Estudos populacionais, assim como modelos animais demonstram que além dos fatores já conhecidos, como uma dieta não balanceada e sedentarismo, insultos nutricionais no período gestacional ou durante a lactação, causam alterações metabólicas importantes que levam ao surgimento da obesidade, Diabetes Mellitus tipo 2 (DM2) e doenças cardiovasculares em longo prazo. Nesse estudo, analisamos o pâncreas de camundongos hiperalimentados adultos (150 dias) e camundongos hiperalimentados jovens (21 e 28 dias). Os camundongos hiperalimentados de 21 dias receberam transplante de células mononucleares de medula óssea (CMO) e o resultado desse transplante foi observado aos 28 dias, quando os animais foram sacrificados. Nós investigamos: a apoptose das células beta através do fator pró-apoptótico Bax; a proliferação das células da ilhota pancreática através do antígeno nuclear de proliferação celular (PCNA); a expressão da citocina TNF-α, relacionado com a resistência à insulina em animais obesos e a expressão de células tronco CD133 com o objetivo de estudar a participação dessa célula na renovação da massa de células beta durante o estabelecimento da DM2. As análises das proteínas citadas no pâncreas foram realizadas através de microscopia de luz, microscopia confocal, microscopia eletrônica e Western blotting. O peso dos animais, a morfometria das ilhotas pancreáticas, bem como os níveis de glicose e insulina plasmáticos também foram determinados. Nossos resultados confirmaram que os camundongos hiperalimentados adultos apresentavam elevados níveis de glicose e insulina plasmática quando comparados ao grupo controle. Além disso, camundongos hiperalimentados adultos apresentaram aumento na expressão de Bax, indicando apoptose das células beta, maior expressão de TNF-α nas ilhotas pancreáticas, e presença de células CD133 nas ilhotas e ductos pancreáticos de camundongos hiperalimentados. Ao analisarmos os animais com 21 dias também observamos elevados níveis de glicose e insulina plasmática no grupo hiperalimentado. Após o transplante de CMO, os camundongos hiperalimentados apresentaram os níveis de glicose e insulina normalizados em relação ao grupo controle, porém os níveis de TNF-α no pâncreas continuavam elevados. A expressão de células CD133 foi observada tanto aos 21 quanto aos 28 dias nas ilhotas pancreáticas nos grupos hiperalimentados. Porém, a expressão estava aumentada no pâncreas dos animais que receberam transplante de CMO aos 28 dias. Portanto, podemos concluir que camundongos hiperalimentados durante a lactação quando adultos se encontram em um estágio inicial do estabelecimento da DM2, com hiperglicemia e hiperinsulinema, ilhotas pancreáticas hipertrofiadas, evidência de apoptose das células beta, neogênese de células beta a partir de células do ducto pancreático, replicação de células beta já existentes e células tronco endógenas marcadas com CD133. Nos camundongos jovens, além dos parâmetros também observados nos animais adultos, observamos os benefícios das CMO para o restabelecimento da glicemia e insulinemia nos camundongos hiperalimentados. Além disso, demonstramos pela primeira vez, células CD133 marcadas com insulina nas ilhotas pancreáticas. O aumento da expressão de células CD133 no pâncreas de camundongos hiperalimentados que mantinham níveis elevados de TNF-α e receberam transplante de CMO, nos sugere a importância dessa citocina para o recrutamento e diferenciação das células tronco CD133 para melhoria do percurso da diabetes. / Population studies as well as animal models show that besides the factors already known as an unbalanced diet and sedentary lifestyle, nutritional insults during pregnancy or during lactation, causes important metabolic changes that lead to the emergence of obesity, type 2 diabetes mellitus (DM2) and cardiovascular diseases in long-term. In this study, we analyzed the pancreas of overfed adult mice (150 days) and young mice (21 and 28 days). At day 21, overfed mice were transplanted with bone marrow mononuclear cells (BMCs) and the results of this transplantation were observed at day 28. We investigated beta-cell apoptosis through pro-apoptotic factor Bax, the proliferation of pancreatic islet cells by proliferating cell nuclear antigen (PCNA), expression of TNF-α which has been linked to insulin resistance in obese animals and the expression of CD133 stem cells, in order to study the participation of this cell on the recovery of beta-cell mass during the establishment of DM2. The protein analysis, were performed using light microscopy, Confocal microscopy, electron microscopy and Western blotting. The animals weight, morphology of pancreatic islets, as well as plasma levels of glucose and insulin were also determined. Our results confirmed that adult overfed mice had high levels of blood glucose and insulin when compared to control mice. Moreover, overfed adult mice showed an increased expression of Bax, indicating apoptosis of beta cells, also confirmed by transmission