Allogeneic Hematopoietic stem cell transplants (HSCT) are used for the treatment of bone marrow aplasias. Allogeneic HSCT is performed by treating the patient with chemotherapy drugs and irradiation and then transplanting hematopoietic stem cells from a healthy donor to restore the immune system and hematopoietic cells. Allogeneic HSCTs has the added benefit of the graft vs leukemia effect (GvL), whereby donor allogeneic T cells are able to mount immune responses against any residual cancer cells. However, alloreactivity towards the mismatched minor and major histocompatibility antigens the patient's healthy tissues leads to graft vs host disease (GvHD). This process is also mediated by Macrophages, Dendritic cells, B cells. Furthermore, a decrease in the number of NK, B, and T regulatory cells exacerbates GvHD. This leads to a state of systemic inflammation, tissue damage and multiorgan fibrosis. Current therapies designed to suppress the immune system have been shown to be efficacious in preventing GvHD but patients become susceptible to infection or experience cancer relapse through the elimination of the GvL response as well. In this thesis, we explore two strategies for targeting T cell activation in two mouse models of GvHD. In the first model, we examined the contribution of donor-derived complement C5 on the induction GvHD. We observed that recipient mice were only protected from GvHD when donor cells were deficient for complement protein C5. Our second strategy involves selective targeting of alloreactive T cells using peptide immunotherapy. For this approach, we first developed a humanized mouse model of GvHD whereby cells from donor mice expressing human class II HLA were reconstituted into recipient mice expressing human class I HLA. We then tested peptide immunotherapy using peptides derived from the human class I HLA. Our initial results were inconclusive and require further optimization. / Thesis / Doctor of Philosophy (PhD) / Graft vs Host Disease is an unwanted side effect of mismatched bone marrow transplant. Donor T cells recognize and attack mismatched tissues of the recipient and this leads to systemic inflammation and tissue scarring. Current treatments primarily target T-cell activation by suppressing the immune system, however, this leaves the patients susceptible to recurrent infections. In this thesis we describe the creation of two mouse models of Graft vs Host Disease and then examine two ways of specifically targeting donor T cell activation that is designed not to affect normal immune responses.

This dissertation outlines a theory of law capable of explaining both the legal systems of domestic states and other types of legal phenomena different from state law that I will call non-state legal phenomena. Central examples of non-state law include indigenous and customary laws, the international legal order, the European Union, and transnational commercial law. This theoretical framework aims to formulate and resolve questions about the common features shared by different types of legality and the distinctive legal character of non-state legal phenomena. It also sets the stage for doctrinal and politico-moral inquiries about these phenomena. My account draws liberally from central themes of HLA Hart’s theory of state law that I deem applicable outside the domestic context. One key idea is the notion of normative order or unified complexes of interrelated rules that regulate specific domains of action. The refined Hartian view that I develop here distinguishes between two kinds of normative orders, sets and systems, which differ in their characteristic features and that allow for different doctrinal and moral inquiries. While these tools can be used to explain both state and non-state normative phenomena, I shall consider as law the normative orders of political communities, i.e. groups whose participants efficaciously employ intense forms of social pressure to secure conformity to norms that regulate pressing politico-moral issues. With these elements in place, the legal domain can be characterized as a constellation of sets and systems that constitute political communities at the state, non-domestic, international, supra-national, and potentially global levels. The argument proceeds as follows. Chapter 1 sets the stage of this inquiry. Chapter 2 explicates the key insights of the Hartian framework. Chapter 3 defends the applicability of this framework to non-state contexts. Chapter 4 illustrates its explanatory virtues by applying it to two regimes of international trade law. The conclusion summarizes the central insights of this view and highlights the avenues for future research. / Thesis / Doctor of Philosophy (PhD) / This dissertation outlines a theory of law capable of explaining both state and non-state legal phenomena. This theoretical framework aims to formulate and resolve questions about the common features shared by different types of legality and the distinctive legal character of non-state legal phenomena, and to help to set the stage for further inquiries about them. My account draws liberally from HLA Hart’s theory of state law. The argument proceeds as follows. Chapter 1 sets the stage of this inquiry. Chapter 2 explicates the key insights of the Hartian framework. Chapter 3 defends the applicability of this framework to non-state contexts. Chapter 4 illustrates its explanatory virtues by applying it to two regimes of international trade law. The conclusion summarizes the central insights of this view and highlights the avenues for future research.

