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<p>Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion <italic>in vivo</italic>. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia. </p><p>To identify the developmental requirements for <italic>cftr</italic> function, we generated <italic>cftr</italic> mutant zebrafish using transcription activator like effector nucleases (TALENs). In <italic>cftr</italic> mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in <italic>cftr</italic> mutants, which prevents directional fluid flow necessary for KV function. To examine <italic>cftr</italic> expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the <italic>cftr</italic> promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for <italic>cftr</italic> during KV morphogenesis and function in the zebrafish. </p><p>In the zebrafish pancreas, we found that loss of <italic>cftr</italic> function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of <italic>cftr</italic> BAC expression in the adult and larval zebrafish pancreata revealed that <italic>cftr</italic> is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult <italic>cftr</italic> mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that <italic>cftr</italic> is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and <italic>cftr</italic> mutant larvae. These results indicate that <italic>cftr</italic>-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of <italic>cftr</italic> function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.</p> / Dissertation

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

<p>Transferrin receptor 1 (Tfr1) serves as a receptor for transferrin, an iron-binding protein in the blood, in its canonical role of iron assimilation. Tfr1 is expressed ubiquitously in many tissues and is believed to be required for iron uptake by most cells. </p><p>The Tfr1 global knockout mouse highlights the requirement for Tfr1 in erythrocyte precursors. The erythron is the tissue with the highest iron requirement, to enable hemoglobin production. Tfr1-null embryos die by embryonic day 12.5 with anemia, which has been assumed to cause lethality of the knockout mice. Due to the embryonic lethality of the mice, the role of Tfr1 has not been well characterized in other tissues in vivo. This thesis examines the role of Tfr1 in other tissues through the generation and characterization of conditional knockout mouse models of Tfr1 deletion in the intestinal epithelium, pancreas, and skin.</p><p>Tfr1 is expressed on the basolateral surface of proliferating cells in the intestinal epithelium. Deletion of Tfr1 specifically in the intestinal epithelium resulted in the loss of intestinal epithelial homeostasis, loss of proliferation, lipid accumulation, gene expression indicating epithelial to mesenchymal transition of intestinal epithelial cells, and early neonatal lethality. These phenotypes were mostly alleviated by forced expression of a mutant Tfr1 allele which is unable to bind to iron-loaded transferrin, suggesting that Tfr1 has a novel role independent of its canonical iron-assimilatory ability.</p><p>Deletion of Tfr1 in the pancreas resulted in juvenile death due to perturbed homeostasis of both endocrine and exocrine tissues, resulting in symptoms associated with pancreatitis and diabetes. No diabetic phenotype was detected in the conditional knockout mouse model of Tfr1 deletion specifically in &#946;-cells, suggesting that the primary effect of the loss of Tfr1 was limited to the exocrine tissue.</p><p>Deletion of Tfr1 in the epidermis of the skin caused neonatal lethality with abnormal hair follicle morphology and a significant reduction in dermal adipocytes.</p><p>These results indicate that the loss of Tfr1 has pleiotropic effects, depending on the cell type affected. Furthermore, Tfr1 appears to have non-canonical functions in the intestinal epithelium, a novel discovery.</p> / Dissertation

