The islets of Langerhans secrets insulin in response to fluctuations of blood glucose level and efficient secretion requires extensive intra-islet communication. Secretory failure from islets is one of the hallmark in progression of type 2 diabetes.  Changes in islet structure and high levels of saturated free fatty acids may contribute to this failure. The aim of this thesis is to study the role of cell-cell interactions and palmitate on β-cells functions. To address the role of cell-cell interactions on β-cells functions MIN6 cells were cultured as monolayers and as pseudoislets. Glucose stimulated insulin secretion was higher in pseudoislets compared to monolayers. Transcript levels of mitochondrial metabolism as well glucose oxidation rate was higher in pseudoislets. Insulin receptor substrate-1 (IRS-1) phosphorylation was altered when cells were grown as pseudoislets. Proteins expression levels related to glycolysis, cellular connections and translational regulations were up-regulated in pseudoislets. We propose the superior capacity of pseudoislets compared to monolayers depend on metabolism, cell coupling, gene translation, protein turnover and differential IRS-1 phosphorylation. To address the role of palmitate on β-cells human islets were cultured in palmitate. Long term palmitate treatment decreased insulin secretion which is associated with up-regulation of suppressor of cytokine signaling-2 (SOCS2) and protein inhibitor of activated STAT-1 (PIAS1). Up-regulation of SOCS2 decreased phosphorylation of Akt at site T308, whereas PIAS1 decreased protein level of ATP- citrate lyase (ACLY) and ATP synthase subunit B (ATP5B). We propose long term palmitate treatment reduces phosphatidylinositol 3-kinase (PI3K) activity, attenuates formation of acetyl-CoA and decreases ATP synthesis which may aggravate β-cells dysfunction.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-12T10:34:15Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) carlos_jose_otoni.pdf: 547684 bytes, checksum: fe5204dc075a407303d639a2b3b9fbda (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-12T10:34:35Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) carlos_jose_otoni.pdf: 547684 bytes, checksum: fe5204dc075a407303d639a2b3b9fbda (MD5) / Made available in DSpace on 2016-01-12T10:34:35Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) carlos_jose_otoni.pdf: 547684 bytes, checksum: fe5204dc075a407303d639a2b3b9fbda (MD5) Previous issue date: 2015 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Banco do Nordeste do Brasil (BNB) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Objetivou-se avaliar o potencial produtivo de duas linhagens de til?pia do Nilo (Oreochromis niloticus), tailandesa e GIFT, nas etapas de desenvolvimento compreendidas entre juvenis e adultos, submetidas a densidades de estocagem, em sistema intensivo com biofiltra??o. Foram realizados dois experimentos, sendo que para o primeiro foi avaliada a densidade de estocagem no cultivo de juvenis de til?pias tailandesa e GIFT. Foram utilizados 2160 juvenis sexualmente revertidos para macho, contados individualmente. Em seguida, os peixes foram transferidos para 24 aqu?rios de 70 L com 30 L de ?gua cada, providos de aera??o constante e fotoper?odo natural. As til?pias foram submetidas a quatro densidades de estocagem: 1 peixe por litro (30 peixes/tanque); 2,3 peixes por litro (69 peixes/tanque); 3,7 peixes por litro (111 peixes/tanque) e 5 peixes por litro (150 peixes/tanque). Foi utilizado um delineamento experimental inteiramente casualizado com tr?s repeti??es cada. O sistema de recircula??o manteve a qualidade de ?gua dentro dos n?veis aceit?veis durante o per?odo experimental. N?o foram observadas intera??es entre as linhagens utilizadas e as densidades de estocagem, como tamb?m nenhuma diferen?a no desempenho entre as linhagens. Para o peso corporal final e ganho de peso foi verificado efeito linear em fun??o da densidade de estocagem. O aumento dessa promoveu uma redu??o no peso final e no ganho de peso. A densidade de 1 peixe por litro ganhou 67,48% mais peso que a densidade