Orientador: João Carlos Pinheiro Ferreira / Resumo: O uso do sêmen refrigerado proporciona maiores taxas de prenhez se comparado ao do sêmen congelado. Essa diferença parece estar relacionada às lesões mais severas das membranas espermáticas desencadeadas pelo processo de congelação. Por sua habilidade de se ligar às proteínas ligadoras de espermatozoides e ao íon cálcio, o caseinato de sódio vem sendo estudado como uma substância capaz de inibir a capacitação espermática precoce, uma importante causa de diminuição da taxa de prenhez quando do uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar a possibilidade de um diluente comercial a base de gema de ovo, destinado à congelação de sêmen bovino, ser empregado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito do uso desse diluente, suplementado com caseinato de sódio, na criopreservação de espermatozoides bubalinos, por meio da avaliação dos espermatozoides, por citometria de fluxo, e da cinética espermática, empregando-se o sistema CASA. Na primeira parte do estudo, quando comparados os resultados das avaliações da cinética espermática e integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação seminal promoveu mais danos celulares que o processo de refrigeração. Na segunda parte do estudo, não foram observados efeitos da adição do caseinato de sódio ao diluente a base de gema de ovo. A partir dos resultados do presente estudo foi possível concluir que o diluente a base de gema de ovo testad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of cooled semen results in higher pregnancy rates compared than the use of frozen semen. This result seems to be related to the more severe damages triggered by the freezing process, when compared to those observed during the refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a ubstance capable to prevent early sperm capacitation, a major cause of decreased pregnancy rate after using frozen semen. The first objective of this study was to evaluate if a commercial egg yolk diluent developed for freezing bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent, added with sodium caseinate, during the procedures of buffalo sperm cryopreservation, using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membranes integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the deleterious effects related to seminal cryopreservation. / Mestre

The parallel and distributed platforms of High Performance Computing available today have became more and more heterogeneous (CPUs, GPUs, FPGAs, etc). Graphics Processing Units (GPU) are specialized co-processor to accelerate and improve the performance of parallel vector operations. GPUs have a high degree of parallelism and can execute thousands or millions of threads concurrently and hide the latency of the scheduler. GPUs have a deep hierarchical memory of different types as well as different configurations of these memories. Performance prediction of applications executed on these devices is a great challenge and is essential for the efficient use of resources in machines with these co-processors. There are different approaches for these predictions, such as analytical modeling and machine learning techniques. In this thesis, we present an analysis and characterization of the performance of applications executed on GPUs. We propose a simple and intuitive BSP-based model for predicting the CUDA application execution times on different GPUs. The model is based on the number of computations and memory accesses of the GPU, with additional information on cache usage obtained from profiling. We also compare three different Machine Learning (ML) approaches: Linear Regression, Support Vector Machines and Random Forests with BSP-based analytical model. This comparison is made in two contexts, first, data input or features for ML techniques were the same than analytical model, and, second, using a process of feature extraction, using correlation analysis and hierarchical clustering. We show that GPU applications that scale regularly can be predicted with simple analytical models, and an adjusting parameter. This parameter can be used to predict these applications in other GPUs. We also demonstrate that ML approaches provide reasonable predictions for different cases and ML techniques required no detailed knowledge of application code, hardware characteristics or explicit modeling. Consequently, whenever a large data set with information about similar applications are available or it can be created, ML techniques can be useful for deploying automated on-line performance prediction for scheduling applications on heterogeneous architectures with GPUs. / As plataformas paralelas e distribuídas de computação de alto desempenho disponíveis hoje se tornaram mais e mais heterogêneas (CPUs, GPUs, FPGAs, etc). As Unidades de processamento gráfico são co-processadores especializados para acelerar operações vetoriais em paralelo. As GPUs têm um alto grau de paralelismo e conseguem executar milhares ou milhões de threads concorrentemente e ocultar a latência do escalonador. Elas têm uma profunda hierarquia de memória de diferentes tipos e também uma profunda configuração da memória hierárquica. A predição de desempenho de aplicações executadas nesses dispositivos é um grande desafio e é essencial para o uso eficiente dos recursos computacionais de máquinas com esses co-processadores. Existem diferentes abordagens para fazer essa predição, como técnicas de modelagem analítica e aprendizado de máquina. Nesta tese, nós apresentamos uma análise e caracterização do desempenho de aplicações executadas em Unidades de Processamento Gráfico de propósito geral. Nós propomos um modelo simples e intuitivo fundamentado no modelo BSP para predizer a execução de funções kernels de CUDA sobre diferentes GPUs. O modelo está baseado no número de computações e acessos à memória da GPU, com informação adicional do uso das memórias cachês obtidas do processo de profiling. Nós também comparamos três diferentes enfoques de aprendizado de máquina (ML): Regressão Linear, Máquinas de Vetores de Suporte e Florestas Aleatórias com o nosso modelo analítico proposto. Esta comparação é feita em dois diferentes contextos, primeiro, dados de entrada ou features para as técnicas de aprendizado de máquinas eram as mesmas que no modelo analítico, e, segundo, usando um processo de extração de features, usando análise de correlação e clustering hierarquizado. Nós mostramos que aplicações executadas em GPUs que escalam regularmente podem ser preditas com modelos analíticos simples e um parâmetro de ajuste. Esse parâmetro pode ser usado para predizer essas aplicações em outras GPUs. Nós também demonstramos que abordagens de ML proveem predições aceitáveis para diferentes casos e essas abordagens não exigem um conhecimento detalhado do código da aplicação, características de hardware ou modelagens explícita. Consequentemente, sempre e quando um banco de dados com informação de \\textit esteja disponível ou possa ser gerado, técnicas de ML podem ser úteis para aplicar uma predição automatizada de desempenho para escalonadores de aplicações em arquiteturas heterogêneas contendo GPUs.

