A maioria dos estudos com ultrassonografia (US), tomografia computadorizada (TC) e ressonância magnética (RM) mostra que as dimensões do pâncreas são reduzidas em pacientes com diabetes, quando comparados com grupo controle. Dados sobre a perfusão pancreática em pacientes com diabetes são escassos na literatura. Essa tese tem por objetivo avaliar características do pâncreas nos exames de imagem que possam trazer uma melhor compreensão da patogênese e da fisiopatologia do diabetes. Primeiramente, realizamos uma revisão sistemática com metanálise de estudos que utilizaram métodos de imagem (US, TC ou RM) para a medida das dimensões – diâmetro, área ou volume - e do conteúdo de gordura do pâncreas em pacientes com diabetes tipo 1 (DM1) ou tipo 2 (DM2). Demonstramos que as dimensões pancreáticas são menores nos pacientes com DM1 ou DM2 em comparação com indivíduos sem diabetes. Além disso, o conteúdo de gordura do pâncreas é maior em pacientes com DM2. Com o intuito de investigar uma possível causa para a redução do volume do pâncreas em pacientes com diabetes, buscamos estudar a vascularização pancreática por meio de TC perfusional. Inicialmente, fizemos um estudo para avaliar a variabilidade intra e interobservador para a medida dos parâmetros de perfusão pancreática por TC (fluxo sanguíneo, volume sanguíneo, tempo de trânsito médio, tempo para o pico de realce), demonstrando que existe uma boa concordância nessas medidas, mesmo entre radiologistas com diferentes níveis de experiência. Em sequência, realizamos um estudo comparando esses parâmetros de perfusão pancreática por TC entre pacientes com DM2 e indivíduos sem diabetes. Mostramos que o volume sanguíneo que perfunde o pâncreas e o seu tempo de trânsito médio pelo órgão são menores em pacientes com DM2 em comparação com indivíduos não diabéticos.

Introduction: The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal and pancreatic cancer. The first part deals with the etiological factors and the importance of polymorphisms in biotransformation enzymes and genetic alterations in the gene CHEK2 in the origin of these malignancies. In the second part, the ABC transporter genes were analyzed as potential prognostic and predictive markers of a treatment's outcome. Materials and methods: The polymorphisms and other genetic alterations were detected using real-time PCR, allelespecific PCR and PCR-RFLP methods in DNA which was extracted from the blood of patients. The frequency of polymorphisms was evaluated and their importance was assessed with regard to the available epidemiological data. Gene expressions were determined by qPCR in paired samples of tumor tissue and adjacent non-tumorous parenchyma. Results: A majority of the observed polymorphisms failed to show a relationship between their presence and the risk of any of these malignancies. CYP2A13 variant allele*7 coding inactive enzyme was found in 7 of 265 controls and in none of 235 pancreatic carcinoma patients. In contrast, GSTP1-codon 105 Val variant allele and GSTT1-null genotype were associated with an elevated...

