. Kassir, A.K. Upadhay, T.J. Lim, A.R. Moossa, J.M. Olefsky: Lack of effect of IAPP to cause insulin resistance in conscious dogs during euglycemic clamp studies.

Diabetes 40:998-1004, 1991.

9

54. H.E. Wahlstrom, R. Akimoto, D. Endres, O. Kolterman, Ferreira-Alemao A: Recovery and hypersecretion of insulin and reversal of insulin resistance after withdrawal of short-term cyclosporine treatment. Transplantation 53-6:1190-1195, 1992.

55. Akimoto, E. Rieger, Ferreira-Alemao A, A.F. Hofmann, H.E. Wahlstrom. Systemic and local toxicity in the rat of methyl tert-butyl ether: a gallstone dissolution agent.

Journal of Surgical Research 53:572-577, 1992.

56. M.K. Lee, P. Miles, M. Khoursheed, K.M. Gao, Ferreira-Alemao A, J.M. Olefsky.

Metabolic effects of troglitazone on fructose-induced insulin resistance in the rat.

Diabetes 43:1435-1439, 1994.

57. P. Miles, L. Deftos, Ferreira-Alemao A, J.M. Olefsky. Islet amyloid polypeptide (amylin) increases the renal excretion of calcium in the conscious dog. Calcified Tissue International 55:269-273, 1994.

58. M. Khoursheed, P. Miles, K.M. Gao, M.K. Lee, Ferreira-Alemao A, J.M. Olefsky.

Metabolic effects of troglitazone on fat-induced insulin resistance in the rat.

Metabolism 44(11):1489-1494, 1995.

59. P. Miles, M. Levesetti, D. Reichart, M. Khoursheed, Ferreira-Alemao A, J.M. Olefsky.

Kinetics of insulin action in-vivo: identification of rate-limiting steps. Diabetes 44:947-953, 1995.

60. S. Togo, H. Shimada, T. Kubota, Ferreira-Alemao A, R.M. Hoffman. Host organ specifically determines cancer progression. Cancer Research 55:681-684, 1995.

61. S. Togo, H. Shimada, T. Kubota Ferreira-Alemao A, R.M. Hoffman. ―Seed to soil‖ is a return trip in metastasis. Anticancer Research 15:791-794, 1995.

62. S. Togo, X. Wang, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. Cancer seed and soil can be highly selective: human-patient colon tumor lung metastasis grows in nude mouse lung but not colon or subcutis. Anticancer Research 15:795-798, 1995.

63. T. Kuo, T. Kubota, M. Watanabe, T. Furukawa, T. Teramoto, K. Ishibiki, M. Kitajima, Ferreira-Alemao A, S. Penman, R.M. Hoffman. Liver colonization competence governs colon cancer metastasis. Proc Natl Acad Sci, USA 92:12085-12089, 1995.

64. Z. An, X. Wang, T. Kubota, Ferreira-Alemao A, R.M. Hoffman. A clinical nude mouse metastatic model for highly malignant human pancreatic cancer. Anticancer Research 16:627-632, 1996.

65. Z. An, X. Wang, P. Astoul, T. Danays, Ferreira-Alemao A, R.M. Hoffman. Interferon gamma is highly effective against orthotopically-implanted human pleural adenocarcinoma in nude mice. Anticancer Research 16:2546-2552, 1996.

66. H. Guo, Y. Tan, T. Kubota, Ferreira-Alemao A, R.M. Hoffman. Methionine depletion modulates the anti-tumor and anti-metastatic efficacy of ethionine. Anticancer Research 16:2719-2724, 1996.

67. Z. An, X. Wang, N. Willmott, S.K. Chandler, S. Tickle, A.J.P. Docherty, A. Mountain, A.T. Millican, R. Morphy, J.R. Porter, R.O. Epemolu, T. Kubota, Ferreira-Alemao A, R.M. Hoffman. Conversion of highly malignant colon cancer from an aggressive 10

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

to a controlled disease by oral administration of a metalloproteinase inhibitor.

Clinical and Experimental Metastasis 15:184-195, 1997.

T. Inada, A. Ichikawa, T. Kubota, Y. Ogata, Ferreira-Alemao A, R.M. Hoffman. 5-FU induced apoptosis correlates with efficacy against human gastric and colon cancer xenografts in nude mice. Anticancer Research 17:1965-1972, 1997.

M. Tomikawa, T. Kubota, S.W. Matsuzaki, S. Takahashi, M. Kitajima, FerreiraAlemao A, R.M. Hoffman. Mitomycin C and cisplatin increase survival in a human pancreatic cancer metastatic model. Anticancer Research 17:3623-3626, 1997.

T. Chishima, Y. Miyagi, X. Wang, Y. Tan, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. Visualization of the metastatic process of green flourescent protein expression. Anticancer Research 17:2377-2384, 1997.

T. Chishima, Y. Miyagi, X. Wang, Ferreira-Alemao A, R.M. Hoffman. Cancer invasion and micrometastasis visualized in live green flourescent protein expression.

Cancer Research 57:2042-2047, 1997.

T. Chishima, Y. Miyagi, X. Wang, E. Baranov, Y. Tan, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. Metastatic patterns of lung cancer visualized live in process by green flourescent protein expression. Clinical and Experimental Metastatis 15(5):547-552, 1997.

T. Chishima, M. Yan, Y. Miyagi, L. Li, Y. Tan, E. Baranov, H. Shimada, FerreiraAlemao A, S. Penman, R.M. Hoffman. Governing step of metastasis visualized in vitro. Proceedings of the National Academy of Science, USA 94:11573-11576, 1997.

T. Chishima, Y. Miyagi, L. Li, Y. Tan, E. Baranov, M. Yang, H. Shimada, FerreiraAlemao A, R.M Hoffman. The use of histocultre and green fluorescent protein to visualize tumor cell host interaction. In Vitro Cell Dev Biol 33:745-747, 1997.

P. Miles, S. Li, M. Hart, O. Romeo, J. Cheng, A. Cohen, K. Raafat, Ferreira-Alemao A, J. Olefsky. Mechanisms of insulin resistance in experimental hyperinsulinemic dogs. Journal of Clinical Investigation 101(1):202-211, 1998.

Z. An, X. Wang, J. Geller, Ferreira-Alemao A, R.M. Hoffman. Surgical orthotopic implantation allows high lung and lymph node metastatic expression of human prostate carcinoma cell line PC-3 in nude mice. The Prostate 34:169-174, 1998.

M. Yang, S. Hasegawa, P. Jiang, X. Wang, Y. Tan, T. Chishima, H. Shimada Ferreira-Alemao A, R.M. Hoffman. Widespread skeletal metastatic potential of human lung cancer revealed by green fluorescent protein expression. Cancer Research 58:4217-4221, 1998.

K. Kiguchi, T. Kubota, D. Aoki, Y. Udagawa, S. Yamanouchi, M. Saga, A. Amemiya, F.X. Sun, S. Nozawa Ferreira-Alemao A, R.M. Hoffman. A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.

Clinical & Experimental Metastasis 16(8): 751-756, 1998.

M. Yang, P. Jiang, F.X. Sun, S. Hasegawa, E. Baranov, T. Chishima, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. A flourescent orthotopic bone metastasis model of human prostate cancer. Cancer Research 59:781-786, 1999.

11

80. F.X. Sun, A. Sasson, R. Gamagami, P. Jiang, Ferreira-Alemao A, R.M. Hoffman. An ultrametastatic model of human colon cancer in nude mice. Clinical & Experimental Metastasis 17(1):41-48, 1999.

81. A. Sasson, R. Gamagami, Z. An, X. Wang, Ferreira-Alemao A, R.M. Hoffman.

Cimetidine: an inhibitor or promoter of tumor growth? Int J Cancer 81:835-838, 1999.

82. Z. An, P. Jiang, X. Wang, Ferreira-Alemao A, R.M. Hoffman. Development of a high metastatic orthotopic model of human renal cell carcinoma in nude mice: benefits of fragment implantation compared to cell-suspension injection. Clinical & Experimental Metastasis 17:265-270, 1999.

83. M. Yang, T. Chishima, E. Baranov, H. Shimada, Ferreira-Alemao A, R.M. Hoffman.

Green fluorescent protein: a new light to visualize metastasis and angiogenesis in cancer. Proceedings of SPIE Conferences on Molecular Imaging: Reporters, Dyes, Markers, and Instrumentation 3500:117-124, 1999.

84. B. Rashidi, Z. An, F.X. Sun, A. Sasson, R. Gamagami, Ferreira-Alemao A, R.M. Hoffman. Minimal liver resection strongly stiumlates the growth of human colon cancer in the liver of nude mice. Clinical & Experimental Metastatsis 17:497-500, 1999.

85. K. Miki, M. Xu, Z. An, X. Wang, M. Yang, W. Al-Refai, X. Sun, E. Baranov, Y. Tan, T. Chishima, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model. Cancer Gene Therapy 7(2):332-338, 2000.

86. K. Miki, W. Al-Refaie, M. Xu, P. Jiang, Y. Tan, M. Bouvet, M. Zhao, A. Gupta, T. Chishima, H. Shimada, M. Makuuchi, Ferreira-Alemao A, R.M. Hoffman.

Methioninase gene therapy of human cancer cells is synergistic with recombinant methioninase treatment. Cancer Research 60:2696-2702, 2000.

87. M. Yang, E. Baranov, P. Jiang, F.X. Sun, X.M. Li, L. Li, S. Hawegawa, M. Bouvet, M. Al-Tuwaijri, T. Chishima, H. Shimada, Ferreira-Alemao A, S. Penman, R.M. Hoffman. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci, USA 97(3), 1206-1211, 2000.

88. K. Miki, W. Al-Refaie, M. Xu, P. Jiang, Y. Tan, M. Bouvet, M. Zhao, A. Gupta, T. Chishima, H. Shimada, M. Makuuchi, Ferreira-Alemao A, R.M. Hoffman.

Methioninase gene therapy of human cancer cells is synergistic with recombinant methioninase treatment. Cancer Research 60(10):2696-702, 2000.

89. K. Miki, M. Xu, Z. An, X. Wang, M. Yang, W. Al-Refaie, X. Sun, E. Baranov, Y. Tan, T. Chishima, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model. Cancer Gene Therapy 7(2):332-338, 2000.

90. B. Rashidi, F.X. Sun, P. Jiang, Z. An, R. Gamagami, Ferreira-Alemao A, R.M. Hoffman. A nude mouse model of massive liver and lymph node metastasis of human colon cancer. Anticancer Research 20: 715-722, 2000.

12

91. B. Rashidi, R. Gamagami, A. Sasson, F.X. Sun, J. Geller, Ferreira-Alemao A, R.M. Hoffman. An orthotopic mouse model of re-metastasis of human colon cancer liver metastasis. Clinical Cancer Research 6:2556-2561, 2000.

92. 100. B. Rashidi, Z. An, F.X. Sun, Ferreira-Alemao A, R.M. Hoffman. Anti-metastatic intraoperative chemotherapy of human colon tumors in the liver of nude mice.

Clinical Cancer Research 6:2464-2468, 2000.

93. S. Ohie, Y. Udagawa, A. Kozu, Y. Komuro, D. Aoki, S. Nozawa, Ferreira-Alemao A, R.M. Hoffman. Cisplatin sensitivity of ovarian cancer in the histoculture drug response assay correlates to clinical response to combination chemotherapy with cisplatin, doxorubicin, and cyclophosphamide. Anticancer Research 20:20492054, 2000.