electron microscopy, increased expression of TNF-α in pancreatic islets when compared with the control group, and interestingly we observed the presence of CD133 cells in the pancreas of overfed mice. By analyzing the animals with 21 days, we also observed high levels of blood glucose and insulin in the overfed group, but we did not observe Bax expression at this lifetime. The expression of TNF-α was also increased in the pancreas of overfed mice at day 21. After BMCs transplantation, the overfed group showed normal levels of blood glucose and insulin when compared with the control group, but the levels of TNF-α remained high. The expression of CD133 cells was observed in the pancreatic islets both at 21 and 28 days in overfed groups. However, the expression of CD133 was increased in the pancreas of animals that received BMCs transplantation at day 28. Therefore, we conclude that overfed mice when adults are at an early stage of stablishment of DM2 with hyperglycemia and hyperinsulinemia, pancreatic islet hypertrophy, evidence of apoptosis and neogenesis of beta cells from pancreatic duct cells, replication of existing beta cells and endogenous stem cells. In young mice, further the parameters observed in adult animals, we observed the benefits of the BMCs for the restoration of glucose and insulin in overfed mice. Moreover, we demonstrated for the first time the CD133 stem cells in pancreatic islets in colocalization with insulin, suggesting that CD133 could be a progenitor beta cell marker in pancreas. Also, the increased expression of CD133 cells in the pancreas of overfed mice with elevated levels of TNF-α after BMCs transplantation, suggests the importance of this cytokine for the recruitment and recovery of CD133 stem cells to improve the course of diabetes.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Camundongos C57BL/6 machos com oito semanas de idade alimentados com diferentes dietas durante 16 semanas: de alta densidade energética (ADE, 26% das calorias de carboidrato, 60% de gordura e 14% de proteína) ou dieta padrão (CO, 76% das calorias de carboidrato, 10% de gordura e 14% de proteína). Comparado ao grupo CO, o grupo ADE apresentou maior ganho de massa e maior depósito de tecido adiposo, bem como maiores níveis plasmáticos de triglicerídeos, LDL-c, ALT, AST e fosfatase alcalina e com maiores níveis de corticosterona plasmática, glicose de jejum e insulina com uma consequente resistência à insulina (avaliado pelo HOMA-IR). No TOTG, a glicose plasmática aumentou ao máximo após 15 min. da administração de glicose oral em ambos os grupos. Entretanto os níveis de glicose foram maiores no grupo ADE que no grupo CO (P<0.0001). O clearance de glicose no grupo ADE foi reduzido, permanecendo aumentado após 120 min. (P<0.001), caracterizando intolerância a glicose no grupo ADE. O teste intraperitoneal de tolerância à insulina mostrou uma rápida redução na glicose plasmática após 15 minutos da administração de insulina em ambos os grupos, mas significativamente aumentada no grupo ADE (P<0.0001), permanecendo desta forma até os 120 min. após a administração. Concluindo, camundongos C57BL/6 respondem a dieta ADE desenvolvimento os sinais e sintomas associados à síndrome metabólica observada em humanos. Por conseguinte, este modelo animal poderá ajudar-nos a compreender melhor as alterações em órgãos alvos associadas com a síndrome metabólica, assim como a possibilidade de tratamentos diferentes. / Eight-week-old male C57BL/6 mice fed different diets during 16 weeks: very high-fat chow (HFC, 26% of calories from carbohydrates, 60% from fat, and 14% from protein), or standard chow (SC, 76% of calories from carbohydrates, 10% from fat, and 14% from protein). Compared to SC mice, the HFC mice showed greater mass gain and greater total visceral fat pads, as well as higher plasma levels of triglycerides, LDL-C, ALT, AST and ALK, and higher plasma corticosterone, fasting plasma glucose and insulin with the consequent insulin resistance (assessed by HOMA-IR). In OGTT, The plasma glucose increased to a maximum after 15 min of glucose oral administration in both groups. However, the glucose level in HFC mice was higher than in SC mice (P<0.0001). The clearance of glucose in HFC mice showed a delay, remaining elevated after 120 min (P<0.001), characterizing an impairment of glucose clearance in HFC mice when compared to SC mice. The insulin tolerance test demonstrated quick decrease of plasma glucose after 15 min of insulin administration in both groups, but plasma glucose remained significantly high in HFC mice when compared to SC mice (P<0.0001) in the following time-points until 120 min. In conclusion, C57BL/6 mice respond to a very high-fat diet developing the aggregate of symptoms and signs associated with the metabolic syndrome seen in humans. Therefore, this animal model could help us better understand the target organ alterations associated with the syndrome and the possibility of different treatments.