A computação paralela permite uma série de vantagens para a execução de aplicações de grande porte, sendo que o uso efetivo dos recursos computacionais paralelos é um aspecto relevante da computação de alto desempenho. Este trabalho apresenta uma metodologia que provê a execução, de forma automatizada, de aplicações paralelas baseadas no modelo BSP com tarefas heterogêneas. É considerado no modelo adotado, que o tempo de computação de cada tarefa secundária não possui uma alta variância entre uma iteração e outra. A metodologia é denominada de ASE e é composta por três etapas: Aquisição (Acquisition), Escalonamento (Scheduling) e Execução (Execution). Na etapa de Aquisição, os tempos de processamento das tarefas são obtidos; na etapa de Escalonamento a metodologia busca encontrar a distribuição de tarefas que maximize a velocidade de execução da aplicação paralela, mas minimizando o uso de recursos, por meio de um algoritmo desenvolvido neste trabalho; e por fim a etapa de Execução executa a aplicação paralela com a distribuição definida na etapa anterior. Ferramentas que são aplicadas na metodologia foram implementadas. Um conjunto de testes aplicando a metodologia foi realizado e os resultados apresentados mostram que os objetivos da proposta foram alcançados. / Parallel computing allows for a series of advantages on the execution of large applications and the effective use of parallel resources is an important aspect in the High Performance Computing. This work presents a methodology to provide the execution, in an automated way, of parallel applications based on BSP model with heterogeneous tasks. In this model it is assumed that the computation time between iterations does not have a high variance. The methodology is entitled ASE and it is composed by three stages: Acquisition, Scheduling and Execution. In the Acquisition step, the tasks\' processing time are obtained; In the Scheduling step, the methodology finds the ideal arrangement to distribute the tasks to maximize the execution speed and, simultaneously, minimize the use of resources. This is made using an algorithm developed in this work; and lastly the Execution step, where the parallel application is executed in the distribution defined in the previous step. The tools used in the methodology were implemented. A set of tests to apply the methodology were made and the results shown that the objectives were reached.