À l'heure actuelle, environ 40 millions de personnes vivent avec le VIH/SIDA et plus de 30 millions de décès ont été constatés depuis le début de cette épidémie, la plupart des victimes vivant dans les pays en développement. Depuis les premières manifestations du SIDA et la découverte du virus d'immunodéficience humaine, des progrès considérables ont été réalisés en ce qui concerne notre compréhension des interactions hôte/pathogène, du système immunitaire et des mesures préventives et sociales à adopter pour contrer la progression du VIH/SIDA. Cependant, malgré tous ces progrès, le nombre d'individus infectés par le virus d'immunodéficience humaine ne cesse de croître, ce qui suggère que les outils thérapeutiques actuels et les approches préventives et sociales déployées à ce jour ne sont pas suffisants. C'est donc dire que d'autres stratégies de traitement doivent être envisagées et développées pour freiner la dissémination du virus et permettre son éradication. À ce sujet, les thèmes de recherche concernant les interactions hôte/pathogène et le rôle de nombreux facteurs cellulaires dans le cycle réplicatif du VIH-1, pourraient mener à l'élaboration éventuelle de telles stratégies. Actuellement, plusieurs études démontrent que des molécules dérivées de la cellule hôte incorporées dans l'enveloppe virale peuvent modifier son cycle de vie en lui conférant les caractéristiques correspondant à leurs fonctions physiologiques normales. D'une façon générale, ces molécules permettent au virus d'échapper à la réponse immunitaire humorale, d'augmenter son attachement aux cellules cibles et d'amorcer des événements signalétiques dans les cellules avec lesquelles il entre en contact. Cette thèse décrit en première partie l'importance de deux de ces molécules, soient les molécules du complexe majeur d'histocompatibilité de classe II (CMH-II) et la molécule de costimulation CD86. Les résultats obtenus à l'aide de plusieurs approches complémentaires démontrent que l'incorporation de ces molécules permet au VIH-1 de présenter un antigène nominal à une cellule lymphocytaire T CD4+ reconnaissant de façon spécifique le peptide logé dans la niche peptidique du CMH-II incorporée dans l'enveloppe virale. Cette présentation antigénique induit des signaux de transduction menant à la translocation nucléaire des facteurs transcriptionnels NF-KB et NFAT, permettant ainsi l'activation de la transcription du génome viral dans les lymphocytes T CD4+. La deuxième partie de cette thèse est consacrée aux myeloid related proteins (MRP). Ces molécules, qui ne sont pas incorporées dans l'enveloppe virale, sont impliquées dans la réponse inflammatoire et on les retrouve en quantité augmentée suite à l'infection par le VIH-1. Nos résultats indiquent que les MRP activent la réplication du VIH-1 dans les cellules lymphocytaires T CD4+ par l'intermédiaire du facteur transcriptionnel NF-KB. Globalement, les travaux présentés dans cette thèse permettent d'approfondir notre compréhension du rôle joué par ces facteurs cellulaires dans la réplication du VIH-1. / At the moment, more than 40 millions individuals Worldwide are affected by the human immunodeficiency virus (HIV) and since the beginning of this pandemic, more than 30 millions deaths have occurred. The important developments regarding HIV pathogenesis, therapeutic strategies, immune System, and preventive measures have not been sufficient to stop or even to slow down the dissemination of HIV/AIDS around the world. Today, research topics regarding the role of cellular factors and host/pathogens interactions on the virus life cycle could eventually lead to the development of efficient strategies to fight the spread of this epidemic. The studies presented in this thesis describe how three types of molecules modulate its replicative cycle. These molecules include the major histocompatibility complex class II molecules and the CD86 costimulatory molecule, both of which are embedded into human immunodeficiency virus envelope, and MRPs, which are proteins involved in inflammatory responses that are not embedded in viral envelope. Using different strategies, we observed that the incorporation of CD86 costimulatory molecule and of the major histocompatibility complex class II molecules enable HIV-1 to present nominal antigens to CD4+ T lymphocytes, activate them through NF-KB and NFAT transcriptional factors, thereby activating viral replication. We also observed that MRPs activate HIV-1 replication in CD4+ T cells via NF-KB transcriptional factor. The work presented here deepens our understanding of how cellular factors can modulatevirus replication.