Introdução: O transplante de pâncreas é uma opção terapêutica para pacientes portadores de diabetes mellitus, quando bem indicado, com objetivo de restaurar a normoglicemia sem necessidade de uso de insulina, sendo que a técnica mais utilizada é o transplante simultâneo pâncreas-rim. Houve aumento da indicação e realização deste procedimento em todo o mundo nos últimos anos, crescendo a importância de se conhecer os aspectos de imagem no pré e pós-transplante de pâncreas. Objetivos: Avaliar e discutir o papel das diferentes modalidades de exames de imagem empregadas na avaliação no pré e pós-transplante de pâncreas enfatizando os achados normais e das possíveis complicações. Propor um fluxograma para realização de exames de imagem para o programa de transplante de pâncreas considerando a casuística apresentada. Metodologia: Levantamento retrospectivo dos métodos de imagem com as indicações e os achados pré e pós-procedimento em todos os pacientes submetidos a transplante de pâncreas no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, da Universidade de São Paulo. Resultados: Foram estudados 14 casos com indicação de transplante simultâneo pâncreasrim, 13 destes submetidos ao transplante no período 2010-2016, os quais apresentaram idade média de 37,6 +/- 6,2 anos (variando entre 24 e 51 anos), sendo 8 (57%) do sexo masculino e 6 (42%) do sexo feminino. Um (7,2%) paciente não pode receber o enxerto pancreático devido a procedimento cirúrgico frustro resultante de diagnóstico intra-operatório de placa ateromatosa extensa. As complicações pós-transplante mais comuns foram: coleções na 7 cavidade abdominal (6/13 casos (46,1%)), pancreatite (6/13 (46,1%)) e complicações vasculares (4/13 (30,8%)). O US com Doppler foi realizado no pós-operatório em todos os casos. Conclusões: há necessidade de avaliação vascular pré-operatória por imagem do receptor, de rotina, para evitar problemas no momento do transplante. O TSPR tem alto risco de complicações, sendo necessária a retirada do enxerto em número relativamente grande dos casos. A avaliação pelos métodos de imagem foi fundamental para o diagnóstico e planejamento do tratamento das complicações, sendo que o US com Doppler se mostrou suficiente como primeiro exame. A correlação com angioTC e(ou) angioRM foi necessária nos casos com suspeita de complicação vascular, permitindo confirmar a suspeita em todos os casos. / Introduction: Pancreas transplantation is a therapeutic option for patients with diabetes mellitus, when well indicated, to restore normoglycemia with no need of insulin, and the most commonly used technique is simultaneous pancreas-kidney transplantation. There has been an increase in the indication and performance of this procedure worldwide in recent years, increasing the importance of knowing the aspects of imaging in the pre- and posttransplantation of pancreas. Objectives: To evaluate and discuss the role of the different imaging modalities used in the assessment in the pre- and post transplantation of the pancreas emphasizing the normal findings and the possible complications. To propose a flowchart to perform imaging tests for the pancreas transplantation program considering the presented case series. Methodology: Retrospective survey of imaging methods with pre and post-procedure indications and findings in all patients undergoing pancreas transplantation at Hospital das Clínicas, Ribeirão Preto Medical School, University of São Paulo. Results: We studied 14 cases with simultaneous pancreas-kidney transplantation, 13 of them undergoing transplantation in the period 2010-2016, which presented a mean age of 37.6 +/- 6.2 years (ranging from 24 to 51 years), of which 8 (57%) were male and 6 (42%) were female. One (7,2%) patient could not receive the pancreatic graft due to the frustrating surgical procedure resulting from an intraoperative diagnosis of extensive atheromatous plaque. The most common post-transplant complications were collections in the abdominal 9 cavity (6/13 cases (46.1%)), pancreatitis (6/13 (46.1%)) and vascular complications (4/13 (30.8% )). The US with Doppler was performed postoperatively in all cases. Conclusions: There is a need for routine preoperative vascular imaging of the recipient to avoid problems at the time of transplantation. TSPR has a high risk of complications, requiring removal of the graft in a relatively large number of cases. The evaluation by the imaging methods was fundamental for the diagnosis and planning of the treatment of the complications, and the US with Doppler was sufficient as the first examination. Correlation with angioTC and (or) angioRM was necessary in cases with suspected vascular complications, allowing confirmation of suspicion in all cases.

Há um número crescente de pessoas e animais com obesidade e sobrepeso, com consequente aumento no número de pacientes resistentes à insulina e portadores de Diabetes mellitus (DM). O fator de crescimento vascular endotelial (VEGF) tem sido caracterizado como uma molécula importante em inúmeros mecanismos fisiopatológicos. Em diabéticos, pesquisas indicam uma redução deste fator em alguns tecidos estudados, sendo esta menor expressão envolvida com o desenvolvimento de hipóxia tecidual e não cicatrização de feridas. Neste contexto, este trabalho teve como objetivos caracterizar um modelo diabético induzido por dieta, avaliar a vascularização, expressão gênica e proteica do VEGFA e seus receptores FLT1 e KDR em pâncreas de camundongos diabéticos e não diabéticos, antes e após a terapia gênica com VEGF. O estudo consistiu de 2 fases para as quais foram utilizados cinquenta camundongos, na primeira fase foram utilizados 28 animais distribuídos em 6 grupos experimentais: submetidos à dieta controle (CT) e dieta hipercalórica (DH) por 