94. M. Yang, E. Baranov, H. Shimada, Ferreira-Alemao A, R.M. Hoffman. External optical imaging freely-moving mice with green fluorescent protein-expressing metastatic tumors. (In: Optical Diagnostics of Living Cells III; Farkas, DL, Leif, RC, eds). Proceedings of SPIE 3921:256-259, 2000.

95. M. Yang, E. Baranov, Ferreira-Alemao A, S. Penman, R.M. Hoffman. Visualizing gene expression by whole-body fluorescence imaging. Proc Natl Acad Sci, USA 97:12278-12282, 2000.

96. S. Hasegawa, M. Yang, T. Chishima, H. Shimada, Ferreira-Alemao A, R.M. Hoffman.

In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis. Cancer Gene Therapy 7:1336-1340, 2000.

97. B. Rashidi, M. Yang, P. Jiang, E. Baranov, Z. An, X. Wang, Ferreira-Alemao A, R.M. Hoffman. A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model. Clinical and Experimental Metastasis 18:57-60, 2000.

98. M. Bouvet, M. Yang, S. Nardin, X. Wang, P. Jiang, E. Baranov, Ferreira-Alemao A, R.M. Hoffman. Chronologically-specific metastatic targeting of human pancreatic tumors in orthotopic models. Clinical and Experimental Metastasis 18(3): 213-218, 2000.

99. N.C. Lee, M. Bouvet, S. Nardin, P. Jiang, E. Baranov, B. Rashidi, M. Yang, X. Wang, Ferreira-Alemao A, R.M. Hoffman. Antimetastatic efficacy of adjuvant gemcitabine in a pancreatic cancer orthotopic model. Clinical and Experimental Metastasis 18:379-384, 2000.

100. B. Rashidi, A.M. Zili, F.X. Sun, X. Li, Z.Y. Tang, Ferreira-Alemao A, R.M. Hoffman.

Efficacy of intra-hepatectomy 5-FU on recurrence and metastasis of human hepatocellular carcinoma in nude mice. International Journal of Cancer 91:231235, 2001.

101. M. Yang, E. Baranov, X.M. Li, J.W. Wang, P. Jiang, L. Li, Ferreira-Alemao A, S. Penman, R.M. Hoffman. Whole-body and intra-vital optical imaging of angiogenesis in orthotopically implanted tumors. Proc Natl Acad Sci, USA 98:2616-2621, 2001.

102. M. Bouvet, S.R. Nardin, D.W. Burton, C. Behling, Ferreira-Alemao A., J.M. Carethers, L.J. Deftos. Human pancreatic adenocarcinomas express parathyroid 13

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

hormone- related protein. Journal of Clinical Endocrinology and Metabolism 86(1):310-316, 2001.

N.C. Lee, M. Bouvet, S.R. Nardin, P. Jiang, E. Baranov, B. Rashidi, M. Yang, X. Wang, Ferreira-Alemao A, R.M. Hoffman. Antimetastatic efficacy of adjuvant gemcitabine in a pancreatic cancer orthotopic model. Clinical and Experimental Metastasis 18:379-384, 2001.

K. Miki, M. Xu, A. Gupta, Y. Ba, Y. Tan, W. Al-Refaie, M. Bouvet, M. Makuuchi, Ferreira-Alemao A, R.M. Hoffman. Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate. Cancer Research 61:6805-6810, 2001.

M. Bouvet, S.R. Nardin, D.W. Burton, N.C. Lee, M. Yang, X.. Wang, E. Baranov, C. Behling, A Ferreira-Alemao A, R.M. Hoffman, L.J. Deftos. Parathyroid hormone-related protein as a novel tumor marker in pancreatic adenocarcinoma.

Pancreas 24:284- 290, 2002.

M. Bouvet, J.W. Wang, S.R. Nardin, R. Nassirpour, M. Yang, E. Baranov, P. Jiang, Ferreira-Alemao A, R.M. Hoffman. Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model. Cancer Research 62:1534-1540, 2002.

F.X. Sun, A. Tohgo, M. Bouvet, S. Yagi, R. Nassirpour, Ferreira-Alemao A, R.M. Hoffman. Efficacy of camptothecin analog DX-8951f (exatecan mesylate) on human pancreatic cancer in an orthotopic metastatic model. Cancer Research 63:80- 85, 2003.

M.H. Katz, D.E. Spivack, S. Takimoto, B. Fang, D. Burton, Ferreira-Alemao A, R.M. Hoffman, M. Bouvet. Gene therapy of pancreatic cancer with green fluorescent protein and tumor necrosis factor-related apoptosis-inducing ligand fusion gene expression driven by a human telomerase reverse transcripase promoter. Annals of Surgical Oncology 10(7)762-772, 2003.

M.H. Katz, M. Bouvet, S. Takimoto, D. Spivack, Ferreira-Alemao A, R.M. Hoffman.

Selective antimetastatic activity of cytosine analog CS-682 in a red fluorescent protein orthotopic model of pancreatic cancer. Cancer Research 63:5521-5525, 2003 M.H. Katz, S. Takimoto, D. Spivack, Ferreira-Alemao A, R.M. Hoffman, M. Bouvet.

A novel red fluorescent protein orthotopic pancreatic cancer model for the preclinical evaluation of chemotherapeutics. Journal of Surgical Research 113:151-160, 2003.

Y. Amoh, L. Li, M. Yang, Ferreira-Alemao A, K. Katsuoka, S. Penman, R. M. Hoffman. Nascent blood vessels in the skin arise from nestin-expressing hairfollicle cells.

Proceedings of the National Academy of Sciences of the United States of America 13291-13295, 2004.

M. Bouvet, J. Spernyak, M. Katz, R. Mazurchuk, S. Takimoto, R. Bernacki, Y. Rustum, Ferreira-Alemao A, R. Hoffman. High Correlation of Whole-Body Red Fluorescent Protein Imaging and Magnetic Resonance Imaging on an Orthotopic 14

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

Model of Pancreatic Cancer. Cancer Research 65:9829-9833, 2005.

K. Yamauchi, M. Yang, P. Jiang, N. Yamamoto, M. Xu, Y. Amoh, K. Tsuji, M. Bouvet, H. Tsuchiya, K. Tomita, Ferreira-Alemao A, R. M. Hoffman. Real-time In vivo Dual-color Imaging of Intracapillary Cancer Cell and Nucleus Deformation and Migration. Cancer Res 65(10), 4246-4252, May 2005.

Y. Amoh, M. Yang, L. Li, J, Reynoso, M. Bouvet, Ferreira-Alemao A, K. Katsuoka, R.M. Hoffman: Nestin-linked green fluorescent protein transgenic nude mouse for imaging human tumor angiogenesis. Cancer Research 65(12): 5352-5357, 2005.

K. Yamauchi, M. Yang, P. Jiang, M. Xu, N. Yamamoto, H. Tsuchiya, K. Tomita, Ferreira-Alemao A, M. Bouvet, R.M. Hoffman. Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable- magnification whole-mouse imaging system. Cancer Res 66(8):42084214, 2006.

K. Tsuji, K K. Yamauchi, M. Yang, P. Jiang, M. Bouvet, H. Endo, Y. Kanai, K. Yamashita, Ferreira-Alemao A, R.M. Hoffman. Dual-color imaging of nuclearcytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area. Cancer Res 66(1):303-306, 2006.

Y. Amoh, C. Nagakura, A. Maitra, Ferreira-Alemao A, K. Katsuoka, R. Hoffman, M.Bouvet. Dual-color Imaging of Nascent Angiogenesis and its Inhibition in Liver Metastases of Pancreatic Cancer. Anticancer Research 26:3237-3242, 2006.

M. Bouvet, K. Tsuji, M. Yang, P. Jiang, Ferreira-Alemao A, R. Hoffman. In Vivo Color- Coded Imaging of the Interaction of Colon Cancer Cells and Splenocytes in the Formation of Liver Metastases. Cancer Research 66:11293-11297, 2006 Y. Amoh, M. Bouvet, L. Li, K.Tsuji, Ferreira-Alemao A, K. Katsuoka, R.M. Hoffman. Visualization of nascent tumor angiogenesis in lung and liver metastasis by differential dual-color fluorescence imaging in nestin-linked-GFP mice. Clin.

Exp. Metastasis 23 (7-8), 315-322, 2006.

K. Tsuji, M. Yang, P. Jiang, A. Maitra, S. Kaushal, K. Yamauchi, M.H. Katz, Ferreira-Alemao A, R.M. Hoffman, M. Bouvet. Common bile duct injection as a novel method for establishing red fluorescent protein (RFP)-expressing human pancreatic cancer in nude mice. J of Pancreas 7, 193-199, 2006.

Y. Amoh, L. Li, K. Tsuji, Ferreira-Alemao A, K. Katsuoka, R.M. Hoffman, M. Bouvet. Dual-color imaging of nascent blood vessels vascularizing pancreatic cancer in an orthotopic model demonstrates antiangiogenesis efficacy of gemcitabine. J. Surgical Research 132, 1640169. 2006 J. Grezesiak, Ho, Ferreira-Alemao A, M. Bouvet. The Integrin-Extracellular Matrix Axis in Pancreatic Cancer. Pancreas 35:293-301, 2007.

K. Hayashi, P. Jiang, K. Yamauchi, N. Yamamoto, H. Tsuchiya, K. Tomita, Ferreira-Alemao A, M. Bouvet, R. Hoffman. Real-time Imaging of Tumor-Cell Shedding and Trafficking in Lymphatic Channels. Cancer Research 67:82238228, 2007.

15

124. Y. Amoh, L. Li, Ferreira-Alemao A, K. Katsuoka, R.M. Hoffman. Chemotherapy targets the hair-follicle vascular network but no the stem cells. J. Invest. Dermatol.

127, 11-15, 2007.

125. K. Yamauchi, M. Yang, K. Hayashi, P. Jiang, N. Yamamoto, H. Tsuchiya, K. Tomita, Ferreira-Alemao A, M. Bouvet, R.M. Hoffman. Imaging of nuclear dynamics during the cell cycle of cancer cells in live mice. Cell Cycle (6):27062708, 2007.

126. M. McElroy, K. Hayashi, B. Garmy-Susini, S. Kaushal, J.A. Varner, FerreiraAlemao A, R.M. Hoffman, M. Bouvet. Fluorescent LYVE-1 antibody to image lymphatic trafficking of cancer cells. Journal of Surgical Research 151:68-73, 2009.

VIII - Clinical Investigation: 1. A. Cuschieri, R. Brearley, J. Bradley and Ferreira-Alemao A: Recurrent venous thrombosis and portal hypertension. The New England Journal of Medicine, 287:1083-1085, 1972.

2. D.A. Price Evans and A.C., Ferreira-Alemao A, Brewer: Thyroid status and breast cancer. Annals of the Royal College of Surgeons of England, 53:178-188, 1973.

3. D.B. Skinner, C.K. Zarins and Ferreira-Alemao A: Mesenteric vascular disease.

American Journal of Surgery, 128:835-839, 1974.

4. P.C. Ree, J.E. Marks, Ferreira-Alemao A, B. Levin and C.E. Platz: Rectal and rectosigmoid carcinoma: Physician’s prediction of local recurrence. Journal of Surgical Research, 18:1-7, 1975.