Cytotoxic T-lymphocytes (CTLs) are important components of the adaptive immune system and function by scanning the intracellular environment so as to detect and de-stroy infected cells. CTL responses play a major role in controlling virus-infected cells such as in HIV or influenza and cells infected with intracellular bacteria such as in tuberculosis. To do so they require the antigens to be presented to them, which is fulfilled by the major histocompatibility complex (MHC), commonly known as human leukocyte antigen or HLA molecules in humans. Recognition of antigenic peptides to Class-1 HLA molecules is a prerequisite for triggering CTL immune responses. Individuals differ significantly in their ability to respond to an infection. Among the factors that govern the outcome of an infection, HLA polymorphism in the host is one of the most important. Despite a large body of work on HLA molecules, much remains to be understood about the relationship between HLA diversity and disease susceptibility. High complexity arises due to HLA allele polymorphism, extensive antigen cross-presentability, and host-pathogen heterogeneity. A given allele can recognize a number of different peptides from various pathogens and a given peptide can also bind to a number of different individuals. Thus, given the plurality in peptide-allele pairs and the large number of alleles, understanding the differences in recognition profiles and the implications that follow for disease susceptibilities require mathematical modelling and computational analysis. The main objectives of the thesis were to understand heterogeneity in antigen presentation by HLA molecules at different scales and how that heterogeneity translates to variations in disease susceptibilities and finally the disease dynamics in different populations. Towards this goal, first the variations in HLA alleles need to be characterized systematically and their recognition properties understood. A structure-based classification of all known HLA class-1 alleles was therefore attempted. In the process, it was also of interest to see if understanding of sub-structures at the binding grooves of HLA molecules could help in high confidence prediction of epitopes for different alleles. Next, the goal was to understand how HLA heterogeneity affect disease susceptibilities and disease spread in populations. This was studied at two different levels. Firstly, modelling the HLA genotypes and CTL responses in different populations and assessing how they recognized epitopes from a given virus. The second approach involved modelling the disease dynamics given the predicted susceptibilities in different populations. Influenza H1N1 infection was used as a case study. The specific objectives addressed are: (a) To develop a classification scheme for all known HLA class-1 alleles that can explain epitope recognition profiles and further to dissect the physic-chemical features responsible for differences in peptide specificities, (b) A statistical model has been derived from a large dataset of HLA-peptide complexes. The derived model was used to identify the interdependencies of residues at different peptide and thereby, rationalize the HLA class-I allele binding specificity at a greater detail, (c) To understand the effect of HLA heterogeneity on CTL mediated disease response. A model of HLA genotypes for different populations was required for this, which was constructed and used for estimating disease response to H1N1 via the prediction of epi-topes and (d) To model disease dynamics in different populations with the knowledge of the CTL response-grouping and to evaluate the effect of heterogeneity on different vaccination strategies. Each of the four objectives listed above are described subsequently in chapters 2 to 5, followed by Chapter 6 which summarises the findings from the thesis and presents future directions. Chapter 1 presents an introduction to the importance of the function of HLA molecules, describes structural bioinformatics as a discipline and the methods that are available for it. The chapter also describes different mathematical modelling strategies available to study host immune responses. Chapter 2 describes a novel method for structure-based hierarchical classification of HLA alleles. Presently, more than 2000 HLA class-I alleles are reported, and they vary only across peptide-binding grooves. The polymorphism they exhibit, enables them to bind to a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern due to multiplicity in their associations. Thus, a powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. The study reports a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification with 7 supergroups was derived, each further categorized to yield a total of 72 groups. An independent clustering scheme based only on the similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explains the basis for the observed clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues that contribute to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants. The results are presented in an interactive web- server http://proline.iisc.ernet.in/hlaclassify. In Chapter 3, the knowledge of structural features