L’adénocarcinome canalaire pancréatique est une maladie agressive et dévastatrice qui est caractérisée par une progression rapide et une résistance aux traitements. Le développement de nouveaux vecteurs à fort potentiel de transfert de gène dans les cellules tumorales pourrait être un outil performant et innovant pour améliorer les traitements existants. Nous avons développé un système de ciblage des cellules tumorales pancréatiques via l’antigène de surface Mucine 4 en utilisant des vecteurs lentiviraux spécifiques permettant le transfert efficace du gène cytotoxique de la Thymidine Kinase (hsv-TK) directement dans des modèles de tumeurs. La transduction lentivirale de cellules tumorales pancréatiques a été possible dans des xénogreffes orthotopiques. Le transfert du gène TK suivi d’un traitement au ganciclovir a montré des résultats encourageants in vivo. L’approche thérapeutique présentée ici semble être plus sécurisé, plus spécifique et aussi efficace qu’un lentivirus à large tropisme pour le transfert de gène dans les tumeurs pancréatiques. La mise en évidence de nouvelles cibles moléculaires dont la dérégulation participe au développement et à l’agressivité des tumeurs pancréatiques pourraient être utilisées pour moduler favorablement les traitements thérapeutiques en cours. De plus, ces nouvelles cibles moléculaires pourraient également servir de facteur de détection précoce ou de marqueurs pronostiques de l’adénocarcinome pancréatique. L’implication des isoformes du récepteur FGFR3 dans les mécanismes moléculaires de l’adénocarcinome pancréatique a été évaluée pour la première fois. Un rôle opposé d’oncogène et de gène suppresseur de tumeur a été découvert pour le FGFR3 en fonction du contexte cellulaire. Des voies de signalisation différentes sont empruntées pour induire ces effets opposés dans les cellules tumorales pancréatiques. Cette nouvelle donnée devra être considérée pour des thérapies ciblées impliquant des inhibiteurs de tyrosine kinases qui, s’ils sont utilisés dans un mauvais contexte cellulaire, pourraient être plus dangereux que bénéfiques. / Pancreatic ductal adenocarcinoma is an aggressive and devastating disease, which is characterized by rapid progression and resistance to treatment. The development of new vectors with high potential of gene transfer in tumor cells could be an innovative and valuable tool. We have developed a strategy of pancreatic tumor cells targeting with the cell surface marker Mucin 4 using specific lentiviral vectors for the efficient transfer of the thymidine kiniase cytotoxic gene within the tumor. Lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. The hsv-TK/GCV anti-cancer system showed promising results in vivo. The approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. Discovering new molecular targets which modulation contributes to the development and aggressiveness of pancreatic tumors could favorably modulate the current therapies. Furthermore, these new molecular targets could also serve as factors of early detection or prognostic markers of pancreatic adenocarcinoma. The involvement of FGFR3 receptor in the molecular mechanisms of pancreatic adenocarcinoma was evaluated for the first time. Opposite roles of both oncogene and tumor suppressor gene have been discovered for FGFR3 depending on the cellular context. Importantly, molecular pathways supporting these effects are different. This new data should be considered for targeted therapies involving tyrosine kinase inhibitors. If used in the wrong cell context they could be more dangerous than beneficial