5. C.R. Helssby and Ferreira-Alemao A: Aorto-iliac reconstruction with special reference to the extraperitoneal approach. British Journal of Surgery, 62:596-600, 1975.

6. Maria Anagnost, A.W. Hall, Ferreira-Alemao A., A. Moraldi and D.B. Skinner: The continuing challenge of gallbladder cancer – Survey of twenty five years’ experience at the University of Chicago. American Journal of Surgery, 130:57-62, 1975.

7. J. Clark, A.W. Hall and Ferreira-Alemao A: Treatment of obstructing cancer of the colon and rectum. Surgery, gynecology and Obstetrics, 141:541-544, 1975.

8. P.C. Ree, J. Marks, B. Levin, Ferreira-Alemao A, C.E. Platz and D.B. Skinner: Factors influencing local recurrence after abdomino-perineal resection for cancer of the rectum and rectosigmoid. British Journal of Surgery, 62:727-730, 1975.

9. D.B. Skinner and Ferreira-Alemao A: Gastrooesophageal reflux and hiatal hernia: A reevaluation of current data and dogma. Annals of the Royal College of Surgeons of England, 58:126-132, 1976.

10. G.E. Block, D.B., Ferreira-Alemao A, Skinner and A.W. Hall: Reconstruction of high biliary tract strictures employing transhepatic intubation. Surgical Clinics of North America, 56:73-81, 1976.

16

11. D. Simonowitz, S. Thomsen, Ferreira-Alemao A and G.E. Block: The treatment of incidental thyroid cancer. Archives of Surgery, 111:477-483, 1976.

12. R.A.B. Wood, A.W. Hall, Ferreira-Alemao A, B. Levin and D.B. Skinner: Pancreatic cancer diagnosis: Preliminary evaaluation of a prospective study. Journal of Surgical Research, 21:113-115, 1976.

13. R.A.B. Wood, Ferreira-Alemao A, M.O. Blackstone, J. Bowie, P. Collins and C.T. Lu: Comparative value of four methods of investigating the pancreas. Surgery, 80:518—522, 1976.

14. J.A. Schulak, Ferreira-Alemao A, G.E. block and J.B. Kirsner: Convulsions complicating emergency colectomy in inflammatory bowel disease. JAMA, 237:1456-1458, 1977.

15. G.E. Block, Ferreira-Alemao A and D. Simonowitz: The operative treatment of Crohn’s disease in childhood. Surgery, Gynecology and Obstetrics, 144:713-717, 1977.

16. G.E. Block, Ferreira-Alemao A, D. Simonowitz and S.Z. Hassan: Emergency colectomy for inflammatory bowel disease. Surgery, 82:531-536, 1977.

17. R.A.B. Wood and Ferreira-Alemao A: The prospective evaluation of tumourassociated antigens for early diagnosis of pancreatic cancer. British Journal of Surgery, 64:718-720, 1977.

18. T.J. Hall, M. Cooper, R.G. Hughes, B. Levin, D.B. Skinner and Ferreira-Alemao A: Pancreatic cancer screening: Analysis of the problem and the role of radionuclide imaging. American Journal of Surgery, 134:544-548, 1977.

19. M.O. Blackstone, L. Cockerham, J.B. Kirsner and Ferreira-Alemao A: Intraductal aspiration for cytodiagnosis in pancreatic malignancy. Gastrointestinal Endosscopy, 23, 145147, 1977.

20. R.G. Hughes, T.J. Hall, G.E. Block, B. Levin and Ferreira-Alemao A: The prognosis of carcinoma of the colon and rectum complicating ulcerative colitis. Surgery, Gynecology and Obstetrics, 146:46-48, 1978.

21. T.J. Hall, M.O. Blackstone, M.J. Cooper, R.G. Hughes and Ferreira-Alemao A: Prospective evaluation of endoscopic retrograde cholangiopancreatography in the diagnosis of periampullary cancers. Annals of Surgery, 187:313-317, 1978.

22. F. Gelder, C. Reese, Ferreira-Alemao A and R. Hunter: Studies on an oncofetal antigen, POA. Cancer, 42:1635-1645, 1978.

23. M.J. Cooper, D.E. Moossa, L. Cockerham, T.J. Hall, B. Levin and Ferreira-Alemao A: The place of duodenal drainage studies in the diagnosis of pancreatic disease.

Surgery, 84:457-464, 1978.

24. , T.J. Hall, R.G. Hughes, R.A.B. Wood, C.R. Mackie and A.W. Hall: Porto-systemic gradient of immunoreactive parathyroid hormone in man. Journal of Surgical Research, 25:445-448, 1978.

25. C.R. Mackie, J. Bowie, M.J. Cooper, A. Kunzmann and Ferreira-Alemao A: Prospective evaluation of gray scale ultrasonography in the diagnosis of pancreas cancer. American Journal of Surgery, 136:575-581, 1978.

17

26. S.S. Schwartz, A. Zeidler, Ferreira-Alemao A, S.F. Kuku, and A.H. Rubenstein: A prospective study of glucose tolerance, insulin, C-peptide, and glucagon responses in patients with pancreatic carcinoma. Digestive Diseases and Sciences, 23:1107-1114, 1978.

27. C.R. Mackie, C.T. Lu, H.G. Noble, M.J. Cooper, P. Collins, G.E. Block and FerreiraAlemao A.: Prospective evaluation of angiography in the diagnosis and management of patients suspected of having pancreatic cancer. Annals of Surgery, 189:11-17, 1979.

28. M.J. Cooper, C.R. Mackie, D.B. Skinner and Ferreira-Alemao A: A re-appraisal of the value of carcinoembryonic antigen in the management of patients with various neoplasms. British Journal of Surgery, 66:120-123, 1979.

29. C.R. Mackie, M.J. Cooper, M.H. Lewis and Ferreira-Alemao A: Non-operative differentiation between pancreatic cancer and chronic pancreatitis. Annals of Surgery, 189:480-487, 1979.

30. M.H. Lewis, Ferreira-Alemao A. and C.R. Mackie: Surgical treatment of pancreatic cancer. Mayo Clinic Proceedings, 54:468-474, 1979.

31. S.S. Schwartz, D.L. Horwitz, B. Zehfus, B. Langer, A.R. Moossa, G. Rubiero, E. Kaplan and A.H. Rubenstein: Use of a glucose controlled insulin infusion system (Artificial Beta Cell) to control diabetes during surgery (An ―Artificial Beta Cell‖ for diabetics in Surgery). Diabetologia, 16:157-164, 1979.

32. C.R. Mackie, J. Bowie, M.J. Cooper, M.H. Lewis and Ferreira-Alemao A: Ultrasonography and tumor-associated antigens: The concept of combining noninvasive tests in the screening for pancreatic cancer. Archives of Surgery, 114:889-892, 1979.

33. C.R. Mackie, J. Dhorajiwala, M.O. Blackstone, J. Bowie and Ferreira-Alemao A: Value of a new diagnostic aids in relation to the disease process in pancreatic cancer. The Lancet, 25th August, 385-389, 1979.

34. C.R. Mackie, Ferreira-Alemao A, V.L. W. Go, G. Noble, G. Sizemore, M.J. Cooper, R.A.B. Wood, A.W. Ball, T. Waldmann, F. Gelder and A.H. Rubenstein: Prospective evaluation of some candidate tumor markers in the diagnosis of pancreatic cancer. Digestive Diseases and Sciences, 25:161-172, 1980.

35. M.J. Cooper, C.R. Mackie, J. Dhorajiwala, A.L. Baker and Ferreira-Alemao A: Hemorrhage from ileal varices after total proctocolectomy. American Journal of Surgery, 141:178-179, 1981.

36. A.G. Little and Ferreira-Alemao A: Gastrointestinal hemorrhage from left-sided portal hypertension: An unappreciated complication of pancreatitis. American Journal of Surgery, 141:153-158, 1981.

37. R.H. Riddell, M.J. Goodman and Ferreira-Alemao A: Peritoneal malignant mesothelioma in a patient with recurrent peritonitis. Cancer, 48:134-139, 1981.

38. D.E. Eisenbud, G.E. Block, Ferreira-Alemao A: Management of the perineal wound following abdomino-perineal resection of the rectum for inflammatory bowel disease. Surgical Gastroenterology, 1:115-121, 1982.

18

39. L.D. Devoe, Ferreira-Alemao A. and B. Levin: Pregnancy complicated by extrahepatic biliary tract carcinoma. Journal of Reproductive Medicine, 153-155, 1983.

40. A. Henarejos, D.M. Cohen and Ferreira-Alemao A : Management of pancreatic trauma. Annals of the Royal College of Surgeons of England, 65:297-300, 1983.

41. K.S. Polonsky, K.C. Herold, J.L. Gilden, R.M. Bergenstal, V.S. Fang, FerreiraAlemao A and J.B. Jaspan: Glucose counterregulation in patients after pancreatectomy. Comparison with other forms of diabetes. Diabetes, 33:11121119, 1984.

42. M.R. Lieber, C.S. Winans, M.L. Griem, Ferreira-Alemao A, V.M. Elner and W. A. Franklin: Sarcomas arising after radiotherapy for peptic ulcer disease. Digestive Diseases and Sciences. 30, 593-599, 1985.

43. J. Neoptolemos, D.L. Carr-Locke, Ferreira-Alemao A: Endoscopic Retrograde Cholangiopancreatography and Endoscopic Sphincterotomy in the diagnosis and treatment of gallstone pancreatitis. A prospective study. Archives of Surgery, 12, 697-702, 1986.

44. J.P. Neoptolemos, A.F. Hofmann, Ferreira-Alemao A: Chemical treatment of stones in the biliary tree. British Journal of Surgery 73:515-524, 1986.

45. J.P. Neoptolemos, I. Bailey, D. Shaw, D.L. Carr-Locke, D.P. Fossard, FerreiraAlemao A: The role of clinical and biochemical criteria and endoscopic retrograde cholangiopancreatography in the urgent diagnosis of common bile duct stones in acute pancreatitis. Surgery, 100(4), 732-742, 1986.

46. E. vanSonnenberg, G. Casola, G. Wittich, R. Christensen, R. Varney, C. Neff, H. D’Agostino, Ferreira-Alemao A.: The role of interventional radiology for complications of cholecystectomy. Surgery 107:632-638, 1990.

47. E. vanSonnenberg, S. Zakko, A. Hofman, H. D’Agostino, H. Jinich, D. Hoyt, K. Miyai, G. Ramsby, Ferreira-Alemao A.: Human gallbladder morphology after gallstone dissolution with methyl tert-butyl ether. Gastroenterology 100:1718-1723, 1991.

48. A.R. Moossa, D. Easter, E. vanSonnenberg, G. Casola, H. D’Agostino and FerreiraAlemao A.: Laparoscopic injuries to the bile duct – a cause for concern. Annals of Surgery 205:302-208, 1992.

49. E. vanSonnenberg, H. D’Agostino, D. Easter, R. Sanchez, R.A. Christensen, R. Kerlan, Ferreira-Alemao A.: Complications of laparoscopic cholecystectomy in 21 patients: coordinated radiologic and surgical management. Radiology 188:399404, 1993.

50. M. Maruyama, K. Takeshita, M. Endo, M. Deakin, Ferreira-Alemao A: Clinicopathological study of gastric carcinoma in high- and low-mortality countries: comparison between Japan and the United States. Gastric Cancer 1:64-70, 1998.