responsible for generating peptide recognition specificities are first analysed and then utilized for predicting T-cell epi-topes for any class-1 HLA allele. Since identification of epitopes is critical and central to many of the questions in immunology, a study of several HLA-peptide complexes is carried out at the structural level and factors are identified that discriminate good binder peptides from those that do not. T-cell epitopes serve as molecular keys to initiate adaptive immune responses. Identification of T-cell epitopes is also a key step in rational vaccine design. Most available methods are driven by informatics, critically dependent on experimentally obtained training data. Analysis of the training set from IEDB for several alleles indicate that sampling of the peptide space is extremely sparse covering only a tiny fraction of all possible nonamer space, and also heavily skewed, thus restricting the range of epitope prediction. A new epitope prediction method is therefore developed. The method has four distinct modules, (a) structural modelling, estimating statistical pair-potentials and constraint derivation, (b) implicit modelling and interaction profiling, (c) binding affinity prediction through feature representation and (d) use of graphical models to extract peptide sequence signatures to predict epitopes for HLA class I alleles . HLaffy is a novel and efficient epitope prediction method that predicts epitopes for any HLA Class-1 allele, by estimating binding strengths of peptide-HLA complexes which is achieved through learning pair-potentials important for peptide binding. It stands on the strength of mechanistic understanding of HLA-peptide recognition and provides an estimate of the total ligand space for each allele. The method is made accessible through a webserver http://proline.biochem.iisc.ernet.in/HLaffy. In chapter 4, the effect of genetic heterogeneity on disease susceptibilities are investigated. Individuals differ significantly in their ability to respond to an infection. Among the factors that govern the outcome of an infection, HLA polymorphism in the host is one of the most important. Despite a large body of work on HLA molecules, much remains to be understood about how host HLA diversity affects disease susceptibilities. High complexity due to polymorphism, extensive cross-presentability among HLA alleles, host and pathogen heterogeneity, demands for an investigation through computational approaches. Host heterogeneity in a population is modelled through a molecular systems approach starting with mining ‘big data’ from literature. The in-sights derived through this is used to investigate the effect of heterogeneity in a population in terms of the impact it makes on recognizing a pathogen. A case study of influenza virus H1N1 infection is presented. For this, a comprehensive CTL immunome is defined by taking a consensus prediction by three distinct methods. Next, HLA genotypes are constructed for different populations using a probabilistic method. Epidemic incidences in general are observed to correlate with poor CTL response in populations. From this study, it is seen that large populations can be classified into a small number of groups called response-types, specific to a given viral strain. Individuals of a response type are expected to exhibit similar CTL responses. Extent of CTL responses varies significantly across different populations and increases with increase in genetic heterogeneity. Overall, the study presents a conceptual advance towards understanding how genetic heterogeneity influences disease susceptibility in individuals and in populations. Lists of top-ranking epitopes and proteins are also derived, ranked on the basis of conservation, antigenic cross-reactivity and population coverage, which pro- vide ready short-lists for rational vaccine design (flutope). Next, in Chapter 5, the effect of genetic heterogeneity on disease dynamics has been investigated. A mathematical framework has been developed to incorporate the heterogeneity information in the form of response-types described in the previous chap-ter. The spread of a disease in a population is a complex process, controlled by various factors, ranging from molecular level recognition events to socio-economic causes. The ‘response-typing’ described in the previous chapter allows identification of distinct groups of individuals, each with a different extent of susceptibility to a given strain of the virus. 