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Le syndrome d'ischémie-reperfusion (IR), inhérent à la transplantation rénale, est caractérisé par un infiltrat leucocytaire important et des lésions tissulaires graves dont les signaux initiateurs restent à ce jour peu décrits. Postulant que la libération d'alarmines par les cellules en nécrose est décisive dans ce processus, l'objectif principal du présent travail a été d'étudier la contribution de l'alarmine IL-33 dans la genèse des lésions tissulaires dans un modèle murin d'IR rénale. Nos résultats montrent que l'IL-33 est rapidement libérée du rein après IR comme protéine circulante, dès une heure de reperfusion. Les souris IL-33gt/gt, déficientes en IL-33, sont moins sensibles aux lésions induites par l’IR, comme l'attestent le maintien de la fonction rénale et des lésions histologiques atténuées avec un recrutement de polynucléaires neutrophiles (PNN) diminué par rapport aux souris contrôles. Ceci est associé à la perte du recrutement de cellules iNKT productrices d'IFN-γ/IL-17A. Parallèlement, les souris Jα18KO, déficientes en cellules iNKT et protégées contre les lésions d'IR, possèdent également des niveaux élevés d'IL-33 circulante. Nous proposons donc que l'IL-33 endogène contribue aux lésions d'IR en favorisant le recrutement de cellules iNKT, conduisant ainsi à un recrutement amplifié de PNN au niveau du rein lésé. Notre étude, en identifiant l'alarmine IL-33 comme un médiateur précoce de la réponse immunitaire innée induite par l'IR rénale, mettant en jeu les cellules iNKT, contribue à la compréhension des mécanismes impliqués dans la genèse des lésions associées à la greffe rénale et permet de proposer de nouvelles stratégies thérapeutiques. / Ischemia-reperfusion (IR) injury in renal transplantation is characterized by leukocyte infiltration and tissue damage. However, the signals that initiate these events remain poorly understood. Assuming that alarmin release by necrotic cells during IRI is critical, the main objective of the present study was to investigate the role of alarmin IL-33 in kidney injury using a mouse model of renal IR. We observed release of IL-33 shortly after kidney IR concomitantly with an increase in plasma levels of IL-33 within one hour of reperfusion. IL-33 deficient mice (IL-33gt/gt) exhibited reduced renal IR-induced injury, as attested by function preservation, reduced histological change and attenuation of neutrophil recruitment compared to control mice. This was associated with the loss of IFN-γ/IL-17A-producing iNKT cell recruitment. In the meantime, iNKT cell-deficient (Jα18KO) mice, also protected against IRI, have increased levels of circulating IL-33.These findings lead us to propose that endogenous IL-33 contributes to kidney IRI by promoting iNKT cell recruitment and cytokine production, thereby promoting amplified neutrophil recruitment to the injured kidney. The present study identifies the nuclear alarmin interleukin (IL)-33 as an important and early mediator of innate immune response, involving iNKT cells, following experimental kidney ischemia-reperfusion in mice. Our findings contribute to a better understanding of IR-induced injury and may lead to new therapeutic insights into renal-induced injury associated with renal transplantation.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-02-24T13:29:14Z No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-02-24T15:36:12Z (GMT) No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) / Made available in DSpace on 2017-02-24T15:36:12Z (GMT). No. of bitstreams: 1 micaelpetersxavier.pdf: 17481766 bytes, checksum: fb76bff140085a73dc148ca7493df8b3 (MD5) Previous issue date: 2013-08-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O desenvolvimento de sistemas computacionais que simulam o funcionamento de tecidos ou mesmo de órgãos completos é uma tarefa extremamente complexa. Um dos muitos obstáculos relacionados ao desenvolvimento de tais sistemas é o enorme poder computacional necessário para a execução das simulações. Por essa razão, o uso de estratégias e métodos que empregam computação paralela são essenciais. Este trabalho foca na simulação temporal e espacial, em uma seção tridimensional de tecido, do comportamento de algumas das células e moléculas que constituem o sistema imunológico humano (SIH) inato. Com o objetivo de reduzir o tempo necessário para realizar a simulação, foram utilizadas múltiplas unidades de processamento gráfico (Graphics Processing Unit, GPUs) em um ambiente de agregados computacionais. Apesar do alto custo de comunicação imposto pelo uso de múltiplas GPUs, as abordagens e técnicas utilizadas neste trabalho para implementar as versões paralelas do simulador mostraram-se efetivas para alcançar o objetivo de redução do tempo de simulação. / The development of computer systems that simulate