A obesidade vem aumentando progressivamente na população canina em decorrência de distúrbios endócrinos, alimentares e de manejo, o que leva ao aparecimento de doenças a ela relacionadas, sendo a Diabetes Mellitus a mais comum. Esta enfermidade ocorre devido a alterações na secreção ou sinalização da insulina em tecidos periféricos, gerando uma resistência periférica à insulina, o que pode levar à completa exaustão beta-pancreática, processo irreversível e incompatível com a vida. Pouco se sabe sobre a patogênese da resistência à insulina causada pela obesidade em cães. A proteína C reativa está relacionada à obesidade em humanos e roedores, mas não existem dados na literatura sobre a sua relação com a obesidade e resistência à insulina em cães. O transportador de glicose GLUT4 tem importante função na entrada de glicose nas células adiposas e musculares. A alteração na expressão desta proteína já foi associada à obesidade em várias espécies, incluindo cães, porém não se sabe se esta alteração está relacionada à resistência à insulina. O objetivo deste trabalho foi elucidar alguns aspectos bioquímicos e moleculares que cercam a resistência à insulina induzida pela obesidade em cães e com isso gerar subsídios para a prevenção da diabetes mellitus na espécie canina. Para tanto, foram realizados três experimentos, onde os dois primeiros tentaram elucidar aspectos bioquímicos da obesidade (efeito sobre os níveis de proteína C-reativa) e resistência à insulina (efeito sobre os níveis de fructosamina), e o último tentou avaliar aspectos moleculares em tecido adiposo e muscular (efeito sobre a expressão de GLUT4). Os resultados mostraram que a proteína C-reativa não se altera com a obesidade canina, e que a fructosamina altera-se com o desenvolvimento da resistência à insulina em cães. Uma modificação no padrão expressão da proteína GLUT4 entre os tecidos muscular e adiposo foi observada na presença da resistência à insulina e uma alteração na translocação foi verificada nos dois tecidos, causada pela obesidade. Com base nos resultados obtidos pode-se concluir que a patogênese da resistência à insulina causada pela obesidade não é similar a outras espécies, e deve ser prevenida com medidas distintas. O presente estudo também gerou informações que podem auxiliar no diagnóstico e prevenção da resistência à insulina e diabetes mellitus na espécie canina. / endocrine, nutritional and management disturbances, what leads to the appearance of related diseases, being Diabetes Mellitus the most common. This illness occurs because of insulin secretion or signaling alterations, generating a peripheral insulin resistance, what can lead to a complete beta-pancreatic exhaustion that is considered an irreversible and life-incompatible process. Little is known about pathogenesis of insulin resistance caused by obesity in dogs. C-reactive protein is related to obesity in human and rodent species, but there is no data in the literature about its relation with obesity and insulin resistance in dogs. GLUT4 glucose transporter has an important role on adipose and muscular cells glucose uptake. Alterations on GLUT4 protein expression have been established in obesity in some species including dogs, but there is no knowledge about this mechanism in insulin resistance in canine species. The aim of this work was to elucidate some biochemical and molecular issues surrounding obesity induced insulin resistance in dogs and thus generate information leading to prevention of diabetes mellitus in canine species. To achieve that, three experiments were accomplished, where the first two tried to elucidate biochemical aspects of obesity (effect on C-reactive protein levels) and insulin resistance (effect on fructosamine levels), and the third attempted to evaluate molecular aspects in fat and muscular tissues (effect on GLUT4 expression). Results showed that C-reactive protein levels do not alter with canine obesity, and that fructosamine levels change with the onset of insulin resistance. A modification on GLUT4 protein expression pattern between adipose and muscular tissues was observed in the presence of insulin resistance and a translocational alteration was detected in both tissues, caused by obesity. Based on the obtained results it can be postulated that the pathogenesis of insulin resistance caused by obesity differs from other species, and must be prevented with distinct measures. Furthermore, the present study generated information that can help on diagnosis and prevention of insulin resistance and diabetes mellitus in canine species.

