Rozprawy doktorskie na temat „HlyA”

E. coli productora de toxina shiga (ECST) y E. coli enterohemorrágica (ECEH) pertenecen a los grupos patógenos de Escherichia coli causantes de enfermedades diarreicas. La presencia de ECST y ECEH en estas enfermedades diarreicas se atribuye principalmente al consumo de vegetales y carnes provenientes de bovinos y ovinos contaminados con estos grupos patógenos y que actualmente se encuentran dentro de las etiologías más importantes del grupo de Enfermedades Trasmitidas por Alimentos (ETA). ECST y ECEH presentan los genes Stx1, Stx2, eaeA y hlyA que codifican a proteínas con cualidades toxicas para el hombre ocasionando graves enfermedades como la colitis hemorrágica (CH), síndrome urémico hemolítico (SUH) y purpura trombocitopénica trombótica (PTT). Por lo tanto, el objetivo de esta investigación es identificar la presencia de los genes Stx1, Stx2, eaeA y hlyA en los aislamientos de E. coli obtenidos de canales de ovinos en rastro municipal de Capulhuac y en carne procesada de ovinos provenientes de la ciudad de Toluca. Se analizaron 71 aislados de E. coli para la identificación genes característicos de los grupos patógenos de ECST y ECEH. Los resultados encontrados indican: los 18 aislamientos de E. coli obtenidas de la carne procesada de ovinos no se identifica la presencia los genes analizados y, por otro lado, de los 53 aislados obtenidos de las canales de ovinos en el rastro municipal de Capulhuac, México, amplificaron los genes Stx1 en 3.7% (2/53), Stx2 en 5.6% (3/53), eaeA en 3.7% (2/53) y hlyA en 3.7% (2/53). De tal manera, que estas canales puede ser una fuente potencial de infección por E. coli productora de toxina shiga y E. coli enterohemorrágica.

The SmpA outer membrane lipoprotein of B. hyodysenteriae has several characteristics that indicate the potential to protect against swine dysentery (SD). It localises to the outer membrane and antibodies directed against SmpA can prevent the growth of B. hyodysenteriae in vitro. There is some variation observed in the distribution and expression of the SmpA lipoprotein, suggesting that vaccination with SmpA may not provide protection against challenge with a heterologous B. hyodysenteriae strain. This study has characterised the variation at the smpA locus, and in the process has identified a novel gene, smpB. There is very low similarity between smpB and smpA, with the exception of an identical lipoprotein signal sequence. This suggests that SmpB may be translocated to the outer membrane of B. hyodysenteriae in a similar fashion to SmpA. The results described in this thesis indicate that strains of B. hyodysenteriae harbour either smpA or smpB, but not both, explaining the earlier results of Turner et al. (1991). The presumed outer membrane location of SmpB lead to further investigations into its potential to protect mice from infection with B. hyodysenteriae. Swine Dysentery is a inflammatory disease of the swine colon. Therefore it is believed that a mucosal immune response may provide increased protection against challenge. In this study, vaccination of mice with recombinant SmpB elicited high levels of serum antibodies, induced the production of Interleukin-4 producing T lymphocytes and decreased the observed histological effects after challenge with virulent B. hyodysenteriae. In efforts to increase the protected conferred by vaccination with SmpB, recombinant Salmonella typhimurium STM-1 vaccines were created to express SmpB or deliver DNA vaccines encoding SmpB. Vaccination with these recombinant Salmonella vectors did not induce a measurable SmpB specific immune response. Macrophage survival and plasmid stability studies indicated that this was due to instability of the expression plasmids in STM-1. Although SmpB will only ever protect against strains of B. hyodysenteriae harbouring smpB, these results indicate that with further research, SmpB (and SmpA) may contribute to protection from SD. Toxin production is an important aspect of the pathogenesis of many pathogenic bacteria. Vaccination with attenuated toxins is commonly used to prevent disease. In this study, the B. hyodysenteriae â-haemolysin HlyA was used to vaccinate mice to determine the protection induced after challenge. Vaccination of mice with recombinant HlyA induced significant levels of serum antibodies and lowered the observed pathological effects after challenge of vaccinated mice with virulent B. hyodysenteriae. In an attempt to increase the mucosal immune response and therefore the protection afforded after vaccination with HlyA, recombinant S. typhimurium STM-1 strains were created to express HlyA or deliver DNA vaccines encoding HlyA. Similar to the recombinant STM-1 vaccines expressing SmpB, a HlyA specific immune response was not observed by ELISA or ELISPOT analysis. Plasmid stability trials revealed that the inability to induce a detectable HlyA specific immune response by recombinant STM-1 vaccination may be due to ins tability of the plasmids. Outer membrane proteins are often important components of vaccines against bacterial and viral pathogens. Considering the variation observed in the smpA locus in this study resulting in the identification of smpB, further investigation into the distribution and conservation of outer membrane encoding genes in B. hyodysenteriae strains was undertaken. In particular, the blpAEFG, vspABCD and vspEFGH clusters were analysed for their distribution. It was demonstrated that genes that are B. hyodysenteriae specific (vspABCD and vspEFGH) displayed higher levels of polymorphism than those that are distributed amongst non-pathogenic species, such as B. innocens (which contains blpAEFG). This suggests that the variation in the vspABCD and vspEFGH clusters amongst B. hyodysenteriae strains may be a result of the exposure to the host immune system. Further investigation was undertaken by PFGE analysis and 2D-gel electrophoresis, to analyse genomic and proteomic variation at a global level. Although strains of B. hyodyse nteriae produced several different electrophoretic types (ET) upon PFGE analysis, only limited correlation between the PFGE ET, the polymorphisms in vspABCD and vspEFGH and the presence of smpA/smpB were observed. 2D-gel electrophoresis analysis of outer membrane preparations of two B. hyodysenteriae strain revealed several distinct differences in the outer membrane between B. hyodysenteriae strains. The observed differences in the proteins contained in the outer membrane of B. hyodysenteriae is important for vaccine design, as the induction of cross protection between strains of B. hyodysenteriae is essential for a effective vaccine.

Le facteur cytotoxique nécrosant-1 (CNF1) et l’hémolysine-alpha (HlyA) sont les deux toxines majeures des Escherichia coli uropathogènes (UPEC), premier agent étiologique des infections du tractus urinaire (ITU) et principale cause de bactériémie à E. Coli. Comme plusieurs autres facteurs de virulence, ces toxines ont été associées à l’ITU en raison de leur grande prévalence dans les UPEC par rapport aux E. Coli fécaux, respectivement 30 % et 0. 9 % pour CNF1. Cependant, leurs rôles dans la physiopathologie de l’infection urinaire reste imprécis. Dans un modèle murin de bactériémie, nous avons pu montrer pour la première fois que CNF1 favorisait la réponse immunitaire innée de l’hôte entraînant l’élimination rapide des bactéries. Nous avons aussi montré que CNF1 présente un effet pro-inflammatoire aussi bien in vitro sur les monocytes qu’in vivo chez la souris. Un effet qui est au détriment de la survie des bactéries dans le sang. Nous montrons que HlyA présente un effet cytotoxique sur les monocytes. La mort des monocytes, induite par HlyA, empêche la production des cytokines pro-inflammatoires induites par CNF1. Nous