the behavior of tissues or even whole organs is an extremely complex task. One of the many obstacles related to the development of such systems is the huge computational resources needed to execute the simulations. For this reason, the use of strategies and methods that employ parallel computing are essential. This work focuses on the spatial-temporal simulation of some human innate immune system (HIS) cells and molecules in a three-dimensional section of tissue. Aiming to reduce the time required to perform the simulation, multiple graphics processing units (GPUs) were used in a cluster environment. Despite of high communication cost imposed by the use of multiple GPUs, the approaches and techniques used in this work to implement parallel versions of the simulator proved to be very effective in their purpose of reducing the simulation time.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-02T17:47:54Z No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-06T19:58:07Z (GMT) No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) / Made available in DSpace on 2017-03-06T19:58:07Z (GMT). No. of bitstreams: 1 pedroaugustoferreirarocha.pdf: 4715587 bytes, checksum: dfef00badf9cc3d7c79c1b4c62d3abfd (MD5) Previous issue date: 2012-08-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dois mecanismos são utilizados pelo Sistema Imunológico Humano (SIH) para defender o organismo contra doenças causadas pelos mais distintos agentes patogênicos: o sistema inato e o sistema adaptativo. O primeiro é composto por células e substâncias químicas que utilizam um mecanismo genérico de defesa para prevenir ou limitar infecções ocasionadas pela maioria dos patógenos. Já o segundo mecanismo é ativado pelo primeiro, baseando-se na habilidade de reconhecer e de recordar agentes patogênicos específicos, colaborando para a montagem de um ataque mais potente a cada vez que o mesmo patógeno é encontrado. Apesar de ser muito estudado, muitas questões sobre o funcionamento do SIH ainda estão em aberto em virtude de sua complexidade e do grande número de interações, nos mais diversos níveis, entre seus distintos componentes. Neste sentido, ferramentas computacionais podem se constituir em um poderoso ferramental para auxiliar nas pesquisas sobre o tema. O presente trabalho está inserido neste escopo, dividindo-se em duas partes. Na primeira parte, o trabalho apresenta os resultados de uma análise de sensibilidade em um modelo matemático-computacional que simula a resposta imunológica inata ao lipopolissacarídeo (LPS), com o objetivo de encontrar os parâmetros mais sensíveis deste modelo. Além disto, a segunda parte do trabalho propõe uma adaptação do modelo original para um modelo tridimensional. As simulações realizadas nas duas partes do trabalho mostraram-se computacionalmente caras, demandando longos períodos de tempo para serem concluídas. Assim, GPGPUs (General Purpose Graphics Processing Units) foram utilizadas para reduzir os tempos de execução. O uso de GPGPUs permitiu que acelerações de 276 vezes para a análise de sensibilidade massiva e de 87 vezes para a computação do modelo em três dimensões fossem obtidas. / Two mechanisms are used by the Humman Immune System (HIS) to protect the body against diseases caused by distinct pathogens: the innate and the adaptive immune system. The first one is composed of cells and chemicals that use a generic mechanism of defense to prevent or limit infections caused by most pathogens. The second mechanism is activated by the first one. It has the ability to recognize and remember specific pathogens, contributing to the assembly of a more powerful attack each time the same pathogen is encountered again. Despite being widely studied, many questions about the functioning of the HIS are still open because of its complexity and the large number of interactions of its components on distinct levels. In this sense, computational tools are a powerful instrument to assist researchers on this field of study. This work is inserted in this scope and it is split into two parts. In the first part, this work presents the results of a sensitivity analysis on a mathematical-computational model that simulates the innate immune response to lipopolysaccharide (LPS). The main objective of the sensitivity analysis was to find the most sensitive parameters of the mathematical model. The second part of this work proposes the extension of the original model to a three-dimensional one. The simulations in the two parts of the work proved to be computationally expensive, requiring long periods of time to complete. Thus, GPGPUs (General Purpose Graphics Processing Units) were used to reduce execution times. The use of GPGPUs allowed speedups of 276 times for sensitivity analysis, when compared to the sequential one, and of 87 times for computations using the three dimensions model.