Adenocarcinoma pancreático é um tumor maligno com mau prognóstico, e por isso há necessidade de aperfeiçoamento ou criação de novas estratégias terapêuticas. A vacinação baseada em DCs é uma das abordagens mais promissoras, uma vez que as DCs são as células apresentadoras de antígenos (APCs) mais potentes e centrais para a indução e manutenção de uma resposta imune. Entretanto, em pacientes com câncer, a geração e a função de DCs podem ser deficientes, impondo um obstáculo para o sucesso de seu uso. Neste trabalho, nós descrevemos a geração in vitro de DCs a partir de células mononucleares do sangue periférico (PBMC) obtidas de pacientes ictéricos com câncer de pâncreas e, também, o efeito do plasma ictérico (PI) sobre as culturas de DCs de doadores saudáveis. PBMCs foram separadas do sangue obtido de 22 pacientes e 22 doadores saudáveis. Células aderentes foram cultivadas com GM-CSF e IL-4 (50ng/mL) por 7 dias. No 5o dia, TNF-<font face=\"symbol\">a (50ng/mL) foi adicionado para a maturação das DCs. Culturas foram realizadas em 10% PI ou plasma normal (PN). Células não aderentes foram coletadas no 7o dia, marcadas com anticorpos monoclonais anti-CD86, CD11c, CD14 e HLA-DR e analisados por citometria de fluxo. Células de pacientes, cultivadas em 10% de PI, quando comparadas com células de doadores saudáveis, cultivadas em 10% PN, apresentaram expressão reduzida (p<0,05) de CD86 e HLADR. É interessante observar que células geradas de PBMCs de pacientes não expressaram CD11c, diferente das células derivadas de doadores saudáveis. A presença de PI nas culturas dos doadores saudáveis causou uma significante diminuição na porcentagem de expressão de células HLA-DR+, CD11c+, CD86. Finalmente, quando PBMCs de pacientes foram cultivadas em PN, houve um aumento na expressão de HLA-DR e CD86 (p<0,05). Os ensaios de proliferação demonstraram também que as células de pacientes ictéricos tiveram capacidade aloestimuladora de linfócitos reduzida, quando comparada a de células de doadores saudáveis. Estes dados indicam uma alteração importante na capacidade das células de pacientes se diferenciarem em DCs in vitro, um fenômeno que parece depender tanto de fatores solúveis presentes no plasma e sobre as próprias células. / Pancreatic adenocarcinoma (PAdc) is an aggressive malignancy with poor prognosis, urging for improved or new therapeutic strategies. DC-based vaccination is one of such promising approaches. DC are the most potent antigen-presenting cells and central to the induction and maintenance of an immune response. However, in cancer patients DC generation and function may be deficient, imposing an obstacle to the success of their use. Here, we describe the in vitro generation of DC from peripheral blood mononuclear cells (PBMC) obtained from jaundiced patients with PAdc and, also, the effect of jaundiced plasma (JP) in the phenomenon. PBMC were separated from blood obtained from 10 patients and 10 healthy controls over a density gradient. Adherent cells were cultured with GM-CSF and IL-4 (50ng/mL) for 7 days. On the 5th day, TNF-<font face=\"symbol\">a (50ng/mL) was added for DC activation. Cultures were performed in 10% JP or normal plasma (NP). Non-adherent cells were harvested at day 7, labeled with FITC- or PE-conjugated monoclonal antibodies against CD86, CD11c, CD14, HLA-DR and analyzed by flow cytometry. Patients\' cells, cultured in 10% JP, compared to healthy donors\' cells, cultured in 10% NP, had a significantly (p<0.05) lower expression of CD86 and HLA-DR. It is noteworthy that cells generated from patients\' PBMC did not express CD11c, while from those derived from healthy donors\' cells did so. The presence of JP in healthy donors\' cells cultures caused a significant decrease in the percentage of HLA-DR+, CD11c+ and CD86+ cells. Finally, when patients\' PBMC were cultured in NP, a significant increase in HLA-DR and in CD86 expression occurred. MLR assays also demonstrated that cells from jaundice patients had decreased capacity to stimulate alloestimulation of lymphocytes when compared to healthy donors. These data indicate a significant alteration in the patients\' PBMC ability to differentiate into DC in vitro, a phenomenon that seems to depend both on soluble factors present in plasma and on the cells, themselves.