Made available in DSpace on 2014-10-09T12:26:12Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:43Z (GMT). No. of bitstreams: 0 / Este trabalho teve por objetivo propor uma racionalização da síntese, da análise e da purificação do radiofármaco 177Lu3+-DOTATATO para uso diagnóstico e terapêutico em tumores neuroendócrinos, bem como avaliar a biodistribuição deste radiofármaco em modelo animal. A reação de complexação para a síntese do radiofármaco foi realizada em tampão acetato de amônio 0,5 M, pH 7,0, à 95 oC, com tempo de reação de 30 minutos. Obteve-se pureza radioquímica > 95%, de acordo com a análise por cromatografia em ITLC-SG, utilizando-se como fase móvel, tampão citrato de sódio 0,1 M, pH 5,0. A razão molar-limite 177Lu3+: DOTATATO utilizada na síntese do radiofármaco 177Lu3+-DOTATATO foi dependente da atividade específica e da procedência do radioisótopo, sendo de 1:3,5 (370 MBq : 2,6 mg) para radioisótopo oriundo do Oak Ridge National Laboratory/EUA e de 1:16 (370 MBq : 11,8 mg) para o radioisótopo oriundo do Nuclear Analytical and Medical Services/Holanda, considerando para ambos, um decaimento de cinco dias a partir da data de produção do radioisótopo. Esta racionalização da síntese do radiofármaco 177Lu3+-DOTATATO permite uma grande economia nos custos de produção. O estudo químico da síntese do radiofármaco também evidenciou a interferência do 177Hf4+, produto de decaimento do 177Lu3+, como competidor do 177Lu3+ pelo DOTATATO. A preparação radiofarmacêutica mostrou-se estável durante 24 horas, a uma atividade de 2775 MBq, com adição de 0,6 mg/mL de ácido gentísico, mantida em gelo seco. Nos estudos de biodistribuição em camundongos Swiss e Nude demonstrou-se a especificidade do radiofármaco pelos tecidos receptor-específicos para somatostatina como pâncreas, estômago, pulmão, adrenais, rins e de células tumorais AR42J. Palavras chave: síntese, complexação, radiofármaco, 177Lu-DOTATATO, tumor neuroendócrino. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Objetivos: Comparar quantitativamente por técnicas de ressonância magnética (RM), a fração de gordura pancreática em indivíduos sadios, obesos e diabéticos (tipos 1 e 2). Secundariamente, buscou-se identificar diferença das medidas do ADC (difusão) no parênquima pancreático. Materiais e Métodos: Estudo retrospectivo com avaliação por dois radiologistas de imagens de RM abdominal de 89 indivíduos (56 controles; 33 diabéticos). Utilizadas três sequências: T1-GRE em fase e fora de fase; difusão (mapa ADC). Calculados e comparados frações de gordura e valores médios do ADC pancreáticos nos grupos. Resultados: Observaram-se diferenças significativas da fração de gordura pancreática entre diabéticos tipo 2 (DM2) e sadios e diabéticos tipo 1 (DM1), com valores de p de 0,01 e 0,02 para homens e 0,02 e 0,01 para mulheres, com confiabilidade interobservador ótima (coeficiente de correlação intraclasse > 0,8). Em obesos não diabéticos, observaram-se frações de gordura pancreática semelhantes às dos diabéticos tipo 2. Observou-se também diferença significativa nos valores de ADC entre DM2 e sadios e DM1 (p: 0,02 e 0,03 no sexo masculino; p: 0,002 e 0,001 no sexo feminino), menores nos DM2. Discussão: Observaram-se frações de gordura pancreáticas significativamente maiores em DM2, comparativamente a indivíduos sadios e DM1, achado que fomenta a hipótese da infiltração gordurosa do órgão como um fator causal associado para a falência de células beta pancreáticas. / Objectives: To compare, through quantitative MRI techniques, the pancreatic fat fraction in healthy, obese and diabetic (type 1 and 2) individuals. Secondarily, weve tried to identify differences in ADC (diffusion) values in the pancreatic parenchyma. Materials and Methods: A retrospective study, with review, by two radiologists, of abdominal MR images of 89 subjects (56 controls; 33 diabetics). Three sequences have been used: T1-GRE in-phase and out-of-phase; diffusion (ADC map). Fat fractions and average values of the ADC in pancreatic parenchyma have been calculated and compared. Results: We observed significant differences between pancreatic fat fractions of diabetics type 2 (DM2) and healthy and diabetic type 1 (DM1) individuals, with p values of 0.01 and 0.02 for men and 0.02 and 0.01 for women, with good interobserver reliability (intraclass correlation coefficients > 0.8). Obese nondiabetic sujects showed high pancreatic fat fraction similar to DM2. There was also a significant difference in ADC values between DM2 and DM1 and healthy individuals (p: 0.02 and 0.03 in males; p: 0.002 and 0.001 in females), lower in DM2. Discussion: We observed significantly higher pancreatic fat fractions in DM2, when compared to healthy and DM1 individuals. This finding favors the hypothesis of fatty infiltration of the organ as an associated causal factor to the pancreatic beta cells failure.