A computação paralela permite uma série de vantagens para a execução de aplicações de grande porte, sendo que o uso efetivo dos recursos computacionais paralelos é um aspecto relevante da computação de alto desempenho. Este trabalho apresenta uma metodologia que provê a execução, de forma automatizada, de aplicações paralelas baseadas no modelo BSP com tarefas heterogêneas. É considerado no modelo adotado, que o tempo de computação de cada tarefa secundária não possui uma alta variância entre uma iteração e outra. A metodologia é denominada de ASE e é composta por três etapas: Aquisição (Acquisition), Escalonamento (Scheduling) e Execução (Execution). Na etapa de Aquisição, os tempos de processamento das tarefas são obtidos; na etapa de Escalonamento a metodologia busca encontrar a distribuição de tarefas que maximize a velocidade de execução da aplicação paralela, mas minimizando o uso de recursos, por meio de um algoritmo desenvolvido neste trabalho; e por fim a etapa de Execução executa a aplicação paralela com a distribuição definida na etapa anterior. Ferramentas que são aplicadas na metodologia foram implementadas. Um conjunto de testes aplicando a metodologia foi realizado e os resultados apresentados mostram que os objetivos da proposta foram alcançados. / Parallel computing allows for a series of advantages on the execution of large applications and the effective use of parallel resources is an important aspect in the High Performance Computing. This work presents a methodology to provide the execution, in an automated way, of parallel applications based on BSP model with heterogeneous tasks. In this model it is assumed that the computation time between iterations does not have a high variance. The methodology is entitled ASE and it is composed by three stages: Acquisition, Scheduling and Execution. In the Acquisition step, the tasks\' processing time are obtained; In the Scheduling step, the methodology finds the ideal arrangement to distribute the tasks to maximize the execution speed and, simultaneously, minimize the use of resources. This is made using an algorithm developed in this work; and lastly the Execution step, where the parallel application is executed in the distribution defined in the previous step. The tools used in the methodology were implemented. A set of tests to apply the methodology were made and the results shown that the objectives were reached.

The parallel and distributed platforms of High Performance Computing available today have became more and more heterogeneous (CPUs, GPUs, FPGAs, etc). Graphics Processing Units (GPU) are specialized co-processor to accelerate and improve the performance of parallel vector operations. GPUs have a high degree of parallelism and can execute thousands or millions of threads concurrently and hide the latency of the scheduler. GPUs have a deep hierarchical memory of different types as well as different configurations of these memories. Performance prediction of applications executed on these devices is a great challenge and is essential for the efficient use of resources in machines with these co-processors. There are different approaches for these predictions, such as analytical modeling and machine learning techniques. In this thesis, we present an analysis and characterization of the performance of applications executed on GPUs. We propose a simple and intuitive BSP-based model for predicting the CUDA application execution times on different GPUs. The model is based on the number of computations and memory accesses of the GPU, with additional information on cache usage obtained from profiling. We also compare three different Machine Learning (ML) approaches: Linear Regression, Support Vector Machines and Random Forests with BSP-based analytical model. This comparison is made in two contexts, first, data input or features for ML techniques were the same than analytical model, and, second, using a process of feature extraction, using correlation analysis and hierarchical clustering. We show that GPU applications that scale regularly can be predicted with simple analytical models, and an adjusting parameter. This parameter can be used to predict these applications in other GPUs. We also demonstrate that ML approaches provide reasonable predictions for different cases and ML techniques required no detailed knowledge of application code, hardware characteristics or explicit modeling. Consequently, whenever a large data set with information about similar applications are available or it can be created, ML techniques can be useful for deploying automated on-line performance prediction for scheduling applications on heterogeneous architectures with GPUs. / As plataformas paralelas e distribuídas de computação de alto desempenho disponíveis hoje se tornaram mais e mais heterogêneas (CPUs, GPUs, FPGAs, etc). As Unidades de processamento gráfico são co-processadores especializados para acelerar operações vetoriais em paralelo. As GPUs têm um alto grau de paralelismo e conseguem executar milhares ou milhões de threads concorrentemente e ocultar a latência do escalonador. Elas têm uma profunda hierarquia de memória de diferentes tipos e também uma profunda configuração da memória hierárquica. A predição de desempenho de aplicações executadas nesses dispositivos é um grande desafio e é essencial para o uso eficiente dos recursos computacionais de máquinas com esses co-processadores. Existem diferentes abordagens para fazer essa predição, como técnicas de modelagem analítica e aprendizado de máquina. Nesta tese, nós apresentamos uma análise e caracterização do desempenho de aplicações executadas em Unidades de Processamento Gráfico de propósito geral. Nós propomos um modelo simples e intuitivo fundamentado no modelo BSP para predizer a execução de funções kernels de CUDA sobre diferentes GPUs. O modelo está baseado no número de computações e acessos à memória da GPU, com informação adicional do uso das memórias cachês obtidas do processo de profiling. Nós também comparamos três diferentes enfoques de aprendizado de máquina (ML): Regressão Linear, Máquinas de Vetores de Suporte e Florestas Aleatórias com o nosso modelo analítico proposto. Esta comparação é feita em dois diferentes contextos, primeiro, dados de entrada ou features para as técnicas de aprendizado de máquinas eram as mesmas que no modelo analítico, e, segundo, usando um processo de extração de features, usando análise de correlação e clustering hierarquizado. Nós mostramos que aplicações executadas em GPUs que escalam regularmente podem ser preditas com modelos analíticos simples e um parâmetro de ajuste. Esse parâmetro pode ser usado para predizer essas aplicações em outras GPUs. Nós também demonstramos que abordagens de ML proveem predições aceitáveis para diferentes casos e essas abordagens não exigem um conhecimento detalhado do código da aplicação, características de hardware ou modelagens explícita. Consequentemente, sempre e quando um banco de dados com informação de \\textit esteja disponível ou possa ser gerado, técnicas de ML podem ser úteis para aplicar uma predição automatizada de desempenho para escalonadores de aplicações em arquiteturas heterogêneas contendo GPUs.