Foram avaliados, com o auxílio de um aparelho dinâmico, bidimensional e transdutores eletrônicos multifreqüênciais (convexo de 4 a 7 MHz e linear de 7 a 12 MHz), os pâncreas de 33 cães da raça Poodle Toy, 17 fêmeas e 16 machos, com idade entre seis e 168 meses, peso corpóreo entre 1,85 a 6,15 quilogramas, sem processos patológicos aparentes. Estudaram-se as características anátomo sonográficas e suas correlações com as variáveis: sexo, idade e peso. A avaliação ultra-sonográfica do pâncreas permitiu identificar pelo menos uma região (lobo direito) e uma estrutura interna pancreática (veia pancreaticoduodenal), em todos os animais. O sexo, a idade e o peso não influenciaram na visibilização da glândula. Os valores métricos, referentes à espessura média e o desvio padrão das diferentes regiões e estruturas internas pancreáticas foram: lobo direito, tanto no plano longitudinal, como no transversal 0,72 &plusmn; 0,10 cm, lobo esquerdo 0,77 &plusmn; 0,11 cm, corpo 0,66 ± 0,09 cm, veia pancreaticoduodenal, em ambos planos, 0,18 &plusmn; 0,03 cm, artéria pancreaticoduodenal, no plano longitudinal 0,13 &plusmn; 0,01 cm, e no plano transversal 0,13 &plusmn; 0,02 cm, ducto pancreático, no plano longitudinal 0,08 &plusmn; 0,01 cm e no plano transversal 0,08 &plusmn; 0,02 cm. Observaram-se correlações positivas entre a espessura do lobo direito e a idade (p = 0,00), e entre essa espessura e o peso (p = 0,00). Correlações positivas foram observadas entre a espessura do lobo esquerdo e a idade (p = 0,04), e entre essa espessura o peso (p = 0,04). O estabelecimento dos padrões anátomo ultra-sonográficos permitiram verificar que a topologia e a arquitetura foram semelhantes em todos os animais. Os contornos foram classificados como pouco definidos ou definidos e não variaram com o sexo, o peso e a idade. A ecogenicidade pancreática, quando comparada ao baço, foi predominantemente hipoecogênica, independente de sexo, idade ou peso; a ecogenicidade em relação ao fígado, apresentou-se isoecogênica ou discretamente hiperecogênica; e em relação à gordura mesentérica foi hipoecogênica ou isoecogênica. A ecotextura mostrou-se homogênea a difusamente heterogênea. Observaram-se correlações positivas entre a ecogenicidade em relação ao fígado e a idade (p = 0,00) e o peso (p = 0,00), e entre a ecogenicidade em relação à gordura mesentérica, e a idade (p = 0,01) e ao peso (p = 0,02). Quanto à ecotextura pancreática, também foram observadas correlações positivas em relação à idade (p = 0,001) e ao peso (p = 0,002). / Pancreas of 33 healthy Toy Poodles, 16 male and 17 bitches, ranging from 6 to 168 months of age and weighing from 1,85 to 6,15 kg where evaluated with a dynamic bidimensional equipament and multifrequency ultrasound (curvilinear array 4-7 MHz and linear array 7-12 MHz) scanner. Ultrasonographic anatomy and correlation with gender, age and weight were studied. Ultrasonography allowed visualization of at least one region (right lobe) and one internal structure (pancreaticoduodenal vein) in all animals. Gender, age and weight had no influence on visualization. Measures of thickness where 0,72 &plusmn; 0,10 cm for the right lobe in longitudinal and transversal cut, 0,77 &plusmn; 0,11 cm for the left lobe and 0,66 &plusmn; 0,09 cm for the body. Diameter of the pancreaticoduodenal vein was 0,18 &plusmn; 0,03 cm in both cuts, diameter of the pancreaticoduodenal artery measured 0,13 &plusmn; 0,01 cm in the longitudinal cut and 0,13 &plusmn; 0,02 cm in the transversal; values obtained for the pancreatic duct where 0,08 &plusmn; 0,01 cm (longitudinal) and 08 &plusmn; 0,02 cm (transversal). Right lobe thickness was positively correlated to age (p = 0,00) and weight (p = 0,00). Left lobe thickness was positively correlated to age (p = 0,04) and weight (p = 0,04). Topology and architecture, similar in all animals, allowed recognition of a pattern. Outline was classified as poorly defined or defined and did not change with gender, weight and age. Echogenicity showed to be predominately hypoechoic compared to the spleen, iso or slightly hyperechoic when compared to liver and iso or hypoechoic when compared to mesenteric fat. Echotexture varied from homogeneous to diffusely heterogeneous. Pancreatic ecogenicity compared to the liver ecogenicity changed positively with age (p = 0,00) and weight (p = 0,00). Echogenicity compared to the mesenteric fat changed positively with age (p = 0,01) and weight (p = 0,02). Pancreatic echotexture showed a positive correlation with age (p = 0,001) and weight (p = 0,002).

