Background: Pancreatic islet transplantation for type-1 diabetes has resulted in considerable success over the past decade. However, the worldwide shortage of pancreatic donors remains a major challenge. In an attempt to expand the donor pool, pancreases from obese organs donors (>30 kg/m²) are now routinely offered to islet transplantation in the UK. In addition, it has been suggested that pancreases from donors with early type-2 diabetes mellitus may also be suitable. However, for both these donor groups, although high islet yields (IEQ) may be obtained, it is unclear whether these islets function optimally. An additional approach to the donor shortage is to minimise the number of donors required per islet recipient. One strategy to achieve this is to use different pharmacological agents to enhance post-transplant islet function. Aims: The aims of this thesis were fourfold; 1) To determine whether islets isolated from high BMI donors function normally in vitro and in vivo; 2) To establish why islet yields are higher from obese donors; 3) To determine whether islets from donors with type-2 diabetes are suitable for islet transplantation; 4) To investigate whether glucagon-like peptide 1 receptor agonist therapy improves post-transplant islet graft function. Results: Stimulated insulin and glucagon secretion, and markers of mitochondrial function (intra-islet ATP content and mitochondrial copy number) were compared in islets isolated from obese (BMI>30kg/m²; n= 27) and non-obese (BMI<28kg/m²; n=25) donors. No differences in secretory function or intra-islet ATP were observed between the two groups. Pancreatic lipid and intra-islet triglyceride concentrations were higher in the obese group. In vivo clinical outcomes of patients transplanted in Oxford and a larger cohort (n=35) in Edmonton, Canada with islets from obese or non-obese donors showed no differences in post-transplant outcomes. Improved islet yields were shown to be a result of improved islet recovery of larger islets, in obese donors. Abnormal insulin and glucagon secretory responses were observed in islets from type-2 diabetic subjects (n=6) and islet amyloid content was significantly higher in diabetes. The glucagon-like peptide 1 receptor agonist, exenatide, administered for 20 weeks, significantly improved graft function in patients whom islet function was impaired. Conclusion: The high lipid environment of islets isolated from donors with high BMI appears not to be deleterious to their function either in vitro or when transplanted, supporting the use of islets from high BMI donors for clinical islet transplantation. However, islet dysfunction and pathological changes indicate that islets from type-2 diabetic donors are unsuitable. Therapeutic options such as exenatide, improve transplanted islet viability and function, and could have a significant role in the future of beta-cell replacement therapy for type-1 diabetes.

Dans les conditions normales, les organismes maintiennent une masse cellulaire endocrine stable tout au long de leur vie. En cas d’obésité, la masse de cellules b pancréatiques est capable de maintenir des taux de glucose plasmatique en augmentant la sécrétion en insuline. L’incapacité de ces cellules à fournir de l’insuline entraîne alors l’apparition d’une hyperglycémie et d’un diabète de type II. Cliniquement, la majorité des individus obèses ne développent pas de diabète car les îlots pallient à cette résistance à l’insuline. La preuve de l’adaptation de la masse d’îlots humains à l’obésité, in vivo, n’a pas été clairement décrite et, de plus, peu d’informations existent sur les mécanismes et les types cellulaires impliqués. Actuellement, la mise en évidence de l’augmentation de la masse des cellules b chez les humains obèses repose uniquement sur des études histologiques.But : Au cours de cette thèse, nous avons cherché, dans un premier temps (partie 1), à évaluer la morphologie des îlots pancréatiques et la distribution des cellules a et b chez des donneurs en état de mort cérébrale normaux, en surpoids et obèses. Dans un second temps (partie 2), nous avons étudié l’adaptation au cours du temps des îlots humains à un environnement obésogène. Nous avons montré ainsi que les îlots humains non diabétiques s’adaptent in vivo à l’obésité en modifiant la masse de cellules b, leur fonction et leur expression génique. En suite (partie 3), on a identifié le mécanisme de transdifférenciation des cellules alpha et beta en utilisant la méthode de lineage tracing. Finalement (partie 4), on a déterminé la différence sur l’expression de gène des ilots humains greffé chez les souris sous régime control ou régime riche en graisse en utilisant les puces d’ARN (Illumina).Methodes et Resultats: Des coupes de pancréas humains inclues en paraffines ont été analysées. Les donneurs obèses étaient caractérisés par une augmentation de la masse endocrine totale, de la taille des îlots, de la graisse intra-pancréatique et du ratio b:a dans les îlots, mais avec une diminution du ratio a:b dans les îlots. Au cours de l’étude longitudinale, des souris Rag2-/- non