O projeto teve como objetivo avaliar o efeito do uso de enzimas exógenas sobre os grânulos de zimógenos no pâncreas. O projeto foi dividido em três experimentos, sendo o experimento I para avaliação do uso da protease via ração nas dosagens de 50, 100 e 150% das recomendações do fabricante. O experimento II, foi realizado avaliando-se o uso da enzima amilase nas dosagens de 50, 100 e 150% das recomendações do fabricante e o experimento III foi realizado avaliando-se as dosagens de amilase, amilase + fitase, protease e protease + fitase sobre os grânulos de zimógenos. Em cada experimento, foi utilizado delineamento inteiramente casualizado sendo um total de nove tratamentos com oito repetições. Aos 21 e 42 dias de idade, as aves foram sacrificadas por deslocamento cervical para coleta e processamento do pâncreas para análise histopatológica e posterior análise pelo programa Image Pro Plus&reg;. Este programa permitiu a mensurar a porcentagem dos grânulos de zimógenos nas imagens obtendo-se um índice matemático da presença dos grânulos de zimógenos no pâncreas, o qual pode ser avaliado estatisticamente e submetidos a regressão polinomial os experimentos I e II e ao teste de Tukey o experimento III. Uma redução significativa (P<0,05) com efeito quadrático na presença dos grânulos de zimógenos pode ser observada nos experimentos I e II. O experimento III apresentou diferença estatística (P<0,05) entre o uso de fitase 100% + Protease 100% quando comparado com o controle. / This research project was conducted in the poultryhouse of the Faculty of Veterinary Medicine USP - Department of Nutrition and Animal Production, Pirassununga Site. Thus, this present study aimed to evaluate the effect of exogenous enzymes on the zymogenes granules of pancreas. It was performed three experimental trials; the first experiment has evaluated the addition of 50, 100 and 150% of protease manufactory recommendation in the feed. The trial two was conducted to evaluate the addition of 50, 100 and 150% of amylase manufactory recommendation in feed, and the experiment three was conducted to evaluated the addition of amylase, amylase + phytase, protease and protease + phytase in the feed on the zymogens granules. Each experiment was designed totally randomized with a total of nine treatments with eight replicates each. The birds were killed by cervical dislocation at days 21 e 42 to collect the pancreas for histopathology analysis followed by the Image Pro Plus&reg; program analysis, this program allowed to transform the image into a mathematic number, which could be used in statistical analysis. At the experiments I and II, the statistical analysis were conducted by the polynomial regression method and the experiment three was evaluated by Tukey test. A significant reduction (P<0,05) with quadratic effect was observed on the zymogens granules at the experiments I and II. The experiment III show a significant difference between the treatment with phytase 100% + Protease 100% and the control group.

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Abstract The carbonic anhydrases (CAs) catalyse the hydration of CO2 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. Carbonic anhydrases are involved in many physiological processes connected with respiration and transport of CO2/bicarbonate between metabolising tissues and the lungs, pH homeostasis and electrolyte secretion in a variety of tissues/organs. The present work was undertaken to study the distribution and expression of CA isoenzymes in the normal and neoplastic gastrointestinal tissues. The expression of CA I, II, IX and XII in the human intestine and colorectal tumours was investigated by immunohistochemistry and western blotting. In the present study, immunohistochemical methods were also used to examine the location of CA IX and XII in the human pancreas and pancreatic tumours. The expression of CA XIV in the murine liver and intestine was studied using immunostaining and northern blotting. The present results suggest that transmembrane CA XII is absent from the small intestine, but is expressed in all segments of the normal large intestine. The positive signal for CA XII was confined to the basolateral plasma membranes of the epithelial cells of the surface epithelial cuff. In tumours, the signal for CA XII became stronger in the deep part of the lesion. The intensity of the immunostaining for CA I and II was clearly found to decrease in benign lesions and became very weak in malignant colorectal tumours. The reciprocal pattern of expression observed for membrane-associated (CA IX and XII) and cytoplasmic (CA I and II) isoenzymes in intestinal samples suggests that CA IX and XII may be functionally involved in tumour progression to malignancy and/or in invasion. CA I and II, which are thought to play important physiological roles in the normal colorectal mucosa, may not be required for growth of colorectal cancers and their expression consistently diminishes with progression to malignancy. In the human pancreas CA IX and XII appeared to be sporadically expressed in the basolateral plasma membrane of the normal acinar and ductal epithelium. The increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis. CA XIV was expressed in the hepatocyte plasma membrane and its localization on both apical and basolateral membrane domains suggests an important role for this isoenzyme in the regulation of ion and pH homeostasis in the liver.