Les architectures parallèles sont de plus en plus présentes dans notre environnement, que ce soit dans les ordinateurs personnels disposant des dizaines d’unités de calculs jusqu’aux super-calculateurs comptant des millions d’unités. Les architectures haute performance modernes sont généralement constituées de grappes de multiprocesseurs, elles même constituées de multi-cœurs, et sont qualifiées d’architecture hiérarchiques. La conception de langages pour de telles architectures est un sujet de recherche actif car il s’agit de simplifier la programmation tout en garantissant l’efficacité des programmes. En effet, écrire des programmes parallèles est, en général, plus complexe tant au point de vue algorithmique qu’au niveau de l’implémentation. Afin de répondre à cette problématique, plusieurs modèles structurés ont été proposés. Le modèle logico-materiel BSP définit une vision structurée pour les architectures parallèles dites plates. Afin d’exploiter les architectures actuelles, une extension adaptée aux architectures hiérarchiques a été proposée : Multi-BSP. Tout en préservant la philosophie BSP, ce modèle garanti efficacité, sécurité d’exécution, passage à l’échelle et prédiction de coût.Cette thèse s’articule donc autour de cette idée et propose de définir Multi-ML, un langage basé sur le modèle logico-materiel Multi-BSP, garantissant les propriétés énoncées ci-dessus. Afin de pouvoir garantir la sécurité d’exécution des programmes Multi-ML, nous proposons une sémantique formelle ainsi qu’un système de type afin d’accepter uniquement des programmes bien formés. De plus, nous proposons une machine abstraite permettant de décrire formellement l’évaluation d’un programme Multi-ML sur une machine Multi-BSP. Une implantation du langage, développé dans le cadre de cette thèse, permet de générer un code exécutable. Il est donc possible d’exécuter, efficacement, des algorithmes Multi-BSP écrits à l’aide de Multi-ML sur diverses machines hiérarchiques / From personal computers using an increasing number of cores, to supercomputers having millions of computing units, parallel architectures are the current standard. The high performance architectures are usually referenced to as hierarchical, as they are composed from clusters of multi-processors of multi-cores. Programming such architectures is known to be notoriously difficult. Writing parallel programs is, most of the time, difficult for both the algorithmic and the implementation phase. To answer those concerns, many structured models and languages were proposed in order to increase both expressiveness and efficiency. Among other models, Multi-BSP is a bridging model dedicated to hierarchical architecture that ensures efficiency, execution safety, scalability and cost prediction. It is an extension of the well known BSP model that handles flat architectures.In this thesis we introduce the Multi-ML language, which allows programming Multi-BSP algorithms “à la ML” and thus, guarantees the properties of the Multi-BSP model and the execution safety, thanks to a ML type system. To deal with the multi-level execution model of Multi-ML, we defined formal semantics which describe the valid evaluation of an expression. To ensure the execution safety of Multi-ML programs, we also propose a typing system that preserves replicated coherence. An abstract machine is defined to formally describe the evaluation of a Multi-ML program on a Multi-BSP architecture. An implementation of the language is available as a compilation toolchain. It is thus possible to generate an efficient parallel code from a program written in Multi-ML and execute it on any hierarchical machine

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.