Die angeborene Immunität ist in der Lage, Pathogene schon beim erstmaligen Eindringen zu erkennen und zu bekämpfen. Haupteffektoren der schnellen, angeborenen Immunantwort sind Makrophagen und polymorphkernige neutrophile Granulozyten. Diese erkennen und phagozytieren Pathogene und koordinieren die weitere Immunantwort durch die Freisetzung von inflammatorischen Mediatoren und Zytokinen. Die Erkennung mikrobieller Bestandteile, wie Lipopolysaccharid (LPS) Gram-negativer Bakterien bzw. Peptidoglykan (PG) und Lipoteichonsäuren (LTA) Gram-positiver Bakterien, führt zur Aktivierung von unterschiedlichen Proteinkinasen, des Transkriptionsfaktors NF-(B und zur Freisetzung von Zytokinen. Mitglieder der Toll-Proteinfamilie, sogenannte Toll-like-Rezeptoren (TLR), wurden kürzlich als Rezeptoren auf Immunzellen identifiziert, die für die Erkennung solcher mikrobieller Bestandteile verantwortlich sind. Während TLR-4 der LPS-Erkennung dient, und TLR-2 und -6 verschiedene Liganden von Gram-positiven Bakterien binden, blieb die Frage der Erkennung von LTA und verwandten Glykolipiden strittig. Sowohl TLR-2 als auch TLR-4 wurden für diese Rolle diskutiert. Zielsetzung dieser Arbeit war, die Rolle von TLRs in der LTA- und Glykolipid-Erkennung zu untersuchten. Glykolipide von zwei eng verwandten Treponemen-Spezies, T. maltophilum (TM) und T. brennaborense (TB), sowie neuartig aufgereinigte Lipoteichonsäuren von Staphylococcus aureus (SA) und Bacillus subtilis (BS) wurden eingesetzt, um die nukleäre Translokation von NF-(B in verschiedenen Zellsystemen zu induzieren. Diese Zellstimulationsexperimente wurden mit verschiedenen TLR-2-negativen Zellinien sowie mit Peritonealexsudatzellen TLR-4-defizienter C3H/HeJ-Mäuse durchgeführt. Weitere Informationen lieferten TLR-2-Überexpressions-Experimente sowie Zellstimulationen unter Verwendung von anti-TLR-4-Antikörpern. Die Aktivierung von NF-(B wurde anhand von Gelshifts nachgewiesen. Mit der Überexpression von dominant-negativen Mutanten verschiedener Moleküle der Signalkaskade, mit Kinase-Hemmstoffen und mit Western Blots wurden die intrazellulären Signaltransduktionswege untersucht. Für Glykolipide von T. maltophilum und beide verwendeten Lipoteichonsäuren ließ sich eine klare TLR-2-Abhängigkeit in der Aktivierung von NF-(B und der Induktion von proinflammatorischen Zytokinen zeigen. Die Glykolipide von T. brennaborense hingegen waren überraschender Weise gleichzeitig auch TLR-4-Liganden. Beide untersuchten Glykolipide sowie beide LTAs aktivierten einen Signalweg unter Einbeziehung des Adaptermoleküls MyD88 und der NF-(B-induzierenden Kinase (NIK). Des weiteren konnte der Einfluß der MAP-Kinasen p42/44 und p38 auf die Treponema-Glykolipid- und LPS-induzierte TNF-(-Ausschüttung dargestellt werden. Zusammenfassend zeigen diese Ergebnisse, daß TLR-2 der Hauptrezeptor von