avons relié cet effet au maintien des UPEC dans le sang. Dans le contexte de la bactériémie, ces deux toxines ont donc des effets opposés sur la survie des bactéries dans le sang. CNF1 apparaît donc comme un facteur d’avirulence dont l’effet est antagonisé par celui de HlyA. La sécrétion de CNF1 n’est pas connue, et on observe une grande divergence dans les résultats des quelques études faites sur ce sujet. Partant de l’observation du lien permanent du gène cnf1 au sein de l‘opéron hly dans les souches CNF1 (+), nous avons émis l’hypothèse que leur système de sécrétion pouvait être commun. Nos résultats montrant l’absence de CNF1 dans le périplasme nous a confortés dans l’idée que CNF1 pourrait être directement secrété dans le milieu extérieur par le système de sécrétion de type-1 décrit pour HlyA. Nous démontrons que l’absence d’HlyB (l’ATPase du système du système de sécrétion de HlyA) a un effet de blocage sur l’induction de mortalité des cellules THP-1 qu’induit CNF1 et inhibe la translocation d’une protéine de fusion CNF1-beta-lactamase dans le cytosol des THP-1. Sachant que la délétion d’HlyB n’affecte pas la production de CNF1, HlyB pourrait intervenir dans la sécrétion de CNF1. Ce travail a permis de mettre en évidence des interactions fonctionnelles entre deux facteurs de virulence toxiques des UPEC (CNF1 et HylA), et aussi le rôle de HlyB dans la translocation de CNF1 dans les THP-1<br>Cytotoxic necrotizing factor 1 (CNF1) and alpha-hemolysin (HlyA) are two major toxins of uropathogenic Escherichia coli (UPEC). UPEC are the major cause of urinary tract infection (UTI) and represent one of the main agents of bacteremia. These two toxins are usually associated to UTI because of their consistent presence in UPEC as compared to commensal strains of E. Coli. Nevertheless, their physiological involvement in UTI remains unclear. Using a mouse model of bacteremia, we showed that CNF1 by its pro-inflammatory property can trigger host immune responses, thus allowing bacterial clearing from the blood by phagocytic cells. We also demonstrated that HylA has a cytotoxic effect on monocytes. This cytotoxic effect will counteract the production of pro-inflammatory cytotoxines induced by CNF1, and will allow the persistence of bacteria in mouse blood. According to this observation, we concluded that during bacteremia, CNF1 and HlyA have an opposite effect on bacterial persistence in the blood. We have developed a technique that allowed us to observe the effect of CNF1 in vitro, by directly infecting monocytes with CNF1 producing UPEC. We thus demonstrated that CNF1 can induce monocytes death during infection. Using a plasmid which produced a chimeric protein (CNF1-bêta-lactamase), we demonstrated that CNF1 can translocate from the bacterial cytoplasmic domain to the monocyte cytosol during infection. This work evidenced a functional interplay between two major virulence factors (CNF1 and HylA) as well as the effect of CNF1 on monocytes

Vibrio cholerae is the cholera causing agent, divided into two biotypes, including the classical biotype and ElTor biotype. Both of these biotypes caused cholera epidemics in the world. The classical biotype caused 6th cholera pandemic (from 1921 to 1961), and ElTor biotype caused 7th cholera pandemic (from 1961 to the 70s). Haemolysin A, a hemolytic protein of V. cholerae ElTor biotype, is encoded by the hlyA gene. This gene is often used for analyzing genetic relationship between strains in the same species or between species in the same Vibrio genus. Results of analyzing nucleotide and amino acid sequences of hlyA gene of V. cholerae strain causing cholera in Vietnam (named hlyA.VN) showed that: the hlyA.VN gene sequence was similar to the hlyA gene sequences of V. cholerae strains of the 6thand 7thcholera epidemics. The hlyA gene of the 6th cholera epidemic strain was deficient in 11 nuleotides (this deficiency leading to the loss of 4 amino acids in the haemolysin A protein) comparing to hlyA.VN gene and hlyA gene of the 7th cholera epidemic strain. The results of genetic distance analysis as well as phylogenetic tree construction also confirmed V. cholerae causing cholera in Vietnam was closely relationship to the strains causing cholera pandemics in the world. It is great significance for the surveillance of molecular epidemiology to prevent cholera effectively.