diabétiques ont été greffées sous la capsule rénale avec des îlots humains issus de donneurs en état de mort cérébrale (donneurs non diabétiques ou donneurs avec un dysfonctionnement métabolique déclaré). Les animaux ont été nourris pendant 2 semaines avec soit un régime contrôle soit un régime riche en graisse (high fat diet HFD). Un suivi du poids, du taux des triglycérides, de la glycémie et du C-peptide a été mis en place. Après sacrifice des souris, les greffons et les pancréas endogènes ont été analysés pour le volume endocrine, la distribution des cellules b et a et les mécanismes de régénération des cellules pancréatiques. Après 12 semaines sous régime gras, les souris montraient toutes les caractéristiques typiques de l’obésité, à savoir, une augmentation du poids, un doublement de la graisse abdominale, des triglycérides, de la glycémie et une sensibilité à l’insuline réduite. De plus, l’apparition sur ces animaux d’un doublement rapide de la quantité de C-peptide humain dans le sérum murin nous indique la mise en place d’une compensation fonctionnelle. Une analyse histologique des greffons a permis de mettre en évidence une adaptation de la masse endocrine des îlots avec une augmentation des cellules b. D’autres analyses ont identifié la prolifération et la néogénèse comme les mécanismes responsables de ce doublement de la masse endocrine humaine.Discussion: Ce nouveau modèle animal permet d’étudier, in vivo sur une longue période, l’adaptation des îlots humains à un environnement obésogène murin. Il peut être utilisé comme un outil dans le décryptage des voies de signalisation impliquées dans l’expansion des cellules b humaines et permettre également l’identification des facteurs prédisposant ces cellules à subir une décompensation. / Under normal healthy conditions, organisms maintain a dynamic endocrinecell mass throughout life. Pancreatic beta cell mass are able to maintain plasma glucose levels increasing insulin secretion in conditions as obesity.Beta cell inability to compensate in insulin demand provokes hyperglycemia and Type 2 Diabetes. Clinically, most obese individuals do not develop diabetes because islets compensate for insulin resistance. Direct evidence that human islet mass adapts longitudinally to obesity in vivo was lacking and, moreover, little information was available on the mechanismsand cell type(s) involved.Current evidence for increased beta cell mass in obese humans (vs lean) is based entirely on postmortem histology.Aim: In this thesis, firstly (Part 1) we performed a descriptive cross sectional study by evaluating the pancreatic islet morphology and alpha and beta cell distribution from our archived human pancreatic sections of obese and normal subjects. Secondly, (Part 2) we explored the longitudinal adaptation of human islets to an obesogenic environment and showed direct evidence that non-diabetic human islets adapt bothendocrine and beta cell mass, function and gene expression to obesity in vivo. Thirdly (Part 3) we performed lineage tracing to determine which cell type alpha or beta give rise to the increase islet mass in obesity. Finally (Part 4) in this diet induced obesity model we developed, we looked at the differential gene expression with Illumina gene chips in a kinetic study on human islets which were laser capture microdissected at 6, 8 and 10 weeks on control or high fat diet.Methods: Archived human pancreatic sections were immunostained for endocrine, beta, alpha, fat. In the obese/immunodeficient mouse model, non-diabetic Rag2–/– mice were transplanted under kidney capsule with human islets from human brain-deceased donors (non-diabetics donors and donors with overt metabolic dysfunction). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analyzed for endocrine volume, distribution of beta and alpha cells, and mechanisms of regeneration.Results: The cross-sectional study, performed on archived human paraffin embedded sections of normal weight, overweight, or obese subjects showed that obese donors were characterized by an increased total endocrine mass, bigger individual islet size, increased intrapancreatic fat, increased &#946; to &#61537; cell ratio and decreased &#61537;:&#946; cell ratio in islets. In the longitudinal study, concomitant with the increased weight gain, doubling of abdominal fat, increased serum triacylglycerol and reduced insulin sensitivity in 12 week HFD animals we reported that human islet grafts showed functional compensation, measured as a more than doubling of fasting human C-peptide in mouse serum, and histological adaptation of islet endocrine mass including increased beta cells. Further analysis of the human grafts revealed proliferation and neogenesis as the responsible mechanisms for the doubling of the human endocrine mass.Discussion: This novel model allows, for the first time, longitudinal studies of human islet adaptation to an obese murine environment and may be instrumental in deciphering pathways involved in human beta cell expansion, as well as in helping to identify factors predisposing human beta cells to undergo decompensation.