Objetivamos identificar os efeitos celulares pelo qual o DHEA melhora a função das ilhotas pancreáticas. Ratos wistar com 12-14 meses de idade receberam uma única injeção subcutânea de DHEA (10mg.kg-1) ou veículo. Após uma semana, ilhotas pancreáticas foram isoladas para realização dos testes: secreção de insulina, respiração mitocondrial, oxidação de glicose, atividade da citrato sintase, fragmentação de DNA, expressão gênica e dosagem plasmáticas de hormônios esteróides sexuais. DHEA apresentou melhoria significativa na secreção de insulina. Notou-se um aumento na oxidação da glicose, respiração mitocondrial, atividade da citrato sintase, expressão do PCNA mRNA e reduzida apoptose. O uso de inibidores de receptores de andrógeno e estrógeno inibiu a secreção de insulina estimulada por glicose. Concluímos que o DHEA é capaz de melhorar a funcionalidade metabólica e reduzir o índice de apoptose da ilhota pancreática, melhorando a capacidade secretora, sendo este papel modulado, provavelmente, pela ligação do DHEA aos receptores de andrógenos e estrógenos. / We sought to identify the cellular effects by which DHEA improves pancreatic islets function. Male Wistar rats, 12-14 month-old, receive one subcutaneous injection of DHEA (10mg.kg-1) or vehicle. After a week, pancreatic islets from these animals were isolated and submitted to the following tests: insulin secretion, mitochondrial respiration, glucose oxidation, citrate synthase activity, DNA fragmentation, gene expression and the dosage of plasmatic steroid hormones. DHEA treatment resulted in significant improvement in insulin secretion. We observed an increase in glucose oxidation, mitochondrial respiration, citrate synthase activity, PCNA mRNA expression, and reduced apoptosis. Glucose-stimulated insulin secretion was inhibited by androgen and estrogen receptor inhibitors. We conclude that DHEA is capable of improving metabolic functionality and reduces apoptosis index in pancreatic islets, improving the secretory capacity stimulated by different secretagogues, with this being probably modulated by the binding of DHEA to androgen and estrogen receptors.

The recent surge in Type 2 Diabetes (T2D) has renewed interest in the study of cellular metabolism – which mitochondria tightly control. Previous work has shown mitochondrial dysfunction plays a critical role in the development of metabolic diseases, such as T2D. The pancreatic β-cell synthesizes and secretes insulin in vivo in response to diverse fuel signals such as glucose, fatty acids, and amino acids; failure or loss of β-cell mass is a hallmark of T2D. Pancreatic β-cell mitochondria are dynamic organelles living a life of fusion, fission, and movement collectively called mitochondrial dynamics. Mitochondrial fusion is impaired in obesity and models of obesity, while basal secretion of insulin is elevated. Previous studies demonstrate that hyperinsulinemia alone is sufficient to induce insulin resistance, yet the relationship between mitochondrial morphology and basal insulin secretion has not yet been studied. Here, we investigated the link between loss of mitochondrial fusion and insulin secretion at basal glucose concentrations by reducing the expression of mitofusin 2 (Mfn2), which controls mitochondrial morphology and metabolism. We found that forced mitochondrial fragmentation caused increased insulin secretion at basal glucose concentrations. In addition, fragmentation of mitochondria enhanced the secretory response of islets to palmitate at nonstimulatory glucose concentrations and increased fatty acid uptake and oxidation in a cell model of pancreatic β-cells. We developed unique solutions to challenges posed by the measurement of mitochondrial dynamics via confocal microscopy by using novel image analysis techniques, including a novel method of mitochondrial segmentation. This technique also revealed novel biology of brown adipose tissue mitochondria dependent on their localization within the cell. Our findings demonstrate that changes to mitochondrial dynamics in the β-cell can lead to increased insulin secretion at basal glucose concentrations. These data support the possibility that hyperinsulinemia and the downstream outcome of insulin resistance can be initiated by altered mitochondrial function in the β-cell independently of other tissues. By uncovering a new process that governs basal insulin secretion, we provide novel targets for regulation, such as mitochondrial morphology or fatty acid induced insulin secretion that may present new approaches to treatment of diabetes.

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