<br>Vibrio cholerae là tác nhân gây bệnh tả, được chia thành hai typ sinh học, đó là typ sinh học cổ điển và typ sinh học ElTor. Cả hai typ này đã từng gây ra các đại dịch tả trên thế giới. Typ sinh học cổ điển đã từng gây ra đại dịch tả lần thứ 6 (từ năm 1921 đến 1961), còn typ sinh học ElTor đã từng gây ra đại dịch tả lần thứ 7 (từ 1961 đến những năm 70). Haemolysin A, một protein có chức năng làm tan máu của V. cholerae typ sinh học ElTor, được mã hóa bởi gen hlyA. Gene này thường được sử dụng cho các phân tích quan hệ di truyền giữa các chủng trong cùng một loài V. cholerae hay giữa các loài trong cùng một chi Vibrio. Kết quả phân tích trình tự nucleotide và axit amin gen hlyA của chủng V. cholerae gâybệnh ở Việt Nam (hlyA.VN) cho thấy: trình tự gen hlyA.VN có sự tương đồng lớn với trình tự gen hlyA của chủng gây đại dịch tả 6 và 7. Gen hlyA của chủng gây đại dịch tả 6 bị thiếu hụt 11 nuleotide (sự thiếu hụt này dẫn tới sự mất đi 4 axit amin trong phân tử haemolysin A) so với gen hlyA.VN và gene hlyA của chủng gây đại dịch tả 7. Kết quả phân tích khoảng cách di truyền cũng như xây dựng cây phát sinh chủng loại cũng đã khẳng định: chủng gây bệnh ở Việt Nam có quan hệ rất gần với các chủng gây đại dịch tả trên thế giới. Nhận định này có ý nghĩa rất lớn đối với công tác giám sát dịch tễ học phân tử để ngăn chặn bệnh tả hiệu quả.

Infectious diarrhea is still a main cause of morbidity and mortality among children from the less developed areas of the world. Among several etiologic agents, a group of diarrheagenic Escherichia coli (DEC) named attaching and effacing E. coli (AEEC), associated to the genesis of the intimin-mediated A/E lesion in enterocytes, should be mentioned. AEEC includes typical (tEPEC) and atypical (aEPEC) subgroups of enteropathogenic E. coli (EPEC) and the pathotype enterohemorrhagic E. coli (EHEC) that also expresses shiga toxin. All of them, mainly aEPEC, are genetically diverse, especially in regard to the expression of genetic markers of virulence. For these reasons, we carried out this investigation aiming to evaluate the distribution of intimin types and hlyA, iha, and toxB, that encode bacterial adhesins and toxins, in 561 AEEC strains. The strains were obtained from 110 children aged up to 69 months with acute diarrhea and without diarrhea, who searched for assistance at Hospital Infantil João Paulo II/FHEMIG, Belo Horizonte, MG, from 2004 to 2007. Beta was the most prevalent intimin genotype in both tEPEC and aEPEC. Gamma was the most common type among EHEC. Intimin kappa was not found in the population studied. Temporal shifts in the distribution of intimin genotypes were observed: type beta prevalence decreased steadly and epsilon intimin emerged from 2006 on. A higher prevalence of intimin iota was detected among children aged 13 to 24 months. Around 10% of AEEC strains could not have their intimin identified. Seasonality and association with gender and diarrhea were not observed. hlyA, iha and toxB were found in less than 50% of the AEEC strains, but were found to be harbored by all AEEC pathotypes. Our findings corroborate the existence of differences in geographic distribution of the virulence factors encoded by eae, hlyA, iha, and toxB among AEEC. They also support the hypothesis that this pathotype comprises a genetically diverse DEC group, specially the subtype aEPEC.