3, 4 e 6 meses. Na segunda fase, 4 grupos experimentais foram avaliados aos 4 meses: CT e DH sem vetor terapêutico (CTPLL e DHPLL) e CT e DH com vetor terapêutico (CTVEGF e DHVEGF). A análise gênica pelo PCR em tempo real e proteica pela imuno-histoquímica evidenciou queda na expressão de VEGF, FLT1 e KDR no grupo DH, sendo que a variável estereológica de densidade de volume vascular (Vv) indicou queda significativa (p<0,05) da vascularização pancreática no grupo DH em relação ao CT aos 3, 4 e 6 meses do estudo. O DM foi caracterizado com queda significativa (p<0,05) na insulinemia após 4 meses com DH. Após a injeção pancreática no grupo DHVEGF do lentivírus contendo a sequência que condifica o VEGF, foram observados aumento na expressão gênica de VEGF , FLT1 e KDR (p<0,05), com aumento de Vv vascular pancreático e aumento na insulinemia. Os resultados obtidos sugerem que é possível obter um modelo animal diabético induzido por dieta, que o VEGF e seus receptores participam da evolução e estabelecimento do quadro diabético, levando a uma redução da vascularização pancreática, e que o aumento na expressão do transgene no pâncreas de camundongos diabéticos possa contribuir para a revascularização pancreática e função das células B. / There is an increasing number of people and pets showing overweight and obesity, with a consequent growth of the number of insulin-resistant and diabetic patients. The vascular endothelial growth factor (VEGF) has been characterized as an important molecule in many physiopathological states. Recent studies indicate a reduction in VEGF content in some tissues of diabetic patients causing tissue hypoxia and impairing cicatrization. In this context, this study aimed to characterize a diet-induced diabetic animal model and to evaluate vascularization, gene and protein expression of VEGFA and its receptors KDR and FLT1, in pancreas of diabetic and non-diabetic mice before and after gene therapy with VEGF. The study was divided in two phases and fifty male mice were used. In the first phase 28 animals were distributed into 6 groups: control diet (CT) and high calorie diet (DH) for 3, 4 and 6 months. In the second phase, four experimental groups were evaluated at 4 months: CT and DH without therapeutic vector (CTPLL and DHPLL) and CT and DH with therapeutic vector (CTVEGF and DHVEGF). The genetic analysis using real time PCR and protein by immunohistochemistry showed decrease in expression of VEGF, FLT1 and KDR in the DH group, and the stereological estimate of vascular volume density (Vv) indicated a significant decrease (p <0,05 ) of vascularization in pancreatic DH group relative to the CT at 3, 4 and 6 months of the study. Diabetic mice were characterized with a significant decrease (p <0,05) in insulin after 4 months with DH. After injection of lentivirus containing the VEGF sequence in DHVEGF´s pancreas, increase in VEGF, FLT1 and KDR gene expression (p <0.05) was observed, accompanied by the increase of vascular Vv and insulinemia. The results suggest that it is possible to obtain a diabetic animal model induced by diet, that VEGF and its receptors participate in the development and establishment of the diabetic state, leading to a reduction of pancreas vascularization, and that the increase of transgene expression in the pancreas of diabetic mice may contribute to the revascularization and function of pancreatic B cells.

INTRODUÇÃO: O câncer do pâncreas apresenta taxas anuais de mortalidade e incidência muito semelhantes, sendo uma das principais causas de morte por câncer no mundo. A agressividade do tumor e o retardo no seu diagnóstico são considerados responsáveis pela sua alta letalidade. O tratamento adjuvante convencional aumenta pouco a sobrevida a longo prazo e a terapia-alvo pode ser uma alternativa ao tratamento deste tipo de tumor. OBJETIVO: O objetivo do presente estudo é avaliar a expressão do receptor do fator de crescimento epitelial e seu eventual valor prognóstico em pacientes portadores de adenocarcinoma pancreático submetidos à ressecção cirúrgica. MÉTODO: Foram estudados retrospectivamente 88 pacientes portadores de adenocarcinoma pancreático operados no Serviço de Cirurgia de Vias Biliares e Pâncreas do HC-FMUSP e no Departamento de Cirurgia Abdominal Hospital A.C. Camargo no período de 1990 a 2006. RESULTADOS: Quarenta e sete (53,4%) pacientes do sexo feminino e 41 (46,6%) do masculino com idade mediana de 60 anos. As cirurgias realizadas foram duodenopancreatectomia com preservação do piloro (55,1%), gastroduodenopancreatectomia (34,8%), pancreatectomia corpo-caudal (6,8%) e gastroduodenopancreatectomia total (2,3%). A ressecção venosa portal foi realizada em 12 pacientes (13,5%). O tamanho tumoral médio foi de 3,75cm. Invasão vascular esteve presente em 31% dos casos e neural em 88,5%. A margem de ressecção estava comprometida em 33 pacientes (37,5%). Cinco (5,7%) pacientes eram do estádio IA, 15(17%) do IB, 19(21,6%) do IIA, 47(53,4) do IIB e dois (2,3%) do III.Observou-se diferença na expressão de EGFR na membrana celular entre o tecido tumoral e o tecido não tumoral (p=0,004); entre o tecido metastático linfonodal e o tecido não tumoral (p=0,02) mas não houve diferença quanto à sua expressão quando comparamos o tecido tumoral e o tecido metastático linfonodal (p=0,28). Dentre as variáveis clínicas e patológicas estudadas, observou-se diferença de expressão do EGFR entre os gêneros feminino e masculino (p=0,03), não havendo diferenças entre as outras variáveis. A sobrevida mediana global foi de 22,9 meses. A sobrevida cumulativa global em 1 ano, 3 anos e 5 anos foi de 48%, 20% e 18%, respectivamente. As sobrevidas cumulativas em 1 ano, 3 anos e 5 anos foram 77%, 30% e 8% no grupo sem expressão do EGFR na membrana tumoral versus 46%, 8% e 