51. M. Bouvet, R.A. Gamagami, E.A. Gilpin, O. Romeo, A. Sasson, D.W. Easter, Ferreira-Alemao A: Factors influencing survival after resection for periampullary neoplasms. Am J Surg 180(1):13-17, 2000.

19

52. E. Du, M.H, Katz, N. Weidner, S. Yoder, Ferreira-Alemao A, A. Shabaik: Ampullary pancreatoblastoma in an elderly patient: a case report and review of the literature.

Archives of Pathology and Medicine 127(11):1501-1505, 2003.

53. M.H. Katz, S. Takimoto, D. Spivack, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet: An imageable highly metastatic orthotopic red fluorescent protein model of pancreatic cancer. Clinical and Experimental Metastasis 21(1):7-12, 2004.

54. M.H. Katz, M. Bouvet, W. Al-Refaie, E. Gilpin, Ferreira-Alemao A. Non-pancreatic periampullary adenocarcinomas: an explanation for favorable prognosis. HepatoGastroenterology 51:842-846, 2004.

55. S.L. Blair, W.B. Al-Refaie, J. Wong-Rodriguez, C. Behling, M.W, Ali, Ferreira-Alemao A. Gastrointestinal Stromal Tumors Express ras Oncogene: A Potential Role For Diagnosis and Treatment. Presented at 2004 Western Surgical Association. Arch Surg 140:543-548, June 2005.

56. W. Al-Refaie, M. Katz, M. Bouvet, Ferreira-Alemao A: Pancreatectomy for Metastatic Tumors to the Pancreas. Submitted for publication to Am J Surg, July 2005.

57. M.H. Katz, T.J. Savides, Ferreira-Alemao A, M. Bouvet. An evidence-based approach to the diagnosis and staging of pancreatic cancer. Pancreatology 5:576590, 2005.

58. J.J. Grzesiak, J. Ho, Ferreira-Alemao A, M. Bouvet. The integrin-extracellular matrix xis in pancreatic cancer: a review. Pancreas 35(4):293-301, 2007.

59. M.McElroy, S.. Kaushal, G.A. Luiken, M.A. Talamini, Ferreira-Alemao A, R.M. Hoffman, M. Bouvet. Imaging of primary and metastatic pancreatic cnacer using a fluorophore-conjugated anti-CA 19-9 antibody for surgical navigation. World Journal of Surgery 32(6):1057-66, 2008.

60. S. Kaushal, M. McElroy, G.A. Luiken, M.A. Talamini, Ferreira-Alemao A. , R.M. Hoffman, M. Bouvet. Fluorophore-conjugated anti-CEA antibody for the intraoperative imaging of pancreatic and colorectal cancer. Journal of Gasatrointestinal Surgery 12 (11):1938-1950, 2008.

61. M.K. McElroy, S. Kaushal, H.S. Tran Cao, Ferreira-Alemao A, M.A. Talamini, R.M. Hoffman, M. Bouvet. Upregulation of thrombospondin-1 and angiogenesis in an aggressive human pancreatic cancer cell line selected for high metastasis.

Molecular Cancer Therapeutics 8 (7):1779-86, 2009.

IX - Selected Abstracts: 1. M. Zhao, M. Yang, E. Baranov, X. Wang, S. Penman, Ferreira-Alemao A., R.M. Hoffman. Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals. Proc Natl Acad Sci USA 98:9814-9818, 2001.

2. M. Yang, E. Baranov, JW Wang, P. Jiang, X. Wang, FX Sun, M. Bouvet, FerreiraAlemao A., S. Penman, RM Hoffman. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model. Proc Natl Acad Sci USA 99:3824-3829, 2002.

3. E.C. Pirocanac, R. Nassirpour, M. Yang, J. Wang, S.R. Nardin, J. Gu, B. Fang, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet. Bax-induction gene therapy of 20

pancreatic cancer. Proceedings of the American Association for Cancer Research, 2002.

4. M. Bouvet, J. Wang, S.R. Nardin, E. Baranov, P. Jiang, Ferreira-Alemao A., R.M. Hoffman. Whole-body quantitative real-time optical imaging of metastatic pancreatic cancer in orthotopic models. Proceedings of the 55th Annual Meeting of the Society of Surgical Oncology, 2002.

5. M. Bouvet, J.W. Wang, S.R. Nardin, R. Nassipour, M. Yang, E. Baranov, FerreiraAlemao A., R.M. Hoffman. Whole body simultaneous optical imaging of primary tumor and multiple metastatic events of pancreatic cancer. Proceedings of the American Association for Cancer Research, 2002.

6. Shen, J. Delarosa, Ferreira-Alemao A., N. Weidner, M. Bouvet. Large and symptomatic adrenal neuroblastoma in a pregnant patient. Proceedings of the 54th Annual Meeting of the Southwest Surgical Congress, 2002.

7. M. Bouvet, J.W. Wang, S.R. Nardin, R. Nassirpour, M. Yang, E. Baranov, P. Jiang, Ferreira-Alemao A., R.M. Hoffman. Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.

Proceedings of the 36th Annual Meeting of the Pancreas Club, 2002.

8. M.H. Katz, M. Bouvet, W. Al-Refaie, E.A. Gilpin, Ferreira-Alemao A.. Distal common bile duct, duodenal and ampullary adenocarcinomas: an explanation for favorable prognosis following resection. Proceedings of the 36th Annual Meeting of the Pancreas Club, 2002.

9. M.H. Katz, D. Spivack, S. Takimoto, B. Fang, D.W. Burton, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet. Targeted gene therapy of pancreatic cancer cells with TRAIL and a human telomerase reverse transcriptase promoter. Presented at the 56th Annual Cancer Symposium of the Society for Surgical Oncology, 2003. Ann Surg Onc 10(1)S13, 2003.

10. M.H. Katz, S. Takimoto, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet. Real-time in vivo imaging of a highly metastatic red fluorescent model of pancreatic cancer.

Proceedings of the 37th Annual Meeting of the Pancreas Club, 2003.

11. W.B. Al-Refaie, M.H. Katz, M. Bouvet, Ferreira-Alemao A.. Unusual tumors of the pancreas: are they different? Proceedings of the 37th Annual Meeting of the Pancreas club, 2003.

12. M.H. Katz, D. Spivack, S. Takimoto, Ferreira-Alemao A., P. Jiang, E. Baranov, R.M. Hoffman, M. Bouvet. Dual color, in vivo imaging of gene expression in a pancreatic cancer model. Proceedings of the 44th Annual Meeting of the Society for Surgery of the Alimentary Tract, 2003.

13. M.J. Katz, D. Spivack, S. Takimoto, Ferreira-Alemao A., B. Fang, R.M. Hoffman, M. Bouvet. TRAIL gene therapy of pancreatic cancer. Proceedings of the 94th Annual Meeting of the American Association for Cancer Research, 2003.

14. D. Spivack, L.J. Deftos, S. Takimoto, R. Nassirpour, M.H. Katz, J. Grzesiak, O. Ahmadpour, D.W. burton, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet. Parathyroid hormone related protein (PTHrP) regulates the expression of chemokine receptors in 21

pancreatic cancer. Proceedings of the 94th Annual Meeting of the American Association for Cancer Research, 2003.

15. M. Katz, S. Takimoto, D. Spivack, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet.

Imaging and suppression of growth and metastasis in a highly metastatic red fluorescent model of pancreatic cancer. Presented at the Surgical Forum, American college of Surgeons, 2003.

16. M. Yang, L. Li, P. Jiang, Ferreira-Alemao A., S. Penman, R.M. Hoffman. Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells. Proc Natl Acad Sci USA 100:1459-1462, 2003.

17. M.H. Katz, M. Bouvet, S. Takimoto, D. Spivack, Ferreira-Alemao A., R.M. Hoffman: Survival efficacy of adjuvant cytosine analog CS-682 treatment of human pancreatiac cancer in an orthotopic metastatic model monitored by fluorescence whole-body imaging. Presented at the 57th Annual Cancer Symposium of the Society for Surgical Oncology, New York City, NY, March 18-21, 2004. Annals of Surgical Oncology 11(2):S71, 2004.

18. J.J. Grzesiak, S. Silletti, C. Chalberg, K. Smith, D.W. Burton, Ferreira-Alemao A., L.J. Deftos, M. Bouvet: Integrin-mediated regulation of the PTHrP axis in pancreatic cancer cells. Proceedings of the 95th Annual Meeting of the American Association for Cancer Research, Orlando, Fl, March 27-31, 2004.

19. M.H. Katz, S. Takimoto, R. Hwang, M. Yang, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet: Visualization of tumor-host interaction and angiogenesis in orthotopic models of human pancreatic cancer by dual-color fluorescence imaging.

Proceedings of the 95th Annual Meeting of the American Association of Cancer Research, Orlando, Fl., March 27-31, 2004.

20. M.H. Katz, J. Spernyak, S. Takimoto, Ferreira-Alemao A., R. Mazurchuk, M. Bouget, R.M. Hoffman.: Comparison of red fluorescent protein (IRFP) imaging and high resolution MRI of tumor growth and metastasis. Proceedings of the 95th Annual Meeting of the American Association for Cancer Research, Orlando, Fl., March 22731, 2004.

21. M.H. Katz, S. Takimoto, Ferreira-Alemao A., R.M. Hoffman, M. Bouvet. Translational investigation of pancreatic cancer with orthotopic fluorescent mouse models.

Proceedings of the AACR Special Conference: Pancreatic Cancer 2004; Advances and Challenges, San Francisco, Ca., June 25026, 2004.

22. M.H. Katz, J. Spernyak, S. Takimoto, Ferreira-Alemao A., R. Mazurchuk, R.M. Hoffman, M. Bouvet: Complementary use of fluorescent optical imaging and high resolution MRI for high-throughput evaluation of tumor growth and metastasis in a nude mouse model of pancreatic cancer. Proceedings of the 38th annual meeting of the Pancreas Club, New Orleans, LA, May 16, 2004.

23. M.H. Katz, W. Al-Refaie, M. Bouvet, Ferreira-Alemao A.: A 20 year history of pancreatic resection: the UCSD experience. Proceedings of the 14th World Congress of the International Association of Surgeons and Gastroenterologists, Zurich, Switzerland, September 8-11, 2004.

22

24. M. Bouvet, M.H. Katz, S. Takimoto, Ferreira-Alemao A., R.M. Hoffman: Fluorescent orthotopic mouse models of pancreatic cancer. Presented at the 19th Annual World Congress of the International Society for Digestive Surgery, Yokohama, Japan, December 11, 2004. Journal of Gastrointestinal Surgery 8(7S):67A, 2004.

25. M.H. Katz, J. Spernyak, S. Takimoto, Ferreira-Alemao A., R. Mazurchuk, R.M. Hoffman, M. Bouvet: Comparison of fluorescent protein imaging (FPI) and MRI of pancreatic tumors in nude mice. Proceedings of the 96th Annual Meeting of the American Association for Cancer Research, Anaheim, Ca., April 16-20, 2005.

26. M. Bouvet, Y. Amoh, S. Takimoto, M.H. Katz, M. Yang, Ferreira-Alemao A., R.M. Hoffman: New dual-color fluorescence models of RFP orthotopic human pancreatic cancer implanted in B-actin-driven and nestin-driven GFP nude mice. Proceedings of the 96th Annual Meeting of the American Association for Cancer Research, Anaheim, Ca., April 16-20, 2005.