de 5 peixes por litro. Para a convers?o alimentar foi verificado efeito linear, havendo aumento com o aumento da densidade. Quanto ao fator de condi??o de Fulton (K) foi observado efeito linear em fun??o da densidade, em que a densidade 1 peixe por litro apresentou maiores resultados e a densidade 5 peixes por litro os menores. Para o ganho em biomassa foi verificado um aumento linear ? medida que se aumentou a quantidade de peixe por tanque. Para os outros par?metros estudados n?o foram verificados efeitos lineares em fun??o da densidade. Com isso, recomenda-se a densidade de 1 peixe por litro (30 peixes/tanque) para proporcionar um maior ganho de peso dos animais, e uma densidade de 2,3 peixes por litro (69 peixes/tanque) para se obter uma melhor convers?o alimentar e melhor ganho em biomassa, podendo ser utilizadas em ambas as linhagens avaliadas. Para o segundo experimento, densidade de estocagem na engorda de til?pias tailandesa e GIFT foram utilizados 1800 juvenis sexualmente revertidos para macho, contados individualmente. Em seguida, as til?pias foram transferidas para 18 caixas de 1000 L com 400 L de ?gua cada, providos de aera??o constante e fotoper?odo natural. Os peixes foram submetidos a tr?s densidades de estocagem: 0,15 peixe por litro (60 peixes/tanque); 0,25 peixe por litro (100 peixes/tanque) e 0,35 peixes por litro (140 peixes/tanque). Foi utilizado um delineamento experimental inteiramente casualizado, com tr?s repeti??es cada. O sistema de recircula??o manteve a qualidade de ?gua dentro dos n?veis aceit?veis durante o per?odo experimental. N?o foram observadas intera??es entre as linhagens utilizadas e as densidades de estocagem, como tamb?m nenhuma diferen?a no desempenho entre as linhagens. Para o peso corporal final e ganho de peso foi verificado efeito linear em fun??o da densidade. O aumento dessa promoveu uma redu??o no peso final e no ganho de peso. A densidade 0,15 peixe por litro ganhou 31,93% (aos 120 dias) e 41,86% (aos 240 dias) mais peso do que a densidade 0,35 peixe por litro. Para a convers?o alimentar tamb?m foi verificado efeito linear, para os dois per?odos, havendo aumento com o aumento da densidade. Quanto ao fator de condi??o de Fulton (K) foi observado efeito linear, para os 120 e 240 dias, em fun??o da densidade, em que a densidade 0,15 peixe por litro apresentou os maiores resultados e a densidade 0,35 os menores. Para o ganho em biomassa foi verificado um aumento linear ? medida que se diminuiu a quantidade de peixes por tanque. Para os outros par?metros estudados n?o foram verificados efeitos lineares em fun??o da densidade. Dessa forma, recomenda-se a densidade de 0,15 peixe por litro (60 peixes/tanque) para melhor ganho de peso e ganho em biomassa, bem como melhor convers?o alimentar, podendo ser utilizada em ambas as linhagens testadas. / Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Sa?de, Sociedade e Ambiente, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2015. / ABSTRACT The productive potential of two strains of tilapia (Oreochromis niloticus), thai and GIFT, in development stages between juvenile and adults, submitted to different stocking densities in intensive system with biofiltration was evaluated. Two trials were conducted, being that the first was evaluated stocking density in Nile tilapia in youth culture, thai and Gift. 2160 sexually fingerlings reverted to male were counted individually. The fish were transferred to 24 L tanks 70 with 30 L of water each provided with constant aeration and natural photoperiod. Tilapias were submitted to four stocking densities: 1 fish/L (30 individuals/tank); 2.3fish/L (69 individuals/tank); 3.7 fish/L (111 individuals/tank) and 5 fish/L (150 individuals/tank). A completely randomized design with three replications was used. The recirculation system maintained the water quality within acceptable levels during the experimental period. There were not interactions between the strains used and the stocking densities. Also there was no difference in performance between the strains. The final weight and weight gain were observed linear effect