<p>It is estimated that 1 in 500 Americans are inflicted with type I diabetes (T1D) with approximately 18,000 children and adolescents diagnosed each year. Islet/β cell replacement with long-lasting glucose-sensing and insulin-releasing functions has the potential to eliminate the need for insulin injections and minimize complications for individuals with T1D. However, limitations remain precluding it from widespread clinical use, including i) limited donor supply, ii) significant loss of functional islet mass upon transplantation, iv) limited functional longevity, and v) need for life-long systemic immunosuppression. To restore glucose-responsive insulin-release back to the patient’s body without the need for systemic immunosuppression, our approach involves a subcutaneous injection using a novel fibril-forming biologic, type I oligomeric collagen (Oligomer). Oligomer protects and in situ encapsulates replacement cells beneath the skin by transitioning from a liquid to a stable collagen-fibril scaffold, within seconds, just like those found in the body’s tissues. Preclinical validation studies in streptozotocin-induced diabetic mice show that replacement of islets at a dose of 500 or 800, results in a rapid (within 24 hours) reversal of hyperglycemia. All animals receiving syngeneic islets maintained euglycemia for beyond 90 days, while >80% of animals receiving allogeneic or xenogeneic (rat) islets remained euglycemia for at least 50 days. Histopathological analysis of Oligomer-islet implants showed normal morphology with no apparent evidence of a foreign body response and immune cell infiltrate. To our knowledge, this is the first report of an injectable subQ islet transplant strategy that yields rapid lowering and extended glycemic control without systemic immunosuppression.</p>

As complicações relacionadas ao diabetes mellitus estão intimamente ligadas à hiperglicemia. Os pacientes que evoluem com grande instabilidade metabólica e progressão das complicações microvasculares apesar do tratamento intensivo com insulina são candidatos ao transplante de pâncreas. Neste contexto, o transplante de ilhotas pancreáticas surge como alternativa por ser menos invasivo e menos imunogênico. No entanto, o processo de digestão do pâncreas e isolamento das ilhotas pancreáticas expõe as células endócrinas a diversos estímulos nocivos, que resultam em diminuição da viabilidade das células isoladas e menor chance de sucesso após o transplante. A geração de espécies reativas de oxigênio (ERO) e o consumo das defesas anti-oxidantes durante o processo de digestão do pâncreas pode contribuir para a perda da viabilidade das ilhotas isoladas. O objetivo deste estudo foi avaliar o impacto da suplementação com glutationa etil mono-éster (GEE), um éster de melhor biodisponibilidade da glutationa (um importante anti-oxidante endógeno) nos resultados do isolamento e do transplante de ilhotas pancreáticas em um modelo animal. GEE foi adicionada na concentração de 10 mM na solução de colagenase durante o isolamento das ilhotas de rato. Após o isolamento, foram realizados estudos in vitro para avaliar a presença de ERO com o ensaio carboxi-H2DCFDA e a viabilidade das ilhotas isoladas com os ensaios JC-1 (integridade mitocondrial) e Sytogreen/brometo de etídio (integridade da membrana celular); viabilidade das células beta-pancreáticas por citometria de fluxo para avaliação de necrose e apoptose, TUNEL para a avaliação do índice de apoptose e secreção de insulina estimulada por glicose. Realizamos também estudos in vivo, com o transplante das ilhotas na cápsula renal de camundongos diabéticos, seguimento dos animais por 30 dias após o transplante e recuperação do enxerto para análise histológica. Quatro grupos de animais foram avaliados: 1) Animais transplantados com número suficiente de ilhotas para reverter o diabetes (500) não isoladas com GEE; 2) Animais transplantados com número suficiente de ilhotas (500) isoladas em presença de GEE; 3) Animais transplantados com número insuficiente de ilhotas (150) não isoladas com GEE e 4) Animais transplantados com número insuficiente de ilhotas (150) isoladas em presença de GEE. A suplementação com GEE na concentração de 10 mM durante o isolamento das ilhotas diminuiu a formação de ERO (Controle 57,0 ± 4,3% versus GEE 47,0 ± 3,9%, p = 0,0034) e aumentou a viabilidade das ilhotas, conforme demonstrado pelo ensaio Sytogreen/brometo de etídio (Controle 70,6 ± 3,4% versus GEE 83,6 ± 4,8%, p= 0,0010) e pela diminuição na porcentagem de células TUNEL-positivas (Controle de 39,2 ± 5,0% versus GEE 29,1 ± 1,9%, p= 0,042) no grupo tratado. O estudo de viabilidade por citometria de fluxo também mostrou um número maior de células