3 different approaches are used for modelling: (i) an SIR model where different response types are considered as partitions of each S, I and R compartment. Initially SIR models are developed, such that the S compartment is sub-divided into further groups based on the ‘response-types’ obtained in the previous chapter. This analysis shows an effect in infection sweep time, i.e., how long the infection stays in the population. A stochastic model incorporates the environmental noise due to random variation in population influx, due to birth, death or migration. The system is observed to show higher stability in the presence of genetic heterogeneity. As the contagion spreads only through direct host to host contact. The topology of the contact network, plays major role in deciding the extent of disease dynamics. An agent based computational framework has been developed for modelling disease spread by considering spatial distribution of the agents, their movement patterns and resulting contact probabilities. The agent-based model (ABM) incorporates the temporal patterns of contacts. The ABM is based on a city block model and captures movement of individuals parametrically. A new concept of system ‘characteristic time’ has been introduced in context of a time-evolving network. ‘Characteristic time’ is the minimum time required to ensure, every individual is connected to all other individuals, in the time aggregated contact network. For any given temporal system, disease time must exceed ‘characteristic time’ in order to spread throughout the population. Shorter ‘characteristic time’ of the system is suggestive of faster spread of the disease. A disease spread network is constructed which shows how the disease spreads from one infected individual to others in the city, given the contact rules and their relative susceptibilities to that viral strain. A high degree of population heterogeneity is seen to results in longer disease residence time. Susceptible individuals preferentially get infected first thereby exposing more susceptible individuals to the disease. Vaccination strategies are derived from the model, which indicates that vaccinating only 20% of the agents, who are hub nodes or highly central nodes and who also have a high degree to susceptible agents, lead to high levels of herd immunity and can confer protection to the rest of the population. Overall, the thesis has provided biologically meaningful classification of all known HLA class-1 alleles and has unravelled the physico-chemical basis for their peptide recognition specificities. The thesis also presents a new algorithm for estimating pep-tide binding affinities and consequently predicting epitopes for all alleles. Finally the thesis presents a conceptual advance in relating HLA diversity to disease susceptibilities and explains how different populations can respond differently to a given infection. A case study with the influenza H1N1 virus identified populations who are most susceptible and those who are least susceptible, in the process identifying important epitopes and responder alleles, providing important pointers for vaccine design. The influence of heterogeneity and response-typing on disease dynamics is also presented for influenza H1N1 infection, which has led to the rational identification of effective vaccination strategies. The methods and concepts developed here are fairly generic and can be adapted easily for studying other infectious diseases as well. Three new web-resources, a) HLAclassify, b) HLaffy and c) Flutope have been developed, which host pre-computed results as well as allow interactive querying to an user to perform analysis with a specific allele, peptide or a pathogenic genome sequence.

This thesis challenges some of the current limits to the grounds for judicial review of legislation accepted by most Canadian jurists. More specifically, it makes a common law-based argument in favour of the priority over legislation of principles which are implied from the Imperial Constitution Acts 1867-1982 and which originally derive from the English constitution – namely implied constitutional principles. The argument faces two main interrelated legal objections: Parliamentary sovereignty and the Framers’ intentions. The first objection is rebutted by arguing that Parliamentary sovereignty possesses an ability to change in a way that can incorporate substantive legal limitations. The most prevalent common law-based theories of change to Parliamentary sovereignty suggest that the courts can authoritatively determine if implied constitutional principles can check legislation. The second objection is rebutted by reference to the notion of progressive interpretation as conceived under Hartian and Dworkinian theories of law and adjudication. Under these theories, progressive interpretation is an aspect of the courts’ best overall interpretation of the constitution, which includes implied constitutional principles. Such progressive interpretation can result in these principles constraining legislative authority. Justification of the progressive interpretation of implied constitutional principles can be based on the rule of law from which derive a number of these principles. One plausible conception of the Canadian rule of law is that it rejects the view that implied constitutional principles can prevail when in conflict with legislation. However, the better conception is that, as an attempt to adapt implied constitutional principles to relevant changes in society and to protect their underlying values, the judiciary should interpret these principles as capable of checking legislation to the extent that they form part of the core content of the rule of law. Such a conception and an operation of implied constitutional principles can properly be explained by Hartian or Dworkinian common law-based progressive interpretation of these principles and by their relationship with legislative authority.