Grades computacionais oportunistas utilizam recursos ociosos de máquinas compartilhadas para executar aplicações que necessitam de um alto poder computacional e/ou trabalham com grandes quantidades de dados. Mas a execução de aplicações paralelas computacionalmente intensivas em ambientes dinâmicos e heterogêneos, como grades computacionais oportunistas, é uma tarefa difícil. Máquinas podem falhar, ficar inacessíveis ou passar de ociosas para ocupadas inesperadamente, comprometendo a execução de aplicações. Um mecanismo de tolerância a falhas que dê suporte a arquiteturas heterogêneas é um importante requisito para estes sistemas. Neste trabalho, analisamos, implementamos e avaliamos um mecanismo de tolerância a falhas baseado em checkpointing para aplicações paralelas em grades computacionais oportunistas. Este mecanismo permite o monitoramento de execuções e a migração de aplicações entre nós heterogêneos da grade. Mas além da execução, é preciso gerenciar e armazenar os dados gerados e utilizados por estas aplicações. Desejamos uma infra-estrutura de armazenamento de dados de baixo custo e que utilize o espaço livre em disco de máquinas compartilhadas da grade. Devemos utilizar somente os ciclos ociosos destas máquinas para armazenar e recuperar dados, de modo que um sistema de armazenamento distribuído que as utilize deve ser redundante e tolerante a falhas. Para resolver o problema do armazenamento de dados em grades oportunistas, projetamos, implementamos e avaliamos o middleware OppStore. Este middleware provê armazenamento distribuído e confiável de dados, que podem ser acessados de qualquer máquina da grade. As máquinas são organizadas em aglomerados, que são conectados por uma rede peer-to-peer auto-organizável e tolerante a falhas. Dados são codificados em fragmentos redundantes antes de serem armazenados, de modo que arquivos podem ser reconstruídos utilizando apenas um subconjunto destes fragmentos. Finalmente, para lidar com a heterogeneidade dos recursos, desenvolvemos uma extensão ao protocolo de roteamento em redes peer-to-peer Pastry. Esta extensão adiciona balanceamento de carga e suporte à heterogeneidade de máquinas ao protocolo Pastry. / Opportunistic computational grids use idle resources from shared machines to execute applications that need large amounts of computational power and/or deal with large amounts of data. But executing computationally intensive parallel applications in dynamic and heterogeneous environments, such as opportunistic grids, is a daunting task. Machines may fail, become inaccessible, or change from idle to occupied unexpectedly, compromising the application execution. A fault tolerance mechanism that supports heterogeneous architectures is an important requisite for such systems. In this work, we analyze, implement and evaluate a checkpointing-based fault tolerance mechanism for parallel applications running on opportunistic grids. The mechanism monitors application execution and allows the migration of applications between heterogeneous nodes of the grid. But besides application execution, it is necessary to manage data generated and used by those applications. We want a low cost data storage infrastructure that utilizes the unused disk space of grid shared machines. The system should use the machines to store and recover data only during their idle periods, requiring the system to be redundant and fault-tolerant. To solve the data storage problem in opportunistic grids, we designed, implemented and evaluated the OppStore middleware. This middleware provides reliable distributed storage for application data, which can be accessed from any machine in the grid. The machines are organized in clusters, connected by a self-organizing and fault-tolerant peer-to-peer network. During storage, data is codified into redundant fragments, allowing the reconstruction of the original file using only a subset of those fragments. Finally, to deal with resource heterogeneity, we developed an extension to the Pastry peer-to-peer routing substrate, enabling heterogeneity-aware load-balancing message routing.

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.

Ce travail prend place au sein d'une recherche sur la modélisation 3d des scènes géologiques. Son apport spécifique est le suivant: 1) une réflexion générale est menée sur la géométrie des principaux types de corps géologiques ; 2) une réflexion est également menée, au vu notamment des travaux réalisés antérieurement ; 3) à la suite de cette réflexion d'ensemble, nous centrons la réflexion sur un problème particulier: celui de la modélisation d'objets de forme quelconque susceptibles d'être déformés ou d'être composés entre eux. Nous montrons que la question essentielle sous-jacente a une modélisation est celle du choix d'une structure de données ; 4) définition d'une structure hybride, l'arbre BSP mixte ; 5) au terme de la réflexion, nous avons construit le logiciel granite.