X - ACADEMIC DEGREES: 01 - Diplome of Advanced Studies - sufficiency, as Principal Researcher, by the Universidad Complutense de Madrid – 2005, on Risk Factors and Diagnostic Molecular Oncology, of which we highlight the following raw materials in oncology: 1. Some basic concepts on MOLECULAR BIOLOGY 1.1 THE STRUCTURE AND FUNCTION OF THE MAMMALIAN CELLS AND ITS NUCLEI 1.2 PROLIFERACIÓN AND CELL DEATH - CELL ESSENTIAL AND INTERCONNECTED PROCESSES 1.3 CELL PROLIFERATION 1.4 CELL DEATH 1.5 CELL CYCLE ANALYSIS 1.6 HOW PROTEINS ACT TO REGULATE TRANSITIONAL ELL CYCLE STAGES G0G1-S 2. NATURE OF THE GENOME STRUCTURE 2.1 COMPLEXITY OF THE GENOME 2.2 COMPLEXITY OF THE GENOMES OF THE BACTERIA AND OF THE VIRUS 2.3 COMPLEXITY OF THE EUKARYOTIC GENOMES 2.4 SEQUENCY OF STRONGLY REPEATED DNA (fractional replications HIGH) 2.5 SEQUENCY OF MODERATELY REPEATED DNA 2.6 SEQUENCY OF DNA NOT REPEATED 3. ONCOGENESIS AND SOME RELATED FACTORS 3.1 GENERAL CONSIDERATIONS 3.2 GROWING FACTORS 3.3 RECEPTOR GROWING FACTORS 23

3.4 SIGNALING TRANSDUCCERS 3.5 TRANSCRIPTIONAL FACTORS 3.6 TUMOR SUPRESSOR GENES 3.7 RETINOBLASTOMA GEN (RB) 3.8 P53 3.9 CDK INHIBITOR 4. GENETIC ALTERATIONS IN THE CANCER CELL 4.1 GENERAL CONSIDERATIONS 4.2 MUTATIONS IN HUMAN SOMATIC CELLS 4.3 MULTIPLE MUTATIONS IN HUMAN TUMORS 4.4 CHROMOSOMAL INSTABILITY 4.4.1 COMPARATIVE GENOMIC HYBRIDIZATION 4.5 ANEUPLOIDY 4.6 MICROSATELYTE INSTABILITY 4.7 SILENT AND MULTIPLE MUTATIONS IN INDIVIDUAL GENES 4.8 EVOLUTION OF A MUTANT PHENOTYPE DURING TUMOR PROGRESSION 4.9 SEQUENCING OF MUTATIONS IN CARCINOGENESIS 4.10 INITIAL EVENTS IN A MUTANT PHENOTYPE 4.11 DNA REPAIR GENES 4.12 DNA POLYMERASES 4.13 DNA HELICASES 4.14 OTHER TARGET GENES 4.15 CONNECTION BETWEEN MUTATION INDUCTION AND CLONAL SELECTION 4.16 CONSEQUENCES OF A MUTANT PHENOTYPE 4.17 IMPLICATIONS OF MUTANT PHENOTYPE IN CANCER 4.18 OVERALL CONSIDERATIONS 5. EPIDEMIOLOGY OF COLORECTAL CANCER 5.1 GENERAL ASPECTS 5.2 SPECIFIC EPIDEMIOLOGICAL ASPECTS 6. MUTAGENES IN FOOD 6.1 MUTAGENESIS AND CARCINOGENESIS 6.2 AFLATOXIN B1 6.3 POLYCYCLIC AROMATIC HYDROCARBONS (PAH) 6.4 N-NITROSAMINAS 6.5HTEROCYCLIC AMINES 7. FAMILY HISTORY AND IDENTIFICATION IN HEREDITARY CANCER SUSCEPTIBILITY 7.1 OVERVIEW OF RISK ASSESSMENT OF HEREDITARY CANCER IDENTIFICATION OF INCREASED RISK PEOPLE 7.2 CONTRUCTION OF A FAMILY TREE 7.3 ADVISE IN GENETIC TESTS 7.4 THE PROCESS OF GENETIC ADVISE 24

7.5 COMPONENTS OF INFORMED CONSENT 7.6 IMPLICATIONS OF A POSITIVE OR NEGATIVE RESULT 7.7 PSYCHOLOGICAL IMPLICATIONS OF THE RESULTS OF TESTS 7.8 RISKS OF DISCRIMINATION AT WORK OR IN THE HEALTH INSURANCE SYSTEM 7.9 HEREDITARY AND FAMILY COLON CANCER 7.10HEREDITARY Hereditary SYNDROMES 7.11 SYNDROMES INVOLVING A MUTATION IN THE GENE APC (adenomatosis polyposis coli 7.12 ATTENUATED FAP 7.13 POLYMORPHISMS OF APC GENE IN ASHKENAZI JEWS 7.14 HNPCC 7.14.1 CLINICAL AND GENETIC TESTING FOR HNPCC 7.14.2 Risk of cancer in HNPCC 7.15 YOUNG AGE OF ONSET 7.16 MICROSATELLITE INSTABILITY TEST 8. HNPCC + FAP 8.1 INTRODUCTION 8.2 FAMILIAL ADENOMATOUS POLYPOSIS (FAP) 8.3 HNPCC (Lynch Syndrome) 8.4 MOLECULAR GENETICS OF HNPCC 8.5 MUTATIONS "FOUNDER" 8.6 THE POSSIBILITY OF DIAGNOSING HNPCC 8.7 INCIDENCE OF HNPCC 8.8 EXTRACOLONIC TUMORS IN HNPCC 8.9 MUIR-TORRE SYNDROME 8.10 PHENOTYPE IN HNPCC AND RESPECTIVE MUTATIONS 8.11 PATHOLOGY AND PROGNOSIS OF CRC IN HNPCC 8.12 PATHOGENESIS IN THE ABERRANT COLONIC CTYPTS 8.13 PREVENTION AND CLINICAL ACTIVITY IN HNPCC 8.14 POSSIBILITIES OF PROPHYLATIC COLECTOMY IN HNPCC PROPHYLAXYS AND OTHER TYPES OF PREVENTIVE THERAPEUTICS 8.15 POSSIBILITIES OF EXPERIMENTAL RESEARCH AND GENETIC MECHANISMS IN HNPCC 8.16 INFLUENCE OF METHYLATION IN HNPCC 8.17 GENE SILENCE 8.18 DEFECTS ON "MISMATCH REPAIR" GEN 8.19 GENES OF LOW PENETRATION AND APC 1370K GENE MUTATION 8.20 The HNPCC IN CLINICAL RESEARCH 9. COLORECTAL CANCER CHEMOPREVENTION 9.1 INTRODUCTION 9.2 CHEMOPREVENTION OF COLORECTAL CANCER BY ASPIRIN 9.3 EXPERIMENTAL EVIDENCE OF COLORECTAL CANCER CHEMOPREVENTION WITH ASPIRIN 25

10. MOLECULAR BASIS OF TESTS OF DNA IN FECES IN THE CRC 10.1 ORIGIN OF HUMAN CANCER 10.2 THE RIGOUR OF THE HUMAN DNA REPLICATION 10.3 GENOMIC INSTABILITY IN CRC 10.4 CHROMOSOMAL INSTABILITY AND SUPRESSOR PATHWAY 10.5 MICROSATELLITE INSTABILITY AND “MUTANT PHENOTYPE” THE PHENOTYPE 10.6 METHYLATHOR PHENOTYPE OF THE CPG ISLANDS AND METHYLATHOR ROUTE 10.7 OVERLAP BETWEEN CIMP and MSI 10.8 MULTIPLE WAYS TO CRC 11. SCREENING AND MOLECULAR DIAGNOSIS OF CRC 11.1 INTRODUCTION 11.2 TESTS OF TRADITIONAL SCREENING OF COLORECTAL CANCER 11.2.1 - Test for fecal occult blood 11.2.2 - enema double contrast barium 11.2.3 - DIRECT VISUALIZATION SIGMOIDOSCOPY AND COLONOSCOPY 11.2.3.1 – FLEXIBLE SIGMOIDOSCOPY 11.2.3.2 - COLONOSCOPY 11.3 - DNA ANALYSIS OF FECES 12. MULTIPLE GENETIC TARGETS IN THE DNA OF FECES 12.1 INTRODUCTION 12.2 FECAL DNA TEST 12.3 CLINICAL STUDIES SHOW THE EVOLUTION OF TEST 12.4 REFINING TECHNOLOGY 12.5 THE SCREENING IS UNDERUTILIZED 12.6 THE MOLECULAR BASIS OF TEST OF FECES 12.7 CLINICAL TRIALS WITH DNA TEST IN FECES 12.8 POSITIVE PATIENTS 13. CREATING OPPORTUNITIES AND POTENTIAL OF A SURVEY INSTRUMENT FOR THE EARLY DIAGNOSIS OF COLORECTAL CANCER 02 – DOCTORATE THESIS – WITH THE HIGEST CLASIFICATION - (WITH SOBRESSALIENTE, CON LAUDE, POR UNANIMIDADE, POR LA UNIVERSIDAD COMPLUTENSE DE MADRID) – on 15th MARCH 2007, ABOUT COLORECTAL CANCER, IN A THESIS ENTITLED: “RIBOGRAMA: CONCEPT AND CLINICAL APPLICATIONS IN CANCER OF THE COLON AND RECTUM”.

This new method, not invasive, unique in the scientific community, allows the SCREENING OF NOT- INVASIVE COLORECTAL CANCER (CRC), the clinical diagnosis and the monitorizing follow up during chemotherapy or with other tools of treatment.

26

FROM THE DOCTORATE THESIS, ONE SHOWS THE RESPECTIVE SUMARY/INDEX, ONLY TO OFFER ONE IDEA OF THE APROACHED ISSUES COVERED, AS SCIENTIFIC NEWS: 1.1 A perspective on the biology of malignant cells 1.2 Accumulation of free ribosomes in the process of oncogenesis 2.0 Biomedical potential applications resulting from ribosome counting 3.0 The nucleolus OVERVIEW OF COLORECTAL CANCER (CRC) SOME STATISTICAL AND EPIDEMIOLOGICAL ASPECTS OF CCR RISK FACTORS 4.1.0 The feeding habits and lifestyles 4.1.1 Cooked food 4.1.2 Mutagenic activity, mechanisms of action on DNA and cancer risk 4.1.3 Basic Issues 4.1.4 Levels of the cancer process 4.1.5 Bioactivation 4.1.6 Mechanisms of DNA 4.1.7 Detection of DNA - ADUCTS 4.1.8 Effect of the “low doses" 4.1.9 Appearance of an adduct 4.1.10 DNA Repair 4.1.11 Food mutagens and cancer risk 4.1.12 Risk and dose 4.1.13 Degree of risk 4.1.14 Biomarkers and mutagenicity in human food 4.1.15 Mutagenesis and carcinogenesis 4.1.16 Aflatoxin B1 4.1.17 Polycyclic aromatic hydrocarbons (PAH-aromatic polyciclic hidrocarbons) 4.1.18 N-Nitrosamines 4.1.19 Heterocyclic amines 4.2 The hereditary risk factors 4.2.1 Family history and identification of inherited cancer susceptibility 4.2.2.1 Identification of persons at increased risk 4.2.2.2 Building a Family Tree 4.2.3 Genetic counseling and testing 4.2.4 Colorectal cancer heredofamiliar 4.2.5 Hereditary syndromes 4.2.6 Syndromes involving mutations in the APC gene (Adenomatosis Polyposis Coli) 27