0% respectivamente no grupo com expressão do EGFR na membrana celular do tumor. Na análise univariada, as seguintes variáveis estiveram associadas a menor sobrevida: sexo masculino, ressecção venosa portal, invasão peri-neural, e vascular, invasão do tecido peri-pancreático, acometimento da margem de ressecção pancreática e expressão positiva de EGFR no tecido tumoral. Na análise multivariada, os fatores associados à sobrevida menor foram: gênero masculino, ressecção venosa portal, invasão vascular e invasão peri-neural. CONCLUSÃO: A expressão do EGFR na membrana celular é significativamente maior no tecido tumoral que no tecido pancreático não tumoral. A expressão do EGFR na membrana celular do tecido tumoral está associada a pior prognóstico (menor sobrevida). / INTRODUCTION: Pancreatic cancer is one of the main cancer related deaths in the world and its incidence is similar to its mortality. Biological aggressiveness and delayed diagnosis are a major concern. Adjuvant treatment has little impact on survival and the expression of potential target molecules has been undertaken in order to increase survival. OBJECTIVE: The aim of the present study is to study the expression of EGFR and its potential prognostic role in tumor, non-tumor and metastatic lymph node tissues of patient with pancreatic adenocarcinoma treated with surgical resection. MATHERIAL AND METHODS: Eighty eight patients with pancreatic adenocarcinoma operated at Serviço de Cirurgia de Vias Biliares e Pâncreas do HC-FMUSP and Departamento de Cirurgia Abdominal do Hospital A.C.Camargo were retrospectively studied between 1990 and 2003. RESULTS: Forty seven females (53,4%) and 41 males (46,6%) with median age of 60 years were studied. Pylorus preserving duodenopancreatectomy was performed in 55%, classical duodenopancreatectomy in 34,8%, distal pancreatectomy in 6,8% and total pancreatectomy in 2,3%. Portal vein resection was performed in 12 patients (13,5%). Mean tumor size was 3,75cm. Vascular and neural invasion were present in 31% and 88,5%, respectively. Positive surgical margin was present in 33 (37,5%) patients. Five (5,7%) patients were stage IA, 15(17%) stages IB, 19(21,6%) stages IIA, 47(53,4%) stages IIB and two (2,3%) stages III. There were difference in the membrane expression of EGFR between tumor and non tumor pancreatic tissue (p=0,004); between metastatic lymph node and non tumor pancreatic tissue (p=0,02); but there were no difference between tumor and metastatic lymph node tissue (p=0,28). Median survival time was 22,9 months. Cumulative one, three and five years survival were 48%, 20% and 18%. Cumulative survival at 1, 3 and 5 years were 77%, 30% and 8% in patients with negative expression of EGFR in tumor membrane and 46%, 8% and 0%, respectively in patients with positive EGFR expression in tumoral membrane. Univariate analysis showed that male gender, portal vein resection, neural, vascular and peri-pancreatic invasion invasion, positive surgical margin and positive membrane EGFR expression in tumoral tissue were correlated with poor survival. Multivariate analysis showed that male gender, portal vein resection, vascular invasion and peri-neural invasion are associated with lower survival after resection. CONCLUSION: EGFR membrane expression is different between tumor tissue and non tumor pancreatic tissue. EGFR membrane expression in tumoral tissue was associated with worst survival.

L’adénocarcinome du pancréas qui représente la quatrième cause de cancer en termes d’incidence reste une tumeur mal comprise et l’ensemble des traitements insuffisamment efficaces. Depuis quelques années, la prise en charge des patients cancéreux s’est individualisée et tente de proposer des traitements à la carte en prenant en compte divers paramètres tumoraux. Pour le cancer du pancréas, la pertinence des hypothèses biologiques pour le pronostic et le traitement des patients, a été très peu évaluée si bien qu’a coté des facteurs cliniques et histologiques, il n’existe pas de marqueurs moléculaires clairement identifiés. <p>Dans la première partie de nos traveaux, nous avons évalué l’intérêt pronostique d’une série de biomarqueurs dont les chimiorécepteurs CXCR4 et CXCR7. CXCR4 est exprimé par les ACP et joue un rôle dans la migration des cellules cancéreuses au travers la matrice extracellulaire, l’adressage métastatique, l’angiogénèse et la vasculogénèse tumorale. CXCR7 est impliqué dans la prolifération cellulaire. Ces deux chimiorécepteurs partagent le même ligand, CXCL12, qui est synthétisé par les cellules stellaires pancréatiques activées et est retrouvé en quantité importante dans le stroma fibrotique tumoral. L’expression de CXCR4 est en partie dépendante du facteur de transcription HIF-1&61537; (hypoxia inducible factor). L’expression de CXCR4, CXCR7, HIF-1&61537; et de Ki-67 à été évaluée par immunohistochimie et confrontée aux facteurs pronostiques classiques (cliniques et anatomopatholologiques). En analyse multivariée, le niveau d’expression de CXCR4 était un facteur pronostique indépendant associé à la survie sans récidive et la survie globale. De plus le niveau d’expression de CXCR4 était associé à la présence de métastase ganglionnaire sur la pièce opératoire et au risque de récidive hépatique.<p>Dans un deuxième temps, les protéines impliquées dans la métabolisation de la gemcitabine ont été évaluées à partir d’une cohorte de patients traités par radiochimiothérapie dans le cadre de deux protocoles cliniques multicentriques de phase II. La gemcitabine pénètre dans la cellule à travers la membrane plasmique soit par diffusion facilitée par des transporteurs nucléosidiques spécifiques appelés hENT (human equilibrative nucleoside transporter), soit par des transporteurs actifs Na+-dépendants ou hCNT (human concentrative nucleoside transporter). Dans la cellule, elle est métabolisée pour générer ses dérivés cytotoxiques actifs. La gemcitabine va tout d’abord être phosphorylée en dérivé monophosphate par la déoxycytidine kinase (dCK). La dCK est considérée comme étant l’enzyme limitante du métabolisme de la gemcitabine. Deux autres étapes de phosphorylation vont suivre faisant tout d’abord intervenir l’uridylate kinase (UMK) puis une troisième kinase qui, elle, est ubiquitaire.