<br>Diarreia infecciosa aguda continua sendo uma das principais causas de morbidade e mortalidade em crianças, especialmente nas regiões menos favorecidas do planeta. Entre os diferentes agentes etiológicos da doença, merece menção, pela prevalência elevada e gravidade da doença, o grupo de Escherichia coli diarreiogência (DEC) denominado attaching and effacing E. coli (AEEC), caracterizado pela formação da lesão A/E (attaching and effacing) nos enterócitos, mediada pela intimina. Este grupo inclui os subgrupos típico e atípico de E. coli enteropatogênica (EPEC) e o tipo patogênico denominado E. coli enteremorrágica (EHEC) que, além de intimina, expressa toxinas shiga. Estes patotipos apresentam grande diversidade genética, inclusive no que se refere à expressão de marcadores de virulência, razões pelas quais este estudo foi desenvolvido com o objetivo de avaliar a distribuição dos tipos genéticos de intimina e dos genes hlyA, iha e toxB, que codificam adesinas e toxina bacterianas. Foram estudadas 561 amostras de AEEC obtidas de 110 crianças com até 69 meses de idade, com diarreia aguda e sem diarreia, atendidos, entre 2004 e 2007, no Hospital Infantil João Paulo II/FEMIG, Belo Horizonte, MG. Intimina beta foi o tipo mais prevalente, detectada principalmente em EPEC típica e atípica. Intimina gama foi a mais comum em EHEC. Intimina kapa não foi detectada na população estudada. Foi observada variação temporal na distribuição dos tipos de intimina, com declínio de beta e emergência de épsilon ao longo do período de estudo. A detecção da intimina iota concentrou-se em crianças com idade entre 13 a 24 meses. Aproximadamente 10% das amostras de AEEC no nosso meio não tiveram sua intimina identificada. Nenhum tipo genético de intimina estava associado com sexo, estação do ano, e presença de diarreia. Os marcadores de virulência hlyA, iha e toxB foram detectados em menos da metade das amostras de AEEC, sendo carreados por todos os tipos patogênicos incluídos neste grupo de DEC. Nossos resultados confirmam a existência de diferenças geográficas na distribuição dos marcadores de virulência de AEEC e a grande diversidade genética deste grupo de DEC, especialmente aEPEC e podem contribuir para o delineamento de estratégias de prevenção e controle da diarreia associada a AEEC.

Thesis (M.S. in Modeling, Virtual Environments and Simulation (MOVES))--Naval Postgraduate School, March 2000.<br>Thesis advisor(s): Zyda, Michael ; Bachmann, Eric. "March 2000." Includes bibliographical references (p. 77). Also available in print.

Au cours de ces 10 dernieres annees, de nombreux genes homologues aux molecules de classes i du complexe majeur d'histocompatibilite (cmh) ont ete mis en evidence. Ils exhibent, pour la plupart, une expression tissulaire restreinte et ne semblent pas avoir pour fonction la presentation peptidique aux cellules t. La famille des genes mic (mhc class i chain-related genes) a recemment ete caracterisee au sein meme de la region de classe i du cmh. Elle se compose de 2 genes fonctionnels (mica et micb) ainsi que de 5 pseudogenes (micc-g). Les travaux presentes dans cette these decrivent le polymorphisme inattendu des genes mica et micb, se rapprochant a terme de celui des genes classiques hla-a, -b et -c, les loci les plus polymorphes du genome humain. Nos investigations ont egalement permis de mettre en evidence un fort desequilibre de liaison entre mica et le gene hla le plus proche, hla-b. De part l'interaction de mica et micb avec certains recepteurs des cellules t et nk, l'evaluation de l'implication du gene mica dans 3 pathologies a caractere autoimmun a ete effectuee. Au vu des resultats obtenus, mica ne semble pas etre implique dans l'etiopathogenese de la sclerose en plaques et dans celle de la maladie de coeliaque, toutefois son role dans la physiopathologie de la maladie de behcet merite approfondissement sur le plan fonctionnel. Enfin, l'analyse phenotypique et genotypique des individus naturellement deficients en mica/b a permis de montrer qu'il n'y a aucune alteration du nombre des lymphocytes b, t et nk en comparaison avec les individus non-deletes ; l'analyse des pseudogenes mic n'indique aucune mutation gain de fonction. L'existence meme de ces individus devrait permettre de dissequer la fonction de ces molecules in vivo.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