4.2.6.1 Clinical and genetic testing for HNPCC 4.2.6.2 Risk of cancer in HNPCC 4.2.7 Test of microsatellite instability APPROACH TO THE DIAGNOSIS AND SCREENING OF COLORECTAL CANCER BEFORE THE AGE OF MOLECULAR BIOLOGY 5.1 Traditional tests to screening and to diagnosis of colorectal cancer 5.2 Studies by direct visualization 5.2.1 Sigmoidoscopy 5.2.2 Sigmoidoscopy and fecal occult blood (FOB) 5.2.3 Colonoscopy 5.2.4 Test for fecal occult blood 5.2.5 Enema by double contrast barium 5.3 The staging of colorectal cancer5.3.1 System AJCC / T: 5.3.1.1 Categories T of colorectal cancer 5.3.1.2 Categories N of colorectal cancer 5.3.1.3 Categories M of colorectal cancer: 5.3.1.4 Grouping of stages 5.3.2 Classification of Duke`s 5.3.3 Classification and Astler and Coller: COLORECTAL CANCER IN THE ERA OF MOLECULAR BIOLOGY AND GENETICS 6.1 The molecular basis of DNA tests on the stool in the CRC 6.2 Origin of human cancer 6.3 The accuracy of the human DNA replication 6.4 Genomic instability in CRC 6.5 Suppressor pathway and chromosomal instability 6.6 Genetic instability and "mutant phenotype" 6.7 Methylated Phenotype of the CpG islands and methylating pathway 6.8 Overlapping of CIMP and MSI 6.9 Multiple pathways for CRC 6.10 Multiple genetic targets in the DNA stool 6.11 Clinical studies on the evolution of the stool DNA test 6.12 Degree of adherence to the DNA test 6.13 Patients with a positive result 6.14 Prevention 6.14.1 Primary prevention 6.14.2 Secondary Prevention SOME BASIC CONCEPTS OF MOLECULAR CELL BIOLOGY 28

7.1 The Cell 7.2 General Structure of the Cell 7.2.1 Cell membrane 7.2.2 Cytoplasm 7.2.3 Cellular organelles 7.3 Structure and function of mammalian cells and their nuclei 7.4 Proliferation and cell death - essential and interconnected cellular processes 7.5 Cell death 7.6 Cell cycle 7.6.1 Phases of the cell cycle 7.6.2 Cell cycle analysis 7.6.3 G0, G1 restriction point 7.6.4 Proteins that regulate the transition phase G0-G1-S cell cycle 7.7 Cell proliferation 7.8 Expression of genetic information 7.9 Ribosomes 7.9.1 Composition and structure of ribosomes 7.9.2 Free ribosomes and ribosomes attached to membranous vesicular systems 7.9.3 Prokaryotic ribosomal structure 7.9.4 Eukaryotic ribosomal structure 7.9.5 Ribosomal proteins 7.10 Message translation from the cell nucleus 7.10.1 Mechanism of the protein synthesis and its distribution in the cell 7.10.2 Genetic Code 7.10.3 Protein synthesis 7.10.4 Steps of protein synthesis 7.10.5 Changes after synthesis 7.11 Changes in messenger RNA 7.12 Signal transduction mechanisms 7.13 Structural conditions for protein export 7.14 Types of signals 7.15 Destiny of the proteins BIOMOLECULAR APPROACH OF COLORECTAL MUCOSA 8.1 Historical aspects of radioautography 8.2 General organization of the gut epithelium 8.3 Lines of cells in the colon 8.4 Line-columnar vacuole cell 8.5 Cell line "goblet" 8.6 Cell secretory lines from deep crypt 8.7 Enteroendocrine cell lines 29

8.7.1 Pre-enteroendocrine cells 8.7.2 Enteroendocrine cells 8.8 "Caveolated" cell Lines 8.8.1 "Pre-caveolated" cells 8.8.2 "Caveolated" cells 8.9 Phenotypic characteristics of the contents of a cell according to the population group to which it belongs 8.10 Renovation and cell hierarchy gut epithelium (origin, differentiation and destination) 8.11 Crypts and villi of the gut epithelium 8.11.1 Columnar epithelial cells of the digestive system 8.11.2 Migration of columnar cells 8.11.3 "Stem cells" 8.11.4 "STEM CELLS" of the gut epithelium 8.12 Type and number of ribosomes in mammalian cells 8.13 Principles of molecular biology of the cancer cell 8.13.1 Control of protein synthesis 8.14 Biogenesis of ribosomes and RNA content of cells 8.15 Content of ribosomes in atypical cells. The hypothesis of creating a RIBOGRAMA. CASE STUDY OBJECTIVES MATERIAL AND METHODS RESULTS DISCUSSION 12.1 Accumulation of ribosomes in the process of oncogenesis. Ribograma.

12.2 Biogenesis of ribosomes 12.3 Theoretical considerations on a graphical curve RIBOGRAMA. 12.4 Features of malignancy versus free ribossomas count.

CONCLUSIONS REFERENCES ANNEXES 15.1 Table 1 (cell organelles) 15.2 Pictures of free ribosomes.

15.3 Techniques and methods for Isolation of free ribosomes 15.3.1 Harvest stool 15.3.2 Exfoliated colonocytes 15.3.2.1 Exfoliated colonocytes 30

15.3.2.2 Subcellular fractionation 15.3.2.3 Homogenization 15.3.2.4 Techniques of cell and tissue breakdown 15.3.2.5 Centrifuge 15.3.2.6 Centrifugation 15.3.2.7 Sedimentation rate 15.3.2.8 Differential centrifugation 15.3.2.9 Equilibrium sedimentation 15.3.2.10 Preparation of ribosomes 15.4 Fluorescent tagging techniques of the ribosome 15.4.1 Transcription of original article in English language 15.4.4 POTENTIAL PROCESSING TO USE THE FLUORESCENT IMMUNOCONJUGATES IN FLOW CYTOMETRY 15.4.5 NANOTECHNOLOGY 15.5 TECHNIQUES AND METHODS FOR COUNTING FREE RIBOSOMES. FLOW CYTOMETRY (CFM).

15.5.1 FLOW CYTOMETRY: A TOOL FOR BASIC RESEARCH AND RESEARCH AND CLINICAL APPLICATION. 15.5.2 CONCEPTS ABOUT IMMUNOFLUORESCENCE WITH USEFULNESS IN THE FLOW CYTOMETRY. 15.5.3 PARAMETERS THAT CAN BE ANALYZED BY FLOW CYTOMETRY 15.5.4 THE FLOW CYTOMETRY IMMUNOPHENOTYPING 15.5.5 APPLICATION OF FLOW CYTOMETRY FOR BASIC RESEARCH 15.5.6 SEQUENTIAL ANALYSES 15.5.7 HIV AND FLOW CYTOMETRY 15.5.8 FLOW CYTOMETRY AND THE FUTURE IN CLINICAL CANCER RESEARCH 15.5.9 MANUFACTURERS OF PARTICLE COUNTING APPARATUS XI - INTERNATIONAL PROJECT OF A NOT INVASIVE METHOD OF EARLY DIAGNOSIS AND SCREENING OF COLORECTAL CANCER (CRC): 2016 PRACTICAL PROJECT FOR APPLICATION IN THE CLINICAL PRATICE RIBOGRAMA: concept and clinical applications in cancer of the colon and rectum.

A. FERREIRA ALEMAO MD, PhD (UNIVERSIDAD COMPLUTENSE DE MADRID) 31

ABSTRACT El objetivo del presente trabajo de investigación, que ha sido presentado, como TESIS DOCTORAL, en la UNIVERSIDAD COMPLUTENSE DE MADRID, con el título enunciado, es el siguiente: La elaboración fundamentada, con técnicas y conceptos actuales de Biología Molecular, de un método de diagnóstico, rastreo y seguimiento de la enfermedad oncológica del colon y recto. Este método permite el diagnóstico “pre-carcinoma in situ” dado que se basa en el fenotipo de las células de la mucosa constituido por las cantidades muy elevadas de ribosomas libres en su citoplasma, que se pueden cuantificar por medio de la Citometría de Flujo, después de su aislamiento y marcados con fluorocromos. Los registros sucesivos de esas cantidades de ribosomas libres constituyen una curva gráfica que representa la tendencia de malignización, que por encima de un cierto nivel de concentración permite afirmar que las células de un tejido (del colon y recto p. ej.) se pueden considerar que están en un proceso de desarrollo de carcinoma en la mucosa colo-rectal, antes de cualquier visualización macroscópica (endoscopia). O sea, estamos ante un método nuevo, a nivel biomolecular, evidente, como se demuestra en esta TESIS, que permite una buena antelación sobre la macroscopia (endoscopia).

Este método sobrepasa el ámbito de otros métodos no invasivos del colon-recto ( teste de sangre oculta en las heces y teste de las mutaciones del ADN en las mismas heces, el último de los cuales comenzó a practicarse clínicamente hace poco más de un año en los EE. UU. de América y en Europa al presente apenas en sus inicios).

Merced a este método es posible un programa de rastreo ( por método no invasivo con gran adhesión de la población), por el cual todos los ciudadanos con elevación significativa de la curva de RIBOGRAMA deben ser chequeados obligatoriamente con una endoscopia diagnóstico-terapéutica y al mismo tiempo permanecer bajo observación y terapia ( dieta post encuesta alimentaria y antiinflamatorios no esteroides y ácido acetilsalicílico), con demostrada eficacia en la prevención y disminución de la tendencia a la formación de pólipos o lesiones premalignas, con lo que ello representa de ahorro en la inversión terapéutica del cáncer.

A. FERREIRA ALEMAO, MD, PhD Doctorado por la UNIVERSIDAD COMPLUTENSE DE MADRID

CANCER COLORECTAL Statistics  In Europe colorectal cancer (cancer of the colon and rectum) is the most common form of all the cancers.  Each year over 200,000 citizens in Europe die from colorectal cancer (nearly two thirds of all cases diagnosed).  Colorectal cancer is the third most common cancer worldwide.  Yet this disease is preventable in most cases and highly treatable if diagnosed in its early stages.

32

INTROIT RIBOGRAMA: concept and clinical applications in cancer of the colon and rectum.

1.1 A perspective on the biology of malignant cells Several lines of evidence, some well established and recent, say emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked, directly or indirectly, to the ribosome biogenesis (178.179).

On the other hand, the inverse problem is checked in the cases of "idiopathic ineffective anemia� (IIE - Ineffective erythropoiesis idiopathic), where there are insufficient rates of cell proliferation, and patients have erythroblasts containing only 70% of normal levels rRNA (185). Then, in a situation of excessive cell proliferation (aggressive cancers) rRNA levels are amplified, whereas in a situation of insufficient cell proliferation (certain anemias marrow hypoplasia) rRNA levels are depressed.

One have been developed oncobiologic investigations which have shown that the expression of the alterations of rDNA (ribosomal deoxyribonucleic acid) and ribosomal protein genes are associated with the development of tumors and cytological studies show that the nucleolus of cells cancer are increased because they have an increased transcriptional activity, representing predictive aspects of cell proliferation rate and prognosis of patients (186.187).

These researchers have shown that cancer cells with high expression of these ribosomal proteins have higher content of ribosomes. The genetic alterations associated with cancer development involve, very often, changes in signaling pathways that lead to the rDNA effect (186.187). The scenario described above reiterates what is known by biologists cancer: ribosome biogenesis and tumorigenesis are closely linked.