Lipoteichonsäuren ist, und TLR-2 und -4 beide Rezeptoren der Treponema-Glykolipide sein können. Diese Ergebnisse sollten dazu beitragen, die molekularen Grundlagen der Reaktionen des Immunsystems auf Gram-positive Bakterien und Treponemen zu verstehen. / The innate immune response to microbial pathogens is able to protect the host after a first pathogen contact. This immediate immune response is largely mediated by macrophages and neutrophils. They recognize and phagocytose pathogens, and coordinate host responses by secreting inflammatory mediators, such as cytokines. The recognition of lipopolysaccharide (LPS) of Gram-negative bacteria, or peptidoglycan (PG) and lipoteichoic acids (LTAs) of Gram-positive bacteria leads to the induction of protein-kinases, the transcription factor NF-(B, and subsequently the release of proinflammatory cytokines. Recently, members of the Toll-protein-family, the so-called Toll-like receptors (TLRs) have been found to be involved in immune cell activation by microbial products. While TLR-4 has been identified as the transmembrane signal transducer for LPS, and TLR-2 and -6 for different ligands originating from Gram-positive bacteria, the molecular basis of recognition of lipoteichoic acids and related glycolipids has not been completely understood: Both, TLR-4 and -2 have been postulated as receptors. In order to determine the role of TLRs in immune cell activation by Treponema glycolipids and LTAs experiments involving TLR-2-negative cell lines, macrophages from TLR-4-deficient C3H/HeJ-mice, cells overexpression TLR-2, and inhibitory TLR-4 antibodies were performed. The induction of NF-(B was assessed by electrophoretic mobility shift assays. Glycolipids of two related Treponema species, T. maltophilum (TM) and T. brennaborense (TB), and LTAs from Staphylococcus aureus (SA) and Bacillus subtilis (BS) were investigated for induction of nuclear translocation of NF-(B in different cell systems. Glycolipids from T. maltophilum and both LTAs studied revealed TLR-2-dependency in induction of NF-(B and proinflammatory cytokines. Surprisingly, glycolipids from T. brennaborense were found to be TLR-4-ligands. Furthermore an involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids was revealed by dominant-negative overexpression experiments. The induction of TNF-( by Treponema glycolipids furthermore was dependent on activation of MAP kinases p42/44 and p38, as indicated by specific kinase inhibitors. Tyrosinephosporylation of the p42/44 kinase induced by Treponema glycolipids were detected by western blots. In summary, the results presented here indicate that TLR-2 is the main receptor for LTAs. Both TLR-2 and -4 serve as receptors for Treponema glycolipids. These results may potentially contribute to explain immune responses to Gram-positive bacteria and treponemes.