Made available in DSpace on 2016-08-10T10:38:33Z (GMT). No. of bitstreams: 1 CAROLINE STEGLICH RAMOS.pdf: 1233030 bytes, checksum: 0c294f9932ce70c185a878b013b4d356 (MD5) Previous issue date: 2012-02-07 / The breast cancer constitutes most common malignancy pathology among women where the knowledge of prognostic aspects suggest the clinical course of disease and patient survival. Detection of HLA-G has been described as prognostic factor for many cancers, this molecule, expresses in ectopic way in certain types of tumors, where induce the immunotolerance and, favors the tumoral immune escape. Thereby, this study aimed to evaluate the expression of the molecule HLA-G in infiltrating ductal breast carcinomas (CDI), as well as evaluate the polyphormism of the gene encoding HLA-G to determine possible correlation between these and the molecule expression. Thus, to meet these objectives, we evaluated the expression of HLA-G protein in forty-five patients with IDC by immunohistochemistry and also the insertion and deletion polymorphism of 14pb gene HLA-G in 80 samples of paraffin tumors, obtained from the Service of Anatomic Pathology at Araújo Jorge Hospital, and a group of 191 healthy people was used as control. All the statistical analyses were made using the programs GraphPad Instat version 5.01 and Genepop version 2.0. Our results showed that the group of patients were inbalance in Hardy-Weinberg (p=0,01), due to the excess of heterozygotes (p=0,0007), there is not statistically significant difference in the allelic frequencies in locus HLA-G, when compared the group of patients with the control group, however, there were difference in the genotypics frequencies when comparing the same groups (p=0,012), being the genotypic frequency deletion/insertion significantly higher in the group of patients. Also, the expression of HLA-G was observed in 62% of the samples analyzed by immunohistochemistry and this expression was observed an mostly in tumor cells and also by intra-tumoral inflammatory infiltrate (p=0,03). The analysis showed that the expression of HLA-G was significantly higher in patients that had shorter survival (log rank = 0,03), after 5 years of follow up and still there was also a correlation between the expression of HLA-G and increased incidence of lymph node metastases (p=0,01). Thus, the results suggest that the HLA-G expression in patients with CDI may be associate to poor clinical evolution, with the reduced survival. / O câncer de mama constitui-se na patologia maligna mais comum entre as mulheres onde o conhecimento dos aspectos prognósticos sugerem o curso clínico da doença e a sobrevida dos pacientes. A detecção da molécula HLA-G tem sido descrita como fator prognóstico para inúmeras neoplasias, esta molécula, expressa de maneira ectópica em certos tipos de tumores, onde induz a imunotolerância e favorece o escape imunológico tumoral. Desse modo, esse estudo objetivou avaliar a expressão da molécula HLA-G em carcinomas ductais infiltrantes da mama (CDI), bem como, avaliar polimorfismo do gene codificador de HLA-G para determinar possível correlação entre estes e a expressão da molécula. Assim, para cumprir tais objetivos, avaliou-se a expressão da proteína HLA-G em quarenta e cinco pacientes com CDI por imunoistoquímica, e ainda, o polimorfismo de deleção e inserção de 14pb do gene HLAG, em 80 amostras de tumores parafinados, obtidas junto ao Serviço de Anatomia Patológica do Hospital Araújo Jorge e um grupo composto por 191 indivíduos saudáveis foi utilizado como controle. Todas as análises estatísticas foram feitas utilizando os programas GraphPad Instat versão 5.01 e Genepop versão 2.0. Os resultados mostraram que o grupo de pacientes encontrava-se em desequilíbrio de Hardy-Weinberg (p= 0,01), devido ao excesso de heterozigotos (p= 0,0007), não havendo diferença estatisticamente significante nas freqüências alélicas no loco HLA-G quando comparado o grupo de pacientes com o grupo controle, no entanto, houve diferença nas freqüências genotípicas ao comparar os mesmos grupos (p= 0,012), sendo a freqüência do genótipo deleção/inserção significantemente maior no grupo de pacientes. Também foi observada a expressão de HLA-G em 62% das amostras totais analisadas por imunoistoquímica e essa expressão foi apresentada principalmente, em células tumorais havendo, no entanto, também expressão pelas células do infiltrado inflamatório intratumoral (p= 0,03). As analises mostram que a expressão de HLA-G foi significantemente maior em pacientes que tiveram menor sobrevida (log rank= 0,03), após 5 anos de seguimento clínico e ainda houve correlação entre a expressão de HLA-G e aumento naincidência de metástases em linfonodos (p= 0,01). Com isso, os resultados sugerem que a expressão de HLA-G em pacientes com CDI pode estar associada à pior evolução clínica dos mesmos havendo redução na taxa de sobrevida.