according to the storage density. The increase of density promoted a reduction in the final weight and weight gain. The density 1 fish/L promoted 67.48% more weight than the density of 5 fish/L. For feed conversion was observed linear effect, getting worse with increasing density. Fulton?s condition factor (K) have a linear effect as a function of density. Biomass gain was observed linear effect with the increase of amount of fish per tank. Other parameters studied do not show an effect in density. Therefore, it is recommended 1 fish/L to provide a greater weight gain of fish, and a density of 2.3 fish/L to obtain a better feed conversion, and biomass gain. For the second trial, the influence of stocking density on the growth of two strains of Nile tilapia (Gift and Thai) was studied. One thousand and eight hundred Nile tilapia sexually reverted to male were used. Tilapias were transferred to 18 boxes with 400L water each provided of constant aeration and natural photoperiod. The fish were submitted to three stocking densities: 0.15 fish/L (60 individuals/tank); 0.25 fish/L (100 individuals/tank) and 0.35 fish/L (140 individuals/tank). A completely randomized design with three replications was used. The recirculation system maintained the water quality within acceptable levels during the experimental period. There were not interactions between the strains used and the stocking densities. Also there was no difference in performance between the strains. Linear effect was observed for final weight and weight gain as a function of density. With the increase of density occurred decrease in final weight and weight gain. The density of 0.15 fish/L increased in 31.93% (at 120 days) and 41.86% (240 days) the gain weight than compared at density of 0.35 fish/L. The feed conversion worse linearly with increase of density for both periods. As to Fulton condition factor (K), linear effect was observed to 120 and 240 days according with the density. For biomass gain, a linear increase was verified as it reduced the number of fish per tank. For the other parameters were not found effects for the two periods as a function of density. Thus, the density of 0.15 tilapia/L promotes better weight gain, biomass gain and feed conversion, which may be used both strains.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

In this thesis, the impact of multihomed clients and multihomed proxy servers on the performance of modern networks is investigated. The network model used in our investigation integrates three main components: the new one-to-any Anycast communication paradigm that facilitates server replication, the next generation Internet Protocol Version 6 (IPv6) that offers larger address space for packet switched networks, and the emerging multihoming trend of connecting devices and smart phones to more than one Internet service provider thereby acquiring more than one IP address. The design of a previously proposed Proxy IP Anycast service is modified to integrate user device multihoming and Ipv6 routing. The impact of user device multihoming (single-homed, dual-homed, and triple-homed) on network performance is extensively analyzed using realistic network topologies and different traffic scenarios of client-server TCP flows. Network throughput, packet latency delay and packet loss rate are the three performance metrics used in our analysis. Performance comparisons between the Anycast Proxy service and the native IP Anycast protocol are presented. The number of Anycast proxy servers and their placement are studied. Five placement methods have been implemented and evaluated including random placement, highest traffic placement, highest number of active interface placements, K-DS placement and a new hybrid placement method. The work presented in this thesis provides new insight into the performance of some new emerging communication paradigms and how to improve their design. Although the work has been limited to investigating Anycast proxy servers, the results can be beneficial and applicable to other types of overlay proxy services such as multicast proxies.