Orientador: Erick da Cruz Castelli<br>Resumo: O complexo gênico de Antígenos Leucocitário Humanos (HLA) é a região mais variável do genoma humano. Os genes HLA de classe I são divididos em clássicos e não clássicos, a depender de suas funções primárias, níveis de expressão e polimorfismos. Assim, HLA-A, HLA-B e HLA-C são loci clássicos e estão associados à apresentação antigênica para células T citotóxicas, enquanto HLA-E, HLA-G e HLA-F são loci não clássicos e estão associados à imunomodulação e inibição de células T e NK. No entanto, o HLA-C também apresenta propriedades imunomodulatórias a partir da interação com o TCR de células T CD8 (ativação) e com receptores KIR de células NK (principalmente inibição). A maioria dos estudos sobre variabilidade dos genes HLA têm como foco suas regiões codificadoras e negligenciam regiões regulatórias – região promotora e região 3’não-traduzida (3’NT). Esta última apresenta um papel essencial na estabilidade do mRNA e no controle pós-transcricional mediado pela ligação de microRNAs (miRNA). Neste trabalho, nós avaliamos a estrutura e a variabilidade das regiões 3’NTs dos genes HLA-A, HLA-C e HLA-G por sequenciamento de nova geração além do padrão de ligação de miRNAs observado para cada gene. A região 3’NT dos genes HLA-A e HLA-C são altamente variáveis, enquanto a de HLA-G apresenta poucos pontos de variação. No entanto, os três genes apresentaram números similares de haplótipos de 3’NT frequentes. Vários miRNAs possuem potencial de regular HLA-A, HLA-C e HLA-G tanto de modo espec... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The Human Leucocyte Antigen (HLA) gene complex is the most variable region of the human genome. The class I HLA genes are classified as classical or nonclassical depending on their primary function, expression levels and polymorphism rate. Thus, HLA-A, HLA-B, and HLA-C are classical loci, associated with antigen presentation to cytotoxic T cells, while HLA-E, HLA-G, and HLA-F are non-classical loci associated with immunomodulation inhibition of both T and NK cells. Nevertheless, HLA-C also presents immumodulatory features by interacting with TCR on CD8 T (activation) cells as well as KIR receptors on NK cells (mostly inhibition). Most studies on HLA variability focus on their coding sequences and neglects their regulatory sequences – promoter and 3’untranslated region (3’UTR). The last one plays a pivotal role on mRNA stability and post-transcriptional control by microRNA (miRNA) binding. Here we evaluated 3’UTR structure and variability for HLA-A, HLA-C, and HLA-G by using massively parallel sequencing in a Brazilian population sample. We also report the miRNA binding pattern observed for each locus. Both HLA-A and HLAC 3’UTR segments are highly variable, while HLA-G 3’UTR presents few variation sites. However, the three loci present a similar number of frequent 3’UTR haplotypes. Several miRNAs are potential regulators of HLA-A, HLA-C, and HLA-G on a specific manner as well as the three of them together. This fact may be associated with the fine regulation of HLA expression ... (Complete abstract click electronic access below)<br>Doutor