LBP vermittelt im Wirtsorganismus die direkte Immunantwort auf bakterielle Liganden wie das Lipopolysaccharid (LPS) von Gram-negativen oder Lipopeptide von Gram-positiven Bakterien. In dieser Arbeit wurde die Funktionsweise von LBP weiter aufgeklärt. Im ersten Teil der Arbeit wurde eine natürlich vorkommende Mutation des LBP (c998t), die an Position 333 zu einem Austausch der Aminosäure Prolin zu Leucin führt, hinsichtlich ihrer Auswirkungen auf Struktur und Funktionalität des Proteins untersucht. Westernblot-Analysen des rekombinant hergestellten Proteins und humaner Seren von Mutationsträgern weisen auf einen Zerfall des mutierten Proteins hin. Es kommt zu einer Beeinträchtigung der Bindung bakterieller Liganden und einer deutlichen Reduktion der LBP-vermittelten Zytokinausschüttung von Immunzellen. Der hier untersuchte Polymorphismus hat eine Allelfrequenz von 0,072 in einer gesunden europäischen Population. Genotypanalysen von Patientengruppen zeigten, dass es durch die Mutation zu einer deutlich erhöhten Mortalität bei Patienten mit septischen Komplikationen und einer durch Gram-negative Erreger verursachten Pneumonie kommt. Unsere Ergebnisse zur eingeschränkten Funktion des LBP-c998t bieten eine erste Erklärung dafür, wie diese Mutation vermutlich die Fähigkeit, Krankheiten zu bewältigen, beeinträchtigt. Innerhalb dieser Arbeit ging es um die Analyse der Bindung von bakteriellen Liganden an LBP. Dabei wurde eine potentiell gemeinsame Bindungsstelle für Liganden untersucht, die von Gram-positiven und Gram-negativen Bakterien stammen und später von den Toll-like Rezeptoren (TLRs) 2 und -4 erkannt werden. Dazu wurden Bindungsversuche zwischen Lipopeptiden und LPS mit einer zweiten LBP-Variante (LBP-E94/95) durchgeführt. Beim LPS führt dies zu einem Bindungsverlust. Auch für die Lipopeptide war durch die Mutationen die Interaktion mit LBP beeinträchtigt, was die These einer gemeinsamen Bindungsstelle von TLR2- und TLR4-Liganden an das Protein weiter unterstützt. / LBP enhances the innate immune reaction against bacterial ligands like LPS from gram negative or lipopeptides from gram positive bacteria in the host. Here we investigated the function of LBP using two recombinant mutants of the protein. The first part of this work examines a natural occurring mutation of LBP (c998t) leading to an amino acid exchange of proline to leucine at position 333 with regard to the impact on structure and function of the protein. Western blot analyses of the recombinant protein and sera obtained from individuals differing in the LBP genotype indicate the disaggregation of the mutated protein. Thereby binding of bacterial ligands to LBP is diminished and the LBP mediated cytokine secretion of immune cells is reduced. The gene polymorphism leading to the occurrence of the mutation is present with an allelic frequence of 0.072. A recent study has shown that this LBP-SNP led to a higher mortality in patients with septic complications and gram negative pneumonia. The results presented here, showing the negative impact on the function of LBP due to the mutation, may therefore be a first explanation on how this mutation affects the ability of people to deal with disease. Within this work binding of ligands to LBP was also explored. It was investigated whether ligands which are later recognized by Toll-like receptors (TLRs) 2 and – 4 share a common binding site on LBP. Assays with immobilized lipopeptides and LPS were performed with a second mutated LBP (LBP-E94/95). LPS binding to LBP is diminished completely. Here we showed that binding of lipopeptide to LBP is affected likewise, furthermore supporting the hypothesis of a common binding site for TLR2- and TLR4- ligands.

Yersinia enterocolitica es un patógeno Gram-negativo que provoca diversos síndromes gastrointestinales y expresa una panoplia de factores de virulencia, la mayoría regulados por la temperatura. El lipopolisacatido (LPS) es uno de los principales factores de virulencia de las bacterias Gram-negativas patógenas. Además, es una de las moléculas reconocidas por el sistema inmune innato y diana de los péptidos antimicrobianos. Por consiguiente, no es de extrañar que las bacterias modifiquen la estructura de su LPS con el fin de resistir a la defensa del sistema inmune. En esta Tesis Doctoral se han identificado los loci responsables de las modificaciones del lípido A de Y. enterocolitica O:8 (YeO8) y se ha demostrado que están reguladas por la temperatura. Se ha definido un circuito regulatorio complejo en el que intervienen los sistemas PhoP/PhoQ y PmrA/PmrB y en el que RovA y H-NS son piezas centrales. Además se demuestra que el lípido A tiene un papel en la virulencia de YeO8. Por último , se han identificado por primera vez en YeO8 la enzima PmrC, encargada de la adición de fosfoetanolamina al lípido A y la enzima LpxR encargada de la deacilación del lípido A.