Protein SUMOylation is a highly dynamic and reversible post-translational modification that targets lysine residues on a wide range of proteins involved in several essential cellular events, including protein translocation and degradation, mitotic chromosome segregation, DNA damage response, cell cycle progression, cell proliferation, and migration. Protein SUMOylation is an ATP-dependent enzymatic process that involves an E1 activating enzyme SAE1/2, a E2 conjugase UBC9, and usually facilitated by SUMO E3 ligases. The SP-RING family is the largest family of SUMO E3 ligases, encompassing seven mammalian protein inhibitor of activated STAT (PIAS) proteins. PIAS family was originally identified as specific inhibitors for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. Recent studies showed that PIAS proteins also play important roles in the regulation of protein stability and signal transduction through the SUMOylation of target substrates. In addition, PIAS-mediated protein SUMOylation is also involved in several cellular processes, including DNA damage repair, immune response, cellular proliferation, and motility. Most notably, PIAS proteins are highly expressed in different cancer types and have been implicated in tumorigenesis. Several reports suggest that PIAS proteins could promote cancer cell growth and progression by regulating the SUMOylation of different substrates. To date, a number of substrates of PIAS ligases have been identified from several individual studies, and hundreds of specific SUMO E3 ligase substrates were identified from a human proteome microarray-based activity screen. However, how these substrates are selected, and which SUMOylation sites are targeted by these PIAS are still unknown. To answer these questions, I started my investigation with PIAS1, one of the most well studied SUMO E3 ligases. By changing the expression level of PIAS1 in HeLa cells using gene overexpression or CRISPR/Cas9 gene knockout, I found PIAS1 had a physiological impact on cell proliferation and migration. I took advantage of the previously developed SUMO proteomics workflow to quantitatively profile global SUMOylome changes upon PIAS1 overexpression in a site-specific manner. I identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, was identified as PIAS1 substrates. Two SUMOylation sites mediated by PIAS1 at Lys-439 and Lys-445 residues were further evaluated and found to be necessary for dynamic disassembly and assembly of vimentin intermediate filaments, which further regulates cell migration and motility. In the second study, I extended my investigation to all PIAS ligases and further found that all PIAS proteins impact cell proliferation and migration of breast cancer cell MDA-MB-231 after CRISPR/Cas9 gene knockout. I further optimized my SILAC-based quantitative SUMO proteomics approach and combined it with transcriptomics to gain a system-level understanding of the functional components involved in PIAS regulatory networks. A large subset of proteins/ genes involved in cell proliferation and migration were commonly regulated by all PIAS proteins, suggesting a redundancy of regulation within the PIAS family. In addition, each PIAS regulated a unique pool of substrates/genes involved in different cellular processes, such as DNA damage repair, chromatin remodeling, and SUMO chain formation, suggesting that each PIAS specifically regulates cellular functions. The trans-scale analyses between proteomics and transcriptomics shed light on the comprehensive pictures of the regulation networks by PIAS proteins beyond their direct enzymatic activity. Overall, the quantitative SUMO proteomics approach provided a robust method for identifying substrates of PIAS SUMO E3 ligases. The combination of proteomic and transcriptomic analyzes made it possible to draw up a global portrait of the regulatory mechanisms governed by the PIAS proteins. / La SUMOylation des protéines est une modification post-traductionnelle se produisant sur des lysines d’un large éventail de protéines cellulaires. Cette modification est dynamique et régit plusieurs évènement cellulaires essentiels, dont la translocation et la dégradation des protéines, la ségrégation chromosomique mitotique, la réparation de l'ADN, la progression du cycle cellulaire, la prolifération cellulaire et la migration. La conjugaison de la protéine SUMO sur son substrat se produit grâce à une triade enzymatique regroupant l’enzyme d’activation E1 SAE 1/2, la conjugase E2 UBC9 et dans la