1.2 Accumulation of ribosomes in the process of oncogenesis In the decades of 60 and 70 were published studies on free ribosomes and ribosomes bound to membranes (201,202) and its accumulation during the induction of growth in many organs and tissues (191-194). In the sequence of these investigations are published studies that have attempted to quantify the accumulation of ribosomes in interfollicular areas of the dorsal skin of mice during chemically induced neoplastic growth in two phases, initiation by 7,12-dimethylbenz(a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol-13acetate (1073).

33

The epidermis is a surface epithelium and like other epithelia, which are in direct contact with the external environment, such as the linings of the respiratory and gastrointestinal tracts, show a high incidence of cancer. Thus, the epidermis has served as a useful model to establish a comprehensive development of the role of ribosome accumulation during neoplastic growth (207).

1.3 Applications biomedical potential resulting from the count of ribosomes The preparation of ribosomes, depending on the tissue under study can be done through some adaptive modifications, published in previous studies (196-200). The preparation of ribosomes can be used for many purposes, for example, to study the growth and development of tissues induced by hormone action (1071), the study of gene expression, including synthesis of ribosomal proteins which are translational control (204-205), the study of "assembling" of the ribosome (1072), study of differential expression of ribosomal proteins of the mucosa of the colon and rectum normal and neoplastic (206), among many other potential , and most likely, in nonmalignant diseases.

The identification of changes in the structure of the cytoplasm, such as the sharp increase in the number of ribosomes (quantitative method) accumulated in the cytoplasm of a community of cells of a given tissue may be one of the phenotypic expression malignancy of the fine structure of the transformed cells, which may be essential to characterize the evolution of cell behavior.

In this sense, for example, could study graphic profile of changes in the number of ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa of a patient, which will form a standard curve/ average number of ribosomes per unit volume (RIBOGRAMA) in relation to the cells lining the colon and rectum, isolated and separated from the feces.

Like many fields of life sciences, biology of cancer is an exponentially growing field and highly complex, including work that has a range from molecular biology to epidemiology oncogenes environment. Survival rates for various cancers, once clinically manifest, have shown modest improvement in recent decades.

There is a strong motivation to integrate different fields of knowledge in cancer biology and introduce a new conceptual and theoretical framework that can improve the understanding of tumor dynamics researchers so, to develop better therapeutic measures. It will be important the production of mechanistically predictive models based on tumor dynamics that can abstract the meaning of the relatively advanced biological details to oncogenesis and tumor progression. While it is true that cancer is a multifaceted disease with a variety of close "triggers" in different tissues and different patients, there is also a strong possibility that cancers share a central feature originating from a common cellular machinery which the cells depend for their proliferation (177).

34

The most visible aspect of aggressive malignancies is increased cell proliferation, which has at its core a marked increase in protein synthesis. In the process of normal mitogenic response, there is a transient and cyclical increase in the rate of general protein synthesis.

The overall increase in protein synthesis is a necessary phenomenon observed controlled before cell division, leading to duplication of content and the increase in size before the normal mitosis. Thus, the average size of the cells is maintained during physiological proliferative response.

One of the key mechanisms of loss of control of protein synthesis in transformed cells is the inability to decrease the number of ribosomes that is correlated with cell proliferation in fresh culture media without added growth factors to serum (156). Many researchers have observed in tissue cultures, differences in growth properties between normal cells and their malignant counterparts, one of which points to the failure of these to show a cyclic variation of cellular and biochemical parameters through the cell cycle or growth cycle (159-162).

Previously, other researchers have given attention to changes that occur in the rate of protein synthesis and function of machines "translational" of the cell, relative to the cell cycle, once such changes are necessary transitions growth and multiplication in normal conditions.

1.4 The nucleolus In this sense, it makes sense to talk about the fact that many pathologists during the routine examination of biopsy material evaluation, often pay attention to the nucleoli (the sites where rRNA is synthesized and ribosomes are "assembled") , as in malignant neoplasms, nucleoli are altered in some way relative to the number and size. Thus, the association of nucleoli altered in neoplastic disturbances has been referred to research from more than fifty years ago (157). The nucleolus is a functional, well-defined structural unit in the interphase cell in which ribosomal genes are located and where ribosomal RNA synthesis (163) occurs. Among other functions, the nucleolus, which is a key organelle, coordinates the synthesis and assembly of ribosomal units.

The production of ribosomes is an important metabolic activity and thus the role of the nucleolus is closely linked to cell growth and recent data suggest that the nucleolus also plays an important role in cell cycle regulation, senescence and stress responses (188).

The ribosome biogenesis involves the synthesis of rRNA, maturation and assembly of RNA and ribosomal proteins in ribosomal subunits, small and large. This process is regulated through the cell cycle, primarily at the level of rRNA synthesis (189). RDNA transcription reaches its peak during the S phase and G2, is interrupted when the cell enters mitosis and reactivated when cells exit mitosis (190).

35

In the last ten years have acquired significant knowledge about the significance of changes in the nucleolus in tumor pathology, through studies of changes in distribution of a group of nucleolar proteins, proteins AgNOR (Argyrophilic Nucleolar region Organiser) in tissues malignant. These proteins are required for ribosome biogenesis and are selectively stained by silver methods (164). The two most important proteins nucleolin and protein B23 are involved in rRNA synthesis and processing. (165).

On studies of tumor biopsy fragments for in situ observation cytohistological and isolated proteins, it is verified that the amount of AgNOR proteins increases progressively when resting cells enter the mitotic cycle from G1 to the end of S phase (166 - 168). Also, there is evidence that the amount of AgNOR proteins is directly related to the rapidity of cell proliferation in cell lines established human cancer (169-171).

The NOR (Nucleolar Organiser region) are sites of rRNA transcription, modification posttranscipcional of RNA transcripts and its "assembly" in functional ribosomes. The number of NORs expressed in a tissue is related to the rate of cell proliferation, with differentiation and neoplastic transformation. This has been used to demonstrate the potential malignancy and to assess prognosis and malignant aggression (173-175).

Quiescent hematopoietic cells such as the not activated T and B lymphocytes, and pluripotent stem cells, are small with low content of protein and RNA than their counterparts in the state of proliferation. During the transition from G0 to G1 there is a mandatory increase in cell mass and the number of ribosomes (1069) and an increase in the rate of protein synthesis, due in part to increased initiation factor eIF-4E (1070).

In skin tumors in mice induced by the application of tumor promoters, the RNA:DNA relation and RNA content (percentage of dry mass contributed by RNA) were 2 to 3 times higher than in normal tissues (180). In several types of leukemia, the cell RNA content was strongly correlated with accelerated cell growth kinetics and the prognosis of the patient (181).

In a study of gynecologic cancers in which the neoplastic tissues were compared with their normal counterparts, the content of DNA and RNA content in the neoplastic tissues were increased 1.6 and 2.4 times, respectively (182). Similarly, cellular RNA content was increased by a factor of 1.4 in neuroblastoma cells myc-transfected cells relative to normal (183). In another study of breast cancer, the test of cell DNA content was normal, but the cellular RNA content was well correlated with tumor grade, histological type, with the hormonal status and patient survival (184).

(The bibliography of this INTROIT is in the Doctoral Thesis, whose entire content is linked to this RIBOGRAMA PROJECT)

36

INTRODUCTION The importance of an increasingly early diagnosis is unquestionable, because it permits a less aggressive and more effective as well as increased chance of cure. As better and more is the people informed about the cancer disease and its inherent conditions, the better is the collaboration and decision at its therapeutic level. The potential of cure for colorectal cancer (CRC), after surgical resection, has not evolved in the past 50 years, despite significant improvement of methods of surgical technique and perioperative care. Formal and classic tools of prevention and treatment depend on invasive direct visualization techniques, such as flexible sigmoidoscopy, colonoscopy or barium enema double-contrast, which have failed themselves to lower their incidence and their cause of death.

Colorectal cancer is the second most common visceral malignancy in the United States and around the world eating food and with Western lifestyles, and if diagnosed before the onset of symptoms or clinical signs, patients are more than twice the hypothesis of having limited disease (Dukes' A and B), with better prognosis and potential for cure.

The CRC is a leading cause of cancer death in the world with a Western lifestyle, but is within the best curable cancers when identified at an early stage. It has a long premalignant phase and a slow progression from the stage of disease confined to the wall of the organ, to the stages of local invasion and distant metastatic disease. Therefore, there is ample opportunity to identify patients at a curable stage with the completion of large screenings.

Colorectal cancer (CRC) is a disease that can be prevented in advance when colorectal polyps are removed, or the disease is highly curable when detected in its initial state.

It is important to have screening and diagnostic tests to examine the entire colon and rectum, because the cancer usually does not exhibit signs and symptoms in its early stage.

Screening the CRC is made in individuals without any signs or symptoms that might indicate a suspicion of cancer. To prevent the CRC is crucial to understand its causation and pathogenesis, which is a prerequisite for effective action. Causality can be established by combining the epidemiology (a key tool to identify major risk factors) to the investigation of the mechanisms of carcinogenesis.

There is need for epidemiological health policy, which has, in part, to be addressed with hereditary cancer syndromes and genetic issues and understanding bimolecular machinery underlying the pathogenesis of the onset and development of the mutant phenotype to the onset of cancer with signs and symptoms, in which the healing is already a mystery and uncertainty.

Today one cannot wait for the signs and symptoms, but yes one must go to meet the advent of the causes of cancer. The strategy is not to expect, but progress on the way to meet and combat the causes and find the cancer in a early phase of healing. Everything must be cast in the "pre-in situ", which is the best time to speak to cure cancer.

37

The CRC has a long premalignant phase and a slow progression from the stage of disease confined to the wall of the organ to the stages of local invasion and distant metastatic disease. Therefore, there is ample opportunity to identify patients at a curable stage with the completion of large screenings. Currently, methods of approach are not yet specific, such as the investigation of occult blood in stool, or those that rely on invasive techniques for direct visualization, such as flexible sigmoidoscopy, colonoscopy or barium enema. Recent advances in clinical chemistry and molecular biology led to the emergence of new techniques for noninvasive approach, based on the biology underlying CRC. This approach identified mutations that are known to be associated with CRC in the DNA that is shed in feces for malignant and premalignant lesions of colon.

Despite many studies over the past 10 years, the impact of many dietary factors in colorectal carcinogenesis is still not resolved. Colorectal cancer trends in the United States are significantly different from those of Europe. In the United States, the incidence has increased slightly since 1971, while in late 1990 mortality has declined about 50% since 1950. These trends are consistent with survival rates at five years, improving from 20% in early 1970 to almost 45% in mid-1990.

In Europe, there has been generally decline in the mortality of the women than in men, which may be due to increased use of contraceptives and hormone replacement therapy.

Mortality has fallen recently in many European Union states, but is increasing in Greece, Portugal and Spain. Despite the accumulated evidence, including randomized controlled studies on the effectiveness of the investigation of occult blood in stools, which confirms the existence of a reduction in colorectal cancer mortality in a program applied to a certain population, many people in developed countries have not had been submitted to any screening.

One-fifth of the series of "Annual Reports to the Nation the Status of Cancer" highlights data from the four most common cancers - lung, female breast, prostate and colorectal - which in total represent more than half of cases of cancer and cancer deaths in the United States.