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.<br>Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2016-02-12T13:36:10Z No. of bitstreams: 1 2015_NicoleSelleski.pdf: 1794617 bytes, checksum: ea6f372597cf25bb16cfdc52ec8a1d29 (MD5)<br>Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2016-02-15T11:43:09Z (GMT) No. of bitstreams: 1 2015_NicoleSelleski.pdf: 1794617 bytes, checksum: ea6f372597cf25bb16cfdc52ec8a1d29 (MD5)<br>Made available in DSpace on 2016-02-15T11:43:09Z (GMT). No. of bitstreams: 1 2015_NicoleSelleski.pdf: 1794617 bytes, checksum: ea6f372597cf25bb16cfdc52ec8a1d29 (MD5)<br>A doença celíaca é uma doença autoimune, de natureza inflamatória, caracterizada por uma intolerância permanente ao glúten em indivíduos geneticamente predispostos. Quase todos os pacientes celíacos possuem genes que codificam os heterodímeros HLA-DQ2.5, DQ8 ou DQ2.2, os quais participam na apresentação de peptídeos específicos derivados do glúten às células T CD4+. O objetivo desse trabalho foi determinar a prevalência das variantes predisponentes para DC em grupos de crianças celíacas (n=100) e não celíacas (n=110) da cidade de Brasília, Brasil. No grupo de crianças celíacas, 51% (n=51) apresentaram unicamente a variante DQ2.5, 22% (n=22) DQ2.5 e DQ2.2, 5% (n=5) somente DQ2.2, 7% (n=7) DQ2.5 e DQ8, 6% (n=6) DQ2.2 e DQ8, 6% (n=6) foram positivas unicamente para DQ8, 1% (n=1) somente para o alelo DQB1*02, 1% (n=1) apresentaram alelos de baixo risco e 1% (n=1) foram negativas para todos os alelos testados. O padrão evidenciado em crianças não celíacas foi: 14,5% (n=16) foram positivas unicamente para a variante DQ2.5, 3,6% (n=4) para DQ2.5 e DQ2.2, 11,8% (n=13) somente para DQ2.2, 0,9% (n=1) para DQ2.5 e DQ8, 0,9% (n=1) para DQ2.2 e DQ8, 13,6% (n=15) unicamente para DQ8, 1,8% (n=2) somente apresentaram o alelo DQB1*02, 26,4% (n=29) foram positivas para alelos de baixo risco e 26,4% (n=29) não apresentou nenhum dos alelos analisados. A prevalência de genótipos predisponentes para doença celíaca encontrada na população de crianças celíacas (DQ2: 80% e DQ8: 19%) foi similar à descrita para populações não europeias. Por outro lado, a proporção total desses alelos achados no grupo de crianças não celíacas (33,6%) mostrou-se semelhante à obtida na população geral europeia. Finalmente, os genes que codificam o heterodímero HLA-DQ2.2 não apresentaram diferenças estatisticamente significativas entre os grupos, o que nos permitiu concluir que a inclusão da variante HLA-DQ2.2 como genótipo predisponente para DC não seria relevante para a população analisada. ______________________________________________________________________________ ABSTRACT<br>Celiac disease is an autoimunne enteropathy triggered by the ingestion of glúten in genetically susceptible individuals. Almost all celiac patients possess immune recognition genes encoding either HLA-DQ2.5, DQ8, or DQ2.2 heterodimers which facilitate CD4+ T-cell recognition of specific gluten-derived peptides. The aim of our study was to determine the prevalence of these predisposing variants in a group of celiac (n=100) and non-celiac (n=110) children from Brasilia, Brazil. Among celiac children, 51% (n=51) were DQ2.5 positives, 22% (n=22) DQ2.5/DQ2.2, 5% (n=5) DQ2.2, 7% (n=7) DQ2.5/DQ8, 6% (n=6) DQ2.2/DQ8 and 6% (n=6) DQ8 positives, 1% (n=1) were positives for DQB1*02, 1% (n=1) were only positives for low risk alleles and 1% (n=1) were negative for all the tested alleles. The HLA-DQ pattern in non-celiac children was: 14.5% (n=16) DQ2.5, 3.6% (n=4) DQ2.5/DQ2.2, 11.8% (n=13) DQ2.2, 0.9% (n=1) DQ2.5/DQ8, 0.9% (n=1) DQ2.2/DQ8 and 13.6% (n=15) DQ8 positives, 1.8% (n=2) were only positives for DQB1*02, 26.4% (n=29) presented low risk alleles and 26.4% (n=29) were negative for all the tested alleles. The prevalence of celiac disease predisposing alleles (DQ2: 80% and DQ8: 19%) was similar to that of non-European populations. However, the total number of alleles found in non celiac children (33.6%) resembles the European parameters for general population. Finally, the prevalence of genes coding for HLA-DQ2.2 heterodimer was not significantly different between celiac and non-celiac children, thus, suggesting that this variant would not be relevant in the studied population.