Pancreas transplantation (PT) is considered a gold-standard cure for brittle insulin dependent diabetes mellitus. In over 90% of cases, this is conducted simultaneously with a kidney transplant, providing concurrent treatment for end-stage renal failure (Simultaneous pancreas and kidney transplantation, SPKT). However, since its inception in the 1960’s, SPKT has been associated with considerable morbidity and mortality. Despite significant recent improvements in graft and patient survival, the multi-factorial nature of the procedure has resulted in persistently high peri-operative morbidity. This thesis has identified four areas to study in the peri-operative assessment and management of these patients, potentially resulting in improved clinical outcomes. 1. Pre-operative risk-prediction scoring systems aide in the consent process and the peri-operative planning of care following major surgery. In PT, multi-system risk-prediction tools are deficient. We therefore assessed the utility of commonly used general surgical risk prediction models in PT recipients. Our finding suggested that The Waterlow Score, a multi-system tool originally developed for predicting the development of decubitus skin ulcers, identified high-risk individuals and has value in predicting outcome following SPKT.2. Peri-operative physiological optimisation (Goal-directed therapy, GDT) is well-recognised to improve outcomes following major general surgery in high-risk individuals. A randomised controlled trial was therefore performed to investigate the benefits of GDT in the peri-operative period following SPKT. The findings demonstrated improved short-term outcomes following GDT in our cohort.3. The temporal evolution of biomarkers following major physiological stresses allow for application in the diagnosis, management surveillance and treatment of diseases. In our cohort the acute evolution of inflammatory and diabetes biomarkers were delineated and correlated to clinical outcome. We identified that cold ischaemic time is significantly negatively related to early pancreatic function and CRP provides an easily measurable predictor of recipient morbidity.4. The final study aimed to evaluate the feasibility and assess the benefits of contrast enhanced ultrasound (CEUS) in the immediate post-operative period following PT. We found CEUS to be a clinically useful adjunct in the post-operative assessment of allograft morphology and perfusion, although further validation and correlation with outcomes is required.

Pancreatic ductal adenocarcinoma (PDAC) has the poorest five-year survival rate of any cancer. Currently, there are no effective diagnostics or chemotherapeutics. Surgical resection is the only curative therapy. However, most patients experience recurrence due largely to challenges in assessing tumor margin status in the operating room. Molecular probes that selectively highlight pancreatic cancer tissue, having the potential to improve PDAC margin assessment intraoperatively, are urgently needed. In this work, a series of red and near-infrared fluorescent probes is reported. Two were found to distribute to normal pancreas following systemic administration. One selectively accumulates in genetically modified mouse models of PDAC, providing cancer-specific fluorescence. In contrast to the small molecule probes reported previously, it possesses inherent affinity for PDAC cells and tissue, and thus does not require conjugation to targeting agents. Moreover, the probe exhibits intracellular accumulation and enables visualization of four levels of structure including the whole organ, tissue, individual cells and subcellular organelles. It can thus promote new strategies for precision image-guided surgery, pancreatic cancer detection, the monitoring of therapeutic outcomes and basic research.