beta pancreáticas viáveis no grupo tratado (Controle 21,4 ± 3,4% versus GEE 33,7 ± 3,9%, p= 0,0156). A manutenção da integridade funcional das ilhotas teve impacto nos resultados dos transplantes, com menor índice de célula TUNEL-positivas (Controle 23,3 ± 2,6% versus GEE 8,3 ± 0,8%, p < 0,0001) nos enxertos recuperados após as primeiras 24 horas do transplante e maior porcentagem de animais normoglicêmicos (Controle 30% versus GEE 65,2%, p = 0,004) após transplante de um número marginal de 150 ilhotas na cápsula renal após seguimento de 30 dias. Em conclusão, estes dados corroboram que a formação de ERO é uma causa relevante de dano celular durante o isolamento de ilhotas pancreáticas e sugerem que o uso do compostos anti-oxidante GEE pode ser uma estratégia para melhorar os resultados dos transplantes de ilhotas / The vascular complications related to Diabetes Mellitus are closely linked to hyperglycemia. Patients who develop metabolic instability and progression of microvascular complications despite intensive insulin therapy are candidates to pancreas transplantation. Pancreatic islet transplant is an alternative approach since it is less immunogenic and minimally invasive. However, the success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during the islet isolation process and early post-transplant period. The increase in reactive oxygen species (ROS) generation and the consumption of antioxidant defenses might be factors related to these injuries. The aim of the present study was to evaluate whether supplementation with glutathione-ethyl-ester (GEE), a compound with higher bioavailability than glutathione (an important endogenous antioxidant), could improve islet viability and efficacy in a marginal islet transplantation model in rodents. GEE was added to a final concentration of 10 mM in collagenase solution during islet isolation. After isolation, in vitro studies were conducted to evaluate the presence of ROS using carboxy-H2DCFDA assay and the viability of isolated islets with JC-1 assay (mitochondrial integrity), Sytogreen/ethidium bromide assay (cellular membrane integrity), fractional beta cell viability assay by flow cytometry, TUNEL assay for apoptosis evaluation and glucose-stimulated insulin secretion. We also performed in vivo studies with islet transplantation under the kidney capsule of diabetic mice, 30 days follow-up after transplantation and recovery of the graft for histological analysis. Four experimental groups were evaluated: 1) animals transplanted with 500 islets, a number considered sufficient to promote diabetes reversion, not isolated in presence of GEE; 2) animals transplanted with 500 islets isolated in presence of GEE; 3) animals transplanted with 150 islets, a number considered insufficient to promote diabetes reversion, not isolated in presence of GEE and 4) animals transplanted with 150 islets isolated in presence of GEE. The addition of GEE at 10 mM concentration during islet isolation was able to decrease ROS content in isolated islets (Control 57.0 ± 4.3% versus GEE 47.0 ± 3.9%, p = 0.0034) and increase islet viability, as demonstrated by the Sytogreen/ethidium bromide assay (Control 70.6 ± 3.4% versus GEE 83.6 ± 4.8%, p = 0.0010) as well as by the reduction in TUNEL-positive cells (Control 39.2 ± 5.0% versus GEE 29.1 ± 1.9%, p = 0.042) in the treated group. The fractional beta-cell viability also showed an improvement in the treated group (Control 21.4 ± 3.4% versus GEE 33.7 ± 3.9%, p = 0.0156). The improved cell viability observed in vitro was translated into better outcomes in vivo, since supplementation of GEE during the isolation process resulted in a significantly lower rate of TUNEL-positive cells (Control 23,3 ± 2,6% versus GEE 8,3 ± 0,8%, p < 0,0001) in the islet grafts recovered after 24h of transplantation and in a higher percentage of normoglycemia (Control 30% versus GEE 65,2%, p = 0,004) after 30 days of follow-up in animals transplanted with the marginal islet mass (150 islets). In conclusion, the current data corroborate that ROS production is a relevant cause of cellular damage during islet isolation and suggest that the use of GEE might be a strategy to improve islet transplantation outcomes