<p>In vitro, dans des lignées cellulaires d’adénocarcinome pancréatique, la résistance intrinsèque à la gemcitabine est associée à une diminution ou à une perte d’expression de hENT1 et de la dCK. En plus de son activité cytotoxique directe, la gemcitabine est un puissant radiosensibilisateur. La capacité de cet analogue pyrimidique à potentialiser les effets de la radiothérapie est en partie liée à sa capacité de pénétration. L’expression de hENT1, de hCNT3 et de la dCK a été évaluée et corrélée à la survie sans récidive et la survie globale des patients. En analyse multifactorielle, hENT1 et la dCK sont des facteurs pronostiques indépendants pour la survie sans récidive et la survie globale alors que hCNT3 est un facteur indépendant pour la survie globale. De plus l’analyse combinée de hENT1 et de hCNT3 s’avèrait plus discriminante dans l’identification des patients à haut rique de récidive que l’analyse de chacun de ces facteurs pris isolémment.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

La spectroscopie RMN Haute Résolution en Rotation à l’Angle Magique (HRMAS) permet la caractérisation métabolomique tissulaire. Nous avons caractérisé par RMN HRMAS le profil métabolomique des glandes parathyroïdiennes hypersécrétantes. Celui de l’hyperparathyroïdie primaire (HPT1) a été comparé à celui des HPT rénales. Au sein des HPT1, la distinction a pu être faite entre pathologie uni- et multi-glandulaire. Le profil métabolomique du tissu pancréatique sain a été comparé à celui du tissu tumoral. Aussi, les patients longs/courts-survivants ont pu être distingués. La relation entre le phénotype métabolique et la survie des patients a été étudiée. Le profil métabolomique des TIPMP a été caractérisé. Les TIPMP non dégénérées et dégénérées ont été comparées. Le risque de dégénérescence a été corrélé au profil métabolomique. Nos résultats montrent que la spectroscopie RMN HRMAS est une technique prometteuse pour l’étude du profil métabolomique des HPT et des pathologies pancréatiques. / High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy allows metabolomics of intact tissues. Metabolomics profiling of hyperfunctioning parathyroid glands were characterized were characterized by using HRMAS NMR spectroscopy. Primary hyperparathy- roidism (PHPT) was compared to renal HPT. Among PHPT, we distinguished single gland disease from multiple gland disease. Pancreatic parenchyma and adenocarcinoma were compared. Thus, long-term and short-term survival patients were distinguished. The relationship between the survival of patients and their metabolic phenotype was studied. Metabolomics profiling of IPMN was also examined. IPMN with no degeneration and de- generated IPMN were compared. Finally, the risk of degeneration was correlated with the metabolomics profile. Our results show that HRMAS NMR spectroscopy is a promising technique in view of studying metabolomic profiling of HPT and pancreatic diseases.

Le pancréas est une glande mixte à composantes endocrine et exocrine. Le tissu endocrine, essentiellement composé de cellules bêta productrices d’insuline, joue un rôle prépondérant dans le maintient de l’homéostasie glucidique. La perte qualitative ou quantitative des cellules bêta conduit au développement de pathologies caractérisées par une hyperglycémie chronique et connues sous le nom de diabète. Le développement de stratégies thérapeutiques innovantes, thérapie cellulaire ou médecine régénérative, pour guérir le diabète repose sur une connaissance précise des mécanismes développementaux impliqués dans la formation des cellules bêta. Ainsi, au delà de l’intérêt cognitif, il est primordial de comprendre au mieux les évènements cellulaires et moléculaires qui régissent l’organogénèse pancréatique pour offrir des thérapies alternatives. Le développement embryonnaire s’effectue dans un environnement où la pression partielle en oxygène (pO2) est faible. Par ailleurs, une étude menée au sein du laboratoire a montré que la pO2 influence la différenciation des cellules bêta pancréatique in vitro. En effet, lorsque des pancréas embryonnaires sont cultivés sur filtre en hypoxie (pO2=3%), le développement des cellules bêta est drastiquement diminué comparativement à une condition de 21% d’O2. Le facteur de transcription HIF-1 (Hypoxia Inducible Factor-1), composé d’une sous-unité alpha sensible au niveau d’oxygène et d’une sous-unité bêta constitutivement présente, permet à la cellule de s’adapter à un environnement pauvre en O2, notamment en favorisant la formation de nouveaux vaisseaux sanguins au cours d’un processus appelé angiogénèse. L’objectif de ma thèse était d’étudier le rôle de HIF-1alpha au cours du développement embryonnaire du pancréas in vivo. Pour cela, nous avons utilisé des lignées murines génétiquement modifiées permettant de stabiliser constitutivement la protéine HIF-1alpha dans l’épithélium pancréatique. En utilisant ce modèle murin, nous avons montré que la différenciation endocrine et le développement des cellules bêta est altéré dans les pancréas mutants comparativement aux contrôles. Par ailleurs, en utilisant une approche pharmacologique in vitro conduisant à l’ablation des cellules endothéliales du pancréas, nous avons pu restaurer une différenciation endocrine comparable aux contrôles. Ce travail a permis