Le sepsis constitue une pathologie fréquente, grevée d’une morbi-mortalité encore élevée. Sa physiopathologie fait notamment intervenir des dysrégulations du système immunitaire inné et adaptatif et des voies de l’apoptose. Ce travail aborde l’expression leucocytaire de deux familles de protéines potentiellement impliquées dans sa physiopathologie :les phosphodiestérases (PDE) et les apolipoprotéines L (apoL). L’étude de l’expression des PDE sous-tend le fait que ces enzymes, qui dégradent les nucléotides cycliques (AMPc et GMPc), sont impliquées dans la modulation de nombreux processus inflammatoires, tant d’origine infectieuse que non-infectieuse. L’expression des PDE après administration de LPS chez l’Homme est cependant mal caractérisée, de même qu’au cours du sepsis. Le présent travail teste l’hypothèse selon laquelle le sepsis, caractérisé par un état de dysrégulation immune complexe, s’accompagne d’une répression de l’expression des PDE au sein des leucocytes circulants, contrairement à ce qui est observé dans des modèles standardisés d’inflammation aiguë (LPS) ;il met également en perspective, dans une démarche observationnelle, l’expression des PDE avec l’expression du complexe HLA-DR, un ensemble protéique permettant la présentation de l’antigène et dont l’expression est partiellement dépendante de l’AMPc. Trois études ont ainsi été menées :étude de l’expression des PDE au cours d’une endotoxinémie chez le volontaire sain (1), et au cours du sepsis au sein de leucocytes totaux circulants (2) ou de sous-populations leucocytaires de l’immunité innée (monocytes CD14+ou granulocytes CD15+) (3). Alors que l’administration intraveineuse de LPS chez le volontaire sain mène à l’induction précoce et transitoire de certaines PDE, de façon similaire aux observations in vitro, les patients septiques présentent au contraire dès leur admission, et jusqu’au 5ème jour, une réduction de l’expression de plusieurs PDE en comparaison aux volontaires sains, tant dans les leucocytes totaux que dans les populations CD14+ et CD15+. L’expression de plusieurs de ces PDE est corrélée aux ratios TNF-α/IL-10 qui sont suggestifs d’un état d’immunodépression, attesté par une réduction significative de l’expression du complexe HLA-DR. L’étude des apoL au cours du sepsis sous-tend quant à elle le fait que cette famille de protéines, qui partage des homologies avec des membres du groupe Bcl-2 impliqué dans l’apoptose, a été associée notamment à l’induction de phénomènes pro-apoptotiques ;or, le sepsis est associé à une apoptose retardée des polynucléaires neutrophiles, un phénomène potentiellement délétère au niveau tissulaire. L’hypothèse d’une répression de l’expression des apoL leucocytaires au cours du sepsis est ainsi posée. Dans le présent travail, une diminution de l’expression des apoL-1, 2, 3 et -6 est observée chez les patients de soins intensifs présentant ou non un sepsis en comparaison à des volontaires sains ;cette réduction, corrélée aux taux de protéine C-réactive, concerne tant les populations leucocytaires totales que les granulocytes CD15+, et intéresse tant les ARNm que l’expression protéique (apoL-6 excepté). Le pourcentage de cellules CD15+ apoptotiques est par ailleurs fortement corrélé aux taux d’ARNm des apoL-1 et -2. Ces observations sont reproduites in vitro en incubant des granulocytes CD15+ de volontaire sain avec du sérum de patients septiques ou non septiques. Ces résultats préliminaires suggèrent ainsi une implication des apoL dans la régulation de l’apoptose des neutrophiles au cours du sepsis. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

<p>Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma <i>in situ</i>.</p>

Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma in situ.