plupart des cas une ligase SUMO E3. Cette cascade enzymatique nécessite une source d’ATP pour son initiation. Parmi la famille des ligases SUMO E3, on retrouve un domaine spécifique nommé SP-RING présent chez une sous population de celles-ci. Parmi ces ligases on retrouve 7 protéines inhibitrices des protéines STAT activées regroupees sous le nom de PIAS. Les ligases PIAS ont été identifiées à l'origine comme des inhibiteurs spécifiques des protéines STAT responsable du signal de transduction et de l’activation de la transcription génique. Des études récentes ont montré que les protéines PIAS jouent également un rôle important sur la stabilité de leurs substrats et la transduction de leur signal. De plus, les substrats SUMOylés par les PIAS sont impliqués dans plusieurs processus cellulaires, notamment la réparation des dommages à l'ADN, la réponse immunitaire, la prolifération et la motilité cellulaire. Ces divers processus cellulaires peuvent être déréglés et entrainer le développement du cancer. Il s’avère que les protéines PIAS sont fortement exprimées dans divers types de cancer et sont impliquées dans la tumorigenèse. Plusieurs rapports suggèrent que les protéines PIAS pourraient favoriser la croissance et la progression des cellules cancéreuses en régulant le niveau de SUMOylation de plusieurs substrats. Initialement, les substrats des ligases PIAS ont été identifiés à partir de plusieurs études individuelles et plus récemment, des centaines de substrats spécifiques de la SUMO E3 ligase ont été identifiés à partir de criblage de micropuces à protéines interrogeant le protéome humain. Cependant, la manière dont ces substrats sont sélectionnés et quels sont les sites de SUMOylation ciblés par ces PIAS demeurent encore méconnus. Afin d’aborder ces questions, j’ai commencé mon étude avec PIAS1, l'une des ligases SUMO E3 les plus étudiées. Pour ce faire, j’ai varié le niveau d'expression de PIAS1 dans des cellules iv HeLa selon l’approche CRISPR/Cas9. Ainsi, deux modèles ont été construit, soit via une surexpression du gène ou via un knockout du gène. Ces mutants ont permis de constater que PIAS1 avait un impact physiologique sur la prolifération et la migration des cellules. J’ai tiré avantage d’une méthode protéomique précédemment développé sur les peptides SUMO pour déterminer les changements de SUMOylation lors de la surexpression de PIAS1. J’ai identifié 983 sites SUMO sur 544 protéines, dont 62 protéines ont été identifiées comme substrats potentiels de PIAS1. Parmi celles-ci, la vimentine (VIM), une protéine de la famille des filaments intermédiaire de type III impliquée dans l'organisation du cytosquelette et la motilité cellulaire, a été reconnu comme un substrat de PIAS1. Afin de valider le rôle de la SUMOylation des lysines Lys-439 et Lys-445 de VIM j’ai effectué des études fonctionelles de motilité cellulaire avec les mutants où ces sites ont été substitués en arginine. Ces expériences m’ont permis de constater que la SUMOylation de VIM aux sites Lys-439 et Lys-445 est nécessaire à l’assemblage et désassemblage dynamique des filaments intermédiaires de VIM, lesquels regulent la migration et la motilité cellulaire. Dans la deuxième étude, j’ai élargi mon recherche sur toutes les ligases PIAS et avons découvert que ces dernières avaient toutes un impact sur la prolifération cellulaire et la migration des cellules du cancer du sein MDA-MB-231 suite à un knockout de ces gènes par CRISPR / Cas9. De plus, j’ai optimisé mon approche de protéomique quantitative SUMO via SILAC et l'avons complémenté d’une analyse transcriptomique. Cette combinaison a permis d’acquérir une compréhension des composants fonctionnels impliqués dans les réseaux de régulation PIAS. Il s’avère qu’un grand sous-ensemble de gènes / protéines impliqués dans la migration et la prolifération des cellules sont régulés par tous les membres de la famille PIAS, et suggère une certaine redondance fonctionnelle parmi ces ligases. De plus, chaque PIAS régule un ensemble unique de substrats / gènes impliqués dans plusieurs processus cellulaires différents, tels que la réparation des dommages de l'ADN, le remodelage de la chromatine et la formation de la chaîne SUMO. Ces résultats suggèrent que chacune des PIASs régule de façon spécifique les fonctions cellulaires. La combinaison des analyses protéomiques et transcriptomiques ont permi de dresser un portrait global des mécanismes de régulation régit par les protéines PIAS et ce au-delà de leur activité enzymatique directe.