d’éclaircir le rôle de HIF-1 et de la vascularisation au cours du développement embryonnaire du pancréas. Nos résultats indiquent que ces paramètres doivent être pris en compte pour améliorer les protocoles actuels permettant de générer des cellules bêta in vitro. / The pancreas is an endoderm-derived organ which is composed by both an exocrine and an endocrine compartment. Within the endocrine tissu, insulin-producing beta-cells are essential for the regulation of glucose homeostasis. The loss of beta-cells can lead to pathologies such as diabetes. Currently, people suffuring from diabetes can be treated but not permanently cured. The development of innovating therapeutical approaches, like cellular therapy or regenerative medecine, relies on the precise knowledge of the mechanisms regulating the ontogenesis of pancreatic beta-cells. Different studies have linked proper embryonic development and low-oxygen tension (pO2). Specifically, when embryonic pancreases are cultured in vitro under a hypoxic condition (pO2=3%), the beta-cells development is impaired compared to a normoxic condition (pO2=21%). Different pathways are involved in the cell adaptation to hypoxia, such as the ubiquitous Hypoxia Inducible Factor 1-alpha (HIF-1alpha). The aim of my PhD project was to elucidate the role of HIF-1alpha during pancreatic development in vivo. To do so, we used genetically modified mice allowing the constitutive stabilization of HIF-1alpha in pancreatic epithelial cells. We have shown that HIF-1alpha stabilization leads to a reduction of endocrine differentiation and beta-cells development. Moreover, using a pharmacological approach in vitro consisting in deleting endothelial cells, we rescued the endocrine differentiation in the mutant pancreases. In conclusion, my data demonstrated the negative influence of both HIF-1 and endothelial cells on endocrine differentiation processes.

[EN] Type 1 diabetes mellitus is a chronic and incurable disease that affects millions of people all around the world. Its main characteristic is the destruction (totally or partially) of the beta cells of the pancreas. These cells are in charge of producing insulin, main hormone implied in the control of blood glucose. Keeping high levels of blood glucose for a long time has negative health effects, causing different kinds of complications. For that reason patients with type 1 diabetes mellitus need to receive insulin in an exogenous way. Since 1921 when insulin was first isolated to be used in humans and first glucose monitoring techniques were developed, many advances have been done in clinical treatment with insulin. Currently 2 main research lines focused on improving the quality of life of diabetic patients are opened. The first one is concentrated on the research of stem cells to replace damaged beta cells and the second one has a more technological orientation. This second line focuses on the development of new insulin analogs to allow emulating with higher fidelity the endogenous pancreas secretion, the development of new noninvasive continuous glucose monitoring systems and insulin pumps capable of administering different insulin profiles and the use of decision-support tools and telemedicine. The most important challenge the scientific community has to overcome is the development of an artificial pancreas, that is, to develop algorithms that allow an automatic control of blood glucose. The main difficulty avoiding a tight glucose control is the high variability found in glucose metabolism. This fact is especially important during meal compensation. This variability, together with the delay in subcutaneous insulin absorption and action causes controller overcorrection that leads to late hypoglycemia (the most important acute complication of insulin treatment). The proposals of this work pay special attention to overcome these difficulties. In that way interval models are used to represent the patient physiology and to be able to take into account parametric uncertainty. This type of strategy has been used in both the open loop proposal for insulin dosage and the closed loop algorithm. Moreover the idea behind the design of this last proposal is to avoid controller overcorrection to minimize hypoglycemia while adding robustness against glucose sensor failures and over/under- estimation of meal carbohydrates. The algorithms proposed have been validated both in simulation and in clinical trials. / [ES] La diabetes mellitus tipo 1 es una enfermedad crónica e incurable que afecta a millones de personas en todo el mundo. Se caracteriza por una destrucción total o parcial de las células beta del páncreas. Estas células son las encargadas de producir la insulina, hormona principal en el control de glucosa en sangre. Valores altos de glucosa en la sangre mantenidos en el tiempo afectan negativamente a la salud, provocando complicaciones de diversa índole. Es por eso que los pacientes con diabetes mellitus tipo 1 necesitan recibir insulina de forma exógena. Desde que se consiguiera en 1921 aislar la insulina para poder utilizarla en clínica humana, y se empezaran a desarrollar las primeras técnicas de monitorización de glucemia, se han producido grandes avances en el tratamiento con insulina. Actualmente, las líneas de investigación que se están siguiendo en relación a la mejora de la calidad de vida de los pacientes diabéticos, tienen fundamentalmente 2 vertientes: una primera que se centra en la investigación en células madre para la reposición de las células beta y una segunda vertiente de carácter más tecnológico. Dentro de esta segunda vertiente, están abiertas varias líneas de investigación, entre las que se encuentran el desarrollo de nuevos análogos de insulina que permitan emular más fielmente la secreción endógena