Estima-se que, em 2013, cerca de 382 milhões de pessoas eram portadoras de diabetes mundialmente. Já o diabetes mellitus do tipo 1 (DMT1) representa de 5-10% desse total de casos, cujo tratamento atual se pauta na administração de insulina exógena. Contudo, desde a publicação do protocolo de Edmonton, o transplante de ilhotas pancreáticas se apresenta como nova técnica no tratamento para o DMT1, inclusive obtendo a independência de insulina em alguns casos. Apesar disso, a escolha do sítio receptor ainda é essencial para diminuir efeitos adversos e permitir o acompanhamento do enxerto. Nesse sentido, destaca-se o transplante de ilhotas para a câmara anterior do olho, pois permite, além do restabelecimento do controle glicêmico, o estudo da fisiologia dos enxertos in vivo. Dessa forma, o objetivo foi estabelecer metodologia de isolamento e transplante de ilhotas de alta reprodutibilidade e baixo custo, utilizando a câmara anterior do olho como sítio receptor. O isolamento foi realizado via injeção de solução de colagenase (1 mg/mL via ducto colédoco) em camundongos machos C57BL/6 hígidos de 8 semanas de idade e posterior transplante dessas ilhotas para camundongos machos da mesma espécie com diabetes induzido por injeção de aloxana (60 mg/kg, i.v.). Esses camundongos foram submetidos a infusão de aproximadamente 250 equivalentes de ilhotas (IEQs) para a câmara anterior do olho e tiveram sua glicemia e alteração de massa corpórea acompanhadas por 14 dias após o transplante. Também foi realizado teste de tolerância a glicose via injeção de solução de glicose (2g/kg i.p.) e realização da curva glicêmica. Obteve-se, na etapa de padronização, que a adição de 0,5% (%p/v) de albumina de soro bovino à solução de colagenase foi capaz de aumentar o número de IEQs isolados por animal. Quanto ao transplante, obteve-se que 50% dos animais submetidos à técnica tiveram diminuição significativa na sua glicemia (172,5 ± 6,4 mg/dL), quando comparados com o grupo controle diabético (582,8 ± 27,5 mg/dL) (p < 0,05). Entretanto, todos os animais tiveram aumento significativo da massa corpórea no período de acompanhamento e glicemia de jejum significativamente menor que os animais diabéticos (p < 0,05). Ademais, a curva glicêmica dos animais que tiveram transplante considerado bem sucedido, no teste de tolerância a glicose, se aproxima da curva do grupo controle sadio. Conclui-se que o modelo de transplante de ilhotas pancreáticas para a câmara anterior do olho foi bem estabelecido neste projeto, confirmado pelos resultados que evidenciam o transplante de ilhotas funcionais capazes de reduzir sensivelmente a glicemia e promover o ganho de peso em camundongos diabéticos. / It is estimated that, in 2013, around 382 million people had diabetes worldwide. Of that number, 5-10% represented cases of T1DM, which treatment is based in the administration of exogenous insulin. However, since the Edmonton protocol was published, islet transplantation presented itself as novel technique for T1DM treatment, achieving insulin independence in some cases. Although, recipient site choice is still essential to diminish side effects and enable graft follow up. In that sense, transplantation to the anterior chamber of the eye stands out, since it allows, beyond the reestablishment of glycemic control, study of islet physiology in vivo. That way, the objective was to establish a low cost and high reproducible model of islet isolation and transplantation, using the anterior chamber of the eye as receptor site. Islet isolation was made by injection of collagenase solution (1 mg/mL via common bile duct) in 8 week old healthy male C57BL/6 mice and followed by transplantation of these islets to male mice of the same age and species with diabetes induced by alloxan injection (60 mg/kg i.v.). These mice were subject of 250 islet equivalents (IEQs) infusion to the anterior chamber of the eye and had their blood glucose and change in body mass monitored for 14 days after transplantation. A glucose tolerance test (GTT) was also made, by injection of glucose solution (2g/kg i.p.) and a glycemic curve was plotted. In the standardization period, was observed that the addition of 0,5% (%w/v) bovine serum albumin is capable of increasing the number of IEQs isolated from each animal. About the transplants, was obtained that 50% of animals subject to transplantation had their blood glucose decreased significantly (172,5 ± 6,4 mg/dL), when compared to the diabetic control group (582,8 ± 27,5 mg/dL) (p < 0,05). However, all animals subject to the procedure had significant body mass increase, when compared to the same control group and fasting blood glucose significantly lower than diabetic animals (p < 0,05). Moreover, the glycemic curve of animals, who had their transplantation considered successful, was similar to that found in healthy control animals, in the GTT. We conclude that the model of transplant to the anterior chamber of the eye is well established in this project, which is confirmed by results that shows transplantation of functional islets, capable of promoting a significant decrease in blood glucose and an increase in total body mass in diabetic animals.