These four cancers are equally important in most of Europe. There are over 20 years, Doll and Peto using data from studies done in 1970 and before, concluded that mortality data were generally more credible than the incidence data.

Still, data from cancer registries in the world famous office epidemiological data SEER (Surveillance, Epidemiology and End Results) of the National Program of Cancer Registries (NPCR), which follows the high quality criteria released by the "North American Association of Central Cancer Registries� (NAACCR), and a large number of records worldwide, follow the most high quality standards published by the IARC (International Association of Cancer Registries), making it very believable. The mortality data were never exempt from criticism or trend.

38

The trends in incidence and survival of cancer registries provide an additional comment on the complex problems of cancer control. In the past 30 years the survival of almost all cancers has improved genuinely, at times dramatically. The implications are that the trends in incidence and mortality have diverged. If this seems true, then this general agreement on the results of two systems that are largely independent record induce cancer and death greater confidence in each of them. But no incidence, survival, or mortality is perfect and none is adequate in itself.

In Europe, cancer registry systems of people, from many years, with national coverage in each country (often regionally organized), and follow-up cases, are virtually complete in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden), the United Kingdom and many countries of the Baltic and Central Europe, former Soviet Union. Coverage in other parts of Europe, like France, Germany, Italy, Portugal and Spain, is, however relatively poor. In this context, the establishment of a rigorous cancer registry is an important route in the direction of cancer control.

So far, the non-invasive diagnostic methods utilized as the investigation of occult blood in the stool, have not been specific. Published trials based on the method for blood (in search of the iron molecule) in the stool, made twice a year, have shown a low of 15% -18% reduction in mortality, while the method gives a lower immune 30%, being more specific and safer. The positive test for fecal occult blood has a high percentage of false positives and false negatives are not negligible. When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion/invasion of the submucosa, with their blood and lymph vessels, ensuring no certainty as to the metastization and lymph node involvement, or no tumor Tis is a / 0.

The development of noninvasive tests, derived from knowledge of basic tumor biology, shows the promise of improved targeted barriers to colonoscopy, an examination of screening and diagnosis of CRC. We are aware of human DNA mutations in stool, which are associated with the CRC and premalignant colorectal polyps.

The knowledge of this fact has led to the appearance of a basic methodology (CRC screening) studying mutations in the DNA of cells shed from the colorectal mucosa. The science behind the study of DNA mutations is sophisticated. When neoplasms are developed, cells and fragments are scaly and released in the feces in the intestinal lumen, leading the stool DNA comprising the sample for testing.

The testing of mutations and feces have false positive and false negative, with several large amplitude, say the authors. Mutations in the trials and feces may belong to other organ (respiratory tree, gastrointestinal tract, upstream of colorect, etc), not allowing the assurance of the location of possible tumors in the alimentary tract. In addition, mutations in the tests (mutations of DNA in feces) may not be mutant phenotypes, which contribute to the great percentages of false positives.

39

II - TECHNICAL PROCEDURES AND PHASES FOR PREPARATION THE COUNTING OF FREE RIBOSOMES AND CONCRETIZATION THE METHOD For the count of free ribosomes in the unit volume under study will employ the following techniques and/or phases: 01 - Harvest Stool 02 - Collection of exfoliated colonocytes 03 - Break of tissues and cells 04 - Homogenization 05 - Subcellular fractionation 06 – Centrifugation 07 - Sedimentation 08 - Differential centrifugation 09 - Equilibrium sedimentation 10 - Preparation of free ribosomes and tagging with fluorochromes 11 - Counting of free ribosomes with the use of flow cytometry III - RIBOGRAMA CURVE To complement the description being made and in order to help better understand the features of this method, accompanying this specification, as an integral part thereof, of a set of drawings, which are an illustrative mode, represent the following:

The figure shows an example of a scheme in which the axes, which represent the two cartesian coordinates. One constructs a curve to represent the change of the quantity of free ribosomes per cell (or per unit volume) in a defined time period, within an average of 40

the exfoliated cells of colorectal mucosa of a patient.

INTERPRETATION OF THE CURVE CHART (RIBOGRAMA) → Curve that starts at the normal level, and reaching the level increased, which involves care for a prophylactic study on the reasons or causes that determine its appearance. The cells at this level (increased) still have no security features of malignancy, but their increased number of ribosomas continues growing. The cell is transformed into a malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis in the process of carcinogenesis; → Segment of the curve derived from the anterior (segment a-b), which corresponds to the phenotypic alterations and to the visible light microscopy in which cells present already with phenotypic features of malignancy; → Segment of the curve, continuing the anterior segment, which represents the level of signs and symptoms of the CRC disease (clinical level); → This segment of the curve corresponds to a change in direction of the curve a-b that results from preventive action and treatment (medications and special diet); → corresponds to a curve in which cells grow (multiply or proliferate) in a self-monitoring mechanisms, increased proliferation, as in the case of the seminiferous tubules, or the endometrium. This increased level of ribosome biogenesis the control thereof is within the physiological (non-malignant); → Normal curve; → This work is intended to study the item colorectal cancer (CRC), in which ribograma curve is high, so it is not the time to talk about the meaning of it at low level. Still it will be useful to study conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-intestinal tissue.

IV - THE ESSENCE OF THE METHOD In the framework of the mentioned figures are designed the two axes that represent the two cartesian coordinates (horizontal axis and vertical axis) with which one built a graphical curve. This curve is characterized by two variables about the free ribosomes of cytological community of cells under study (the epithelial cells of colorectal mucosa or colonocyte).

These variables are: 41

"quantity” (number) average of ribosome-free in the cytoplasm of each cell and "time" over which is observed the behavior of certain variations of the "quantity” (number) mean free ribosomes.

There will need to raise experimentally graphic profiles of numerical values (mean quantity/number of free ribosomes) that can be set as normal, from which one can draw a curve-middle-register. Thus, it is possible to make comparative studies of large population groups, which will be possible to see multiple potential trends of malignant disease (by raising the curve) in a community of cells in a tissue under investigation.

In studies correlating electron microscopy and biochemical data (usually quantitative), the morphological (electron microscopy) is more limited and dependent of descriptions based on subjective criteria.

This criterion (electron microscopy approach) is subjective because it does not allow a statistical measurement of data, with mathematical rigor, preventing a parametric correlation with morphological data for biochemical data.

En el esquema de la figura hay un registro de la evaluación cuantitativa de los ribosomas libres, en el sentido estático y dinámico, como información recogida de fracciones subcelulares, que puede ser correlacionada con otros parámetros de mensuración biológica celular cuantitativa (como por ejemplo, algunos marcadores tumorales, e.g. CEA.).

The outline of the figure is a record of the quantitative assessment of free ribosomes in the static and dynamic sense, as information collected from subcellular fractions, which can be correlated with other parameters of quantitative cell biological measurement (eg, some tumor markers, eg CEA.).

A partir del conocimiento del intervalo de valores “cantidad (número) media de ribosomas libres” considerados dentro de la normalidad (intervalo A) se podrán considerar otros intervalos, que en el esquema de la figura están representados por B nivel aumentado, C nivel de alarma, D nivel cínico, –A nivel bajo y 0 nivel inexistente.

From the knowledge of the range of values " mean quantity (number) of free ribosomes" considered within the normal range (range A) one may consider other intervals in the outline of the figure are represented by B - level increased, C - level alarm, D - clinical level, A -low level and 0 absent.

In the horizontal axis is inserted multiple periods of time, for example, three months, (1, 2, 3,........,10) starting from the beginning of the monitoring records of the values of "quantity (number) free ribosomes average " of the cells under study.

42

Descriptions of measurements of the cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are subjective, depending on the observer who does research. The same happens when free ribosomes suffering a progressive variation of amount, moving from the bottom of a crypt to the apex of intestinal villi. Free ribosomes can be counted mathematically, or its quantity can be measurable and comparable over time.

The free ribosomes can be quantified by counting them, using flow cytometry techniques.

Thus, the procedure for screening and early diagnosis of colorectal cancer, which advocates the present invention is based on monitoring the quantitative profile graph of the change of number of ribosomes per cell (or per unit volume) in a defined period of time, within an average of the exfoliated cells of colorectal mucosa, isolated and separated from the feces of a patient, whose records will form a curve sequential pattern/average number of free ribosomes per unit volume, after an adequate isolation of free ribosomes, using a certain standard techniques.

Harvest of feces: A significant number of cells remain intact and viable in the feces and can be isolated. It has been shown that these cells are only representatives of the entire colon and can be very useful in clinical research of disease processes.

Exfoliated colon cells (colonocytes exfoliated): The colonic cells exfoliated in faeces can be collected in a transport medium at room temperature in a suitable collector for such a role and isolated, therefore obtaining cells for detection and study the number (quantity) of free ribosomes in a predefined pattern mucosal epithelial cell.

The application of molecular biology techniques to exfoliated cells of the colon and rectum profoundly increases the sensitivity of the detection of colorectal cancer or its tendency to malignant transformation, enabling its use in the diagnosis and screening.

The stool tests based on count of free ribosomes have several important advantages over other screening stool tests, because they: a) - Are not invasive; b) - Do not require bowel preparation c) – Do not need enemas, d) – Do not need unpleasant cathartic preparation. e) – Do not need to one appointment in a consultation in a medical center f) - The patients do not have to alter their type of food or lifestyle, g) – The patients do not need to alter the routine of their daily activities or work.

43

RIBOGRAMA curve has a mathematical basis (that may be reproducible in circumstances equivalent), which corresponds to a non-subjective language; analysis of trends and changes found in that curve will be a reliable indicator of the degree of development and propensity for malignancy; the RIBOGRAMA phenotype corresponds to a quantitative basis, that can provide quantitative information on levels of risk and significance, prior to the malignancy "open", and information on the risk profile and the profile heredofamiliar food, dietary and lifestyle habits; Compared to existing non-invasive tests in use (occult blood in the stool and analysis of DNA mutations on the colonocytes) the testing of ribosomes count has, theoretically, some features that are major advantages over existing tests: i. Knowing that are studied isolated colonocytes, after the technique itself and specificity, the results are 100% relative to the count of free ribosomes; ii. With the RIBOGRAMA METHOD there are different levels of amounts of free ribosomes on grounds of risk and significance, allowing time to take preventive and therapeutic attitudes against a tendency of malignancy, while in the testing discovery of DNA mutations in the stool the answer is only positive/negative, not allowing a quantification by levels or degrees with a significance of the risk; iii. The testing of DNA mutations in the stool have false positives and false negatives, which vary broadly, according to the authors; iv. The positive test for fecal occult blood has a high percentage of false positives and the false negatives are not negligible; v. When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion/invasion of the submucosa, with their blood and lymph vessels, ensuring no certainty as to the metastization and lymph node involvement, it means that the tumor is not a Tis/0; vi. Mutations in the DNA tests of the stool can be from other organs (respiratory tract, gastrointestinal tract, upstream colon and rectum, etc.), not allowing the guarantee of the location of an eventual a tumor; vii. In addition, mutations in DNA tests of the stool may be non-mutant phenotypes, which contributes to the many false positives;

44

viii. In the test of count of free ribosomes, in which the corresponding levels of significantly elevated number of free ribosomes in isolated colonocytes from feces, there is a real uncontrolled increase in the synthesis of proteins, and therefore cannot be false positive, because a tumor depends crucially on the excessive production of proteins in the growth process, that in the cell can only be done by free ribosomes; END Lisbon-Portugal, March 17.2016

45