del páncreas, el desarrollo de monitores continuos de glucosa no invasivos, bombas de insulina capaces de administrar distintos perfiles de insulina y la inclusión de sistemas de ayuda a la decisión y telemedicina. El mayor reto al que se enfrentan los investigadores es el de conseguir desarrollar un páncreas artificial, es decir, desarrollar algoritmos que permitan disponer de un control automático de la glucosa. La principal barrera que se encuentra para conseguir un control riguroso de la glucosa es la alta variabilidad que presenta su metabolismo. Esto es especialmente significativo durante la compensación de las comidas. Esta variabilidad junto con el retraso en la absorción y actuación de la insulina administrada de forma subcutánea favorece la aparición de hipoglucemias tardías (complicación aguda más importante del tratamiento con insulina) a consecuencia de la sobreactuación del controlador. Las propuestas presentadas en este trabajo hacen especial hincapié en sobrellevar estas dificultades. Así, se utilizan modelos intervalares para representar la fisiología del paciente, y poder tener en cuenta la incertidumbre en sus parámetros. Este tipo de estrategia se ha utilizado tanto en la propuesta de dosificación automática en lazo abierto como en el algoritmo en lazo cerrado. Además la principal idea de diseño de esta última propuesta es evitar la sobreactuación del controlador evitando hipoglucemias y añadiendo robustez ante fallos en el sensor de glucosa y en la estimación de las comidas. Los algoritmos propuestos han sido validados en simulación y en clínica. / [CAT] La diabetis mellitus tipus 1 és una malaltia crònica i incurable que afecta milions de persones en tot el món. Es caracteritza per una destrucció total o parcial de les cèl.lules beta del pàncrees. Aquestes cèl.lules són les encarregades de produir la insulina, hormona principal en el control de glucosa en sang. Valors alts de glucosa en la sang mantinguts en el temps afecten negativament la salut, provocant complicacions de diversa índole. És per això que els pacients amb diabetis mellitus tipus 1 necessiten rebre insulina de forma exògena. Des que s'aconseguís en 1921 aïllar la insulina per a poder utilitzar-la en clínica humana, i es començaren a desenrotllar les primeres tècniques de monitorització de glucèmia, s'han produït grans avanços en el tractament amb insulina. Actualment, les línies d'investigació que s'estan seguint en relació a la millora de la qualitat de vida dels pacients diabètics, tenen fonamentalment 2 vessants: un primer que es centra en la investigació de cèl.lules mare per a la reposició de les cèl.lules beta i un segon vessant de caràcter més tecnològic. Dins d' aquest segon vessant, estan obertes diverses línies d'investigació, entre les que es troben el desenrotllament de nous anàlegs d'insulina que permeten emular més fidelment la secreció del pàncrees, el desenrotllament de monitors continus de glucosa no invasius, bombes d'insulina capaces d'administrar distints perfils d'insulina i la inclusió de sistemes d'ajuda a la decisió i telemedicina. El major repte al què s'enfronten els investigadors és el d'aconseguir desenrotllar un pàncrees artificial, és a dir, desenrotllar algoritmes que permeten disposar d'un control automàtic de la glucosa. La principal barrera que es troba per a aconseguir un control rigorós de la glucosa és l'alta variabilitat que presenta el seu metabolisme. Açò és especialment significatiu durant la compensació dels menjars. Aquesta variabilitat junt amb el retard en l'absorció i actuació de la insulina administrada de forma subcutània afavorix l'aparició d'hipoglucèmies tardanes (complicació aguda més important del tractament amb insulina) a conseqüència de la sobreactuació del controlador. Les propostes presentades en aquest treball fan especial insistència en suportar aquestes dificultats. Així, s'utilitzen models intervalares per a representar la fisiologia del pacient, i poder tindre en compte la incertesa en els seus paràmetres. Aquest tipus d'estratègia s'ha utilitzat tant en la proposta de dosificació automàtica en llaç obert com en l' algoritme en llaç tancat. A més, la principal idea de disseny d'aquesta última proposta és evitar la sobreactuació del controlador evitant hipoglucèmies i afegint robustesa. / Revert Tomás, A. (2015). Robust strategies for glucose control in type 1 diabetes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/56001 / TESIS

:.In our work we wanted to confirm our clinical experience with therapy of biliary and pancreatic duct injuries from the endoscopic retrograde cholangiopancreatography (ERCP) which was done in 267. Children and infants with a variety of biliary tract disorders and traumatic injuries in the area of biliary and pancreatic duct.. Pressure of the bile plays the key role in the therapy of biliary tract injuries Therefore we have measured the pressure in biliary tract and duodenum before and after the sphincterotomy of Oddi sphincter. Thea aim of our study was to confirm the insertion of drainage into the biliary and pancreatic duct in children with injury in this area. Our results showed significant differences between biliary duct pressure and duodenal pressure in the patients before and after sphincterotomy of Oddi sphincter. This results on theoretical basis confirmed, that it is necessary in children after traumatic rupture of biliary duct to provide ERCP and insert a biliary drainage after sphincterotomy. With this procedure the biliary tract injury is healed ad integrum without surgical liver resection. To provide only papilosphincterotomy without biliary drainage is not sufficient. This new miniinvasive procedure plays a fundamental role in the therapy of blunt abdominal injuries in a children and infants...