In contrast to mammals, adult zebrafish are able to regenerate motor neurons and regain swimming ability within 6 weeks after a spinal cord injury. During this regenerative process, a range of developmental signals such as dopamine and serotonin are found to be re-deployed. This makes the research of embryonic signals become essential for the promotion of regeneration in the future. In my research, I am interested in identifying genes that are important for motor neuron development and motor axon differentiation. I also aimed to study the ability of zebrafish larvae to regenerate spinal motor neurons, and whether they can be used to study the essential developmental cues and the mechanisms underlying successful functional recovery. Motor axons grow out of the spinal cord in a motor neuron subtype specific manner and innervate different muscle groups to facilitate locomotor movements. To find genes and important pathways involved in motor neuron generation and axon development in zebrafish, we conducted an ENU-induced mutagenesis screen in islet-1:GFP transgenic zebrafish, in which a subset of dorsally projecting motor neurons are labelled. We have discovered 6 mutants displaying delayed or inhibited appearance of secondary motor neurons and/or motor axon deficits among 111 F2 families screened. Through subsequent mutant phenotypical analysis, I focused my study in two mutant lines manifesting a lack of islet-1:GFP motor neurons, and an absence of islet-1:GFP motor axons. I used various molecular markers to characterise the mutant phenotypes and observed several additional anatomical defects. I also initiated the study of causative mutation analysis based on the candidate gene list generated from Next Generation Sequencing (NGS). To gain an insight of the genes’ role in motor neuron development and axonal differentiation, I started functional analyses in order to confirm genes that are responsible for the observed motor neuron/axon phenotypes, and I have achieved some promising preliminary results. Motor neurons are generated from the motor neuron progenitor domain (pMN). This neurogenesis process sharply declines at 48 hours post-fertilisation (hpf), while pMN progenitor cells continue to proliferate to produce oligodendrocytes. By inflicting a mechanical lesion in the spinal cord of zebrafish larvae, we demonstrated that they are capable of regenerate new motor neurons and achieve full functional recovery within 48 hours following the injury, sharing similar mechanisms to that of the adult zebrafish. I further studied oligodendrocyte generation and found that pMN domain is able to switch from oligodendrogenesis to motor neuron generation after a spinal lesion. This demonstrates the high plasticity of the pMN domain. Interestingly, the generation of dorsal Pax2-positive interneurons was not altered after the lesion, suggesting that the regenerative potential differs in different progenitor domains. This study showed that the motor neuron regenerative process in zebrafish larvae is robust and they can be used for studying motor neuron regeneration. Taken together, the discovery of the genes from our screen will provide insights to the developmental cues that are involved in motor neuron generation and axon growth. Furthermore, spinal cord lesion in larval zebrafish larvae is established as a regenerative model that can be utilized to dissect the roles and mechanisms of these signals and pathways in the promotion of motor neuron regeneration.

La transplantation d’îlots pancréatiques est une des thérapies proposées aux diabétiques de type 1. Cependant, la perte d’un nombre considérable d’îlots durant le processus est un frein à l’expansion de la thérapie (en culture : anoïkis et hypoxie ; lors de la greffe : réactions inflammatoires intenses déclenchées lors de l’infusion intra-portale). L’objectif de ce travail était de valider un biomatériau permettant d’optimiser la transplantation d’îlots. Nous avons pu démontrer l’efficacité du plasma réticulé sur la survie des îlots en culture via une diminution drastique de l’anoïkis. Nous avons également mis au point une technique de greffe intra-tissulaire à l’aide d’HPMC combinée à du plasma, permettant de reverser le diabète de manière semblable à la greffe hépatique. Ces travaux ont donc permis de valider l’importance d’un support de culture adapté, mais aussi du site receveur de la greffe et placé l’omentum comme site de choix pour ce type de greffe. / Pancreatic islet transplantation is one of the therapies proposed to type 1 diabetic patients. However, the considerable loss of islets during the process is an obstacle to the expansion of this therapy (culture: anoïkis and hypoxia; at graft time: intense inflammatory reactions triggered by intra-portal islet infusion). The objective of this work was to validate a biomaterial that can optimize islet transplantation. We were able to demonstrate the effectiveness of cross-linked plasma on the islet survival in culture through a drastic reduction of anoikis. We also design a new intra-tissular grafting technique using HPMC combined with plasma, which reversed diabetes in a manner similar to the liver. This work allowed validating the importance of an adapted culture support, but also the one of the recipient site. Also, this work placed the omentum as an excellent recipient site for this kind of transplant.

A crescente incidência de disfunções metabólicas, como resistência à insulina e diabetes mellitus tipo 2 (T2DM), está correlacionada com a elevação da ocorrência de obesidade e sobrepeso. Em busca da melhora da saúde e de um corpo ideal segundo os padrões estéticos propagados atualmente, um número cada vez maior de indivíduos adere às dietas da moda que prometem rápida redução do peso corporal, ao invés de adotar uma alimentação balanceada e a prática regular de exercícios físicos. Uma dieta bastante divulgada e até recomendada por profissionais da saúde é o jejum intermitente (JI), que consiste em alternar períodos de jejum de até 24 horas com períodos de ingestão alimentar. Diversos estudos experimentais têm relatado alterações metabólicas em consequência do JI, como modificações da glicemia e da tolerância à glicose, porém, os resultados encontrados na literatura são conflitantes e, além disso, o impacto do jejum intermitente, em longo prazo, sobre as ilhotas pancreáticas ainda não foi devidamente elucidado. Desta forma, o presente estudo teve como objetivo caracterizar os impactos de doze semanas de JI em ratas Wistar. Para tal, ratas Wistar com 30 dias de idade foram distribuídas aleatoriamente em dois grupos: controle, com livre acesso à ração balanceada; e jejum intermitente, submetido a 24 horas de jejum intercalado com 24 horas de livre acesso à ração balanceada. Foi observado que os animais submetidos ao JI, apresentaram menor ganho de peso corporal, redução do comprimento da tíbia e da distância naso-anal, e alteração da composição corporal, incluindo diminuição da massa muscular e aumento do tecido adiposo. Em média, o consumo de ração do grupo JI foi menor, porém, no dia que era disponibilizado alimento, os animais apresentaram hiperfagia o que resultou em grande aumento das dimensões do estômago. O jejum intermitente reduziu os valores plasmáticos do colesterol total, triglicérides, LDL, HDL e glicemia, e aumentou a concentração basal da insulina plasmática, bem como a secreção da insulina após o estímulo com glicose. Foi observada redução significativa da massa de ilhotas pancreáticas e aumento da porcentagem de células dispersas de ilhotas em apoptose. Ainda nas células dispersas de ilhotas pancreáticas, houve aumento do conteúdo de espécies reativas de oxigênio mitocondrial e total, e do peróxido de hidrogênio (H2O2), além de aumento da expressão do sistema antioxidante. Assim, nossos dados sugerem que esse protocolo estudado de 24 horas de jejum intercalados com 24 horas de alimentação à vontade não seja saudável em longo prazo. Mais estudos em longo prazo são necessários para investigar qual seria o melhor protocolo de jejum intermitente de forma a reduzir os efeitos colaterais e melhorar a saúde, para então o JI ser considerado uma boa alternativa para perda e manutenção do peso. / The increasing incidence of metabolic dysfunctions, such as insulin resistance and type 2 diabetes mellitus (T2DM), is correlated with increased occurrence of obesity and overweight. In pursuit of improved health and an ideal body according to today\'s aesthetic standards, an increasing number of individuals adhere to fad diets that promise a rapid reduction of body weight, instead of adopting a balanced diet and regular practice of physical exercises. A well-publicized diet and even recommended by health professionals is intermittent fasting (IF), which consists of alternating fasting periods of up to 24 hours with periods of food intake. Several experimental studies have reported metabolic changes as a consequence of IF, such as changes in glucose and glucose tolerance, but the results found in the literature are conflicting and, in addition, the impact of intermittent fasting in the long term on pancreatic islets has not yet been properly elucidated. Thus, the present study aimed to characterize the effects of twelve-week IF on Wistar rats. For this, 30-day-old Wistar rats were randomly assigned to two groups: control, with free access to balanced chow; and intermittent fasting, subjected to 24-hour fast intercalated with 24 hours of free access to the balanced chow. It was observed that the animals submitted to IF presented lower body weight gain, reduced tibia length and naso-anal distance, and altered body composition, including decreased muscle mass and increased adipose tissue. On average, the dietary intake of the IF group was lower, but on the day that food was available, the animals presented hyperphagia which resulted in a large increase in the stomach size. Intermittent fasting reduced plasma levels of total cholesterol, triglycerides, LDL, HDL, and glycaemia, and increased basal plasmatic insulin level, as well as insulin secretion after stimulation with glucose. We observed significant reduction in pancreatic islet mass and increase in percentage of islet-dispersed cells in apoptosis. Still in islet-dispersed cells, there was an increase in mitochondrial and total reactive oxygen species content, and of hydrogen peroxide (H2O2), in addition to an increase in expression of antioxidant system. Thus, our data suggest that this protocol of 24-hour fasting intercalated with 24-hour feed at will is not healthy in the long run. More long-term studies are needed to investigate the best intermittent fasting protocol in order to reduce side effects and improve health, so IF be considered a good alternative for weight loss and maintenance.

<p>Transplantation of islets of Langerhans is a potential treatment of type 1 diabetes that aims to restore normal glucose homeostasis. Microencapsulation of islets could enable transplantation in the absence of immunosuppression, which would be beneficial as the side effects associated with immunosuppression outweigh the potential benefits of islet transplantation. Alginate is a polysaccharide that can be harvested from brown algae and is often used for microencapsulation of cells.</p><p>The aim of this study was to evaluate alginate/poly-L-lysine/alginate capsules with regard to their biocompatibility and permeability to cytokines. Moreover, the function of microencapsulated islets was studied <i>in vitro</i> as well as their ability to reverse hyperglycaemia in diabetic mice.</p><p>Microencapsulated rodent islets functioned well <i>in vitro</i>, with similar insulin release rates and glucose oxidation rates as naked islets. However, when cultured with interleukin-1β and tumour necrosis factor-α, microencapsulated islets were functionally suppressed, showing that the capsules are permeable to these cytokines. The biocompatibility of capsules varied depending on their composition. The presence of poly-L-lysine in the capsule decreased the biocompatibility. However, the biocompatibility of the capsules was improved when the coating alginate had been epimerised, i.e. enyzmatically tailored. Transplantation of microencapsulated allogeneic islets to immune competent mice lowered blood glucose concentrations up to 1 month after implantation. The success of the microencapsulated islet graft depended on the composition of the alginate/poly-L-lysine/alginate capsule used, as capsules that had poor biocompatibility failed to reverse hyperglycaemia more than transiently in athymic nude mice.</p><p>In conclusion, alginate/poly-L-lysine/alginate capsules can protect islets of Langerhans from allogeneic rejection in mice. However, the composition of the capsule is of critical importance in the success of transplantation. Epimerised alginates may provide a novel capsule with ideal properties for microencapsulation of islets of Langerhans.</p>

<p>Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). </p><p>In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function <i>in vitro</i>, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, <i>in vivo</i>. </p><p>Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, <i>in vitro</i>. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ.</p><p>In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. </p><p>Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model. </p>