Submitted by Gislaine Aparecida Querino (gislainequerino@hotmail.com) on 2018-09-28T01:32:53Z No. of bitstreams: 1 Querino,GA.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5)<br>Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-09-28T12:55:41Z (GMT) No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5)<br>Made available in DSpace on 2018-09-28T12:55:41Z (GMT). No. of bitstreams: 1 querino_ga_dr_bot.pdf: 869186 bytes, checksum: 86a067f5db0e67b546c8ad7691a3d530 (MD5) Previous issue date: 2018-07-25<br>A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae que pode se manifestar sob diferentes formas clínicas a depender da resposta imune celular do hospedeiro. Vários marcadores genéticos têm sido definidos e, devido à participação dos alelos HLA na resposta imune, estes têm sido amplamente estudados na hanseníase. O HLA-DP constitui uma das moléculas clássicas de classe II, com poucos estudos em hanseníase. O objetivo deste trabalho foi conduzir um estudo de associação genética de base populacional em hanseníase para os genes HLA-DPA1 e HLA-DPB1 com duas amostras caso-controle: a primeira de Rondonópolis-MT, uma região hiperendêmica para hanseníase, e a segunda do Estado de São Paulo, uma região com índices epidemiológicos controlados. Foram realizados estudos com 9 tag SNPs na região dos genes HLA-DPA1 e HLA-DPB1 na população de Rondonópolis–MT (411 casos e 357 controles). O SNP rs9277341 que apresentou dados de associação com significância estatística após a correção de Bonferroni foi então testado na população de São Paulo (570 casos e 380 controles). Para avaliar o efeito funcional deste marcador foi determinada estudoa produção das citocinas IL-10 e TNF após estímulo com antígeno sonicado de M. leprae em 21 controles saudáveis. Na população de Rondonópolis-MT foi realizada a tipificação dos alelos HLA-DPA1* e HLA-DPB1* através da técnica de PCR-SSO empregando-se o kit Labtype-SSO (One Lambda, CA, USA). Os resultados de genotipagem mostraram associação com hanseníase per se para o marcador rs9377341 do gene HLA-DPA1 e dois marcadores do gene HLA-DPB1 (rs1431402 e rs9277469). Associações de proteção para a forma multibacilar da hanseníase foram encontradas para os marcadores rs9277341 e rs2301220 do gene HLA-DPA1 e para o marcador rs31350221 do gene HLA-DPB1. O SNP rs9277341, com dados significativos após a correção de Bonferroni e testado na população do Estado de São Paulo, não apresentou associação com hanseníase. A avaliação funcional mostrou que os carreadores do alelo G do rs9277341 apresentaram níveis maiores de IL-10 e TNF. Na tipificação dos alelos HLA, o alelo HLA-DPB1*03 foi associado à hanseníase per se e o genótipo HLA-DPB1*02/03 foi associado à hanseníase per se e à proteção para a forma MB. Esses dados sugerem a participação dos genes HLA-DPA1 e HLA-DPB1 nos desfechos da hanseníase.<br>Leprosy, a chronic infectious disease caused by Mycobacterium leprae, displays different clinical manifestations depending on the immune response of the host. Several genetic markers have been defined and due to the involvement of the HLA alleles in the immune response they were extensively studied in leprosy. However, HLA-DP is a classical class II molecule with few studies on leprosy. The objective of this study was to conduct a population-based genetic association study in leprosy for the HLA-DPA1 and HLA-DPB1 genes with two case-control samples: the first from Rondonópolis - Mato Grosso (MT), a hyperendemic region in leprosy and the second from the state of São Paulo, a region with controlled epidemiological indices. Studies were carried out with 9 Tag Single Nucleotide Polymorphism (SNP) in HLA-DPA and HLA-DPB genes in the population from Rondonópolis – MT (411 cases and 357 controls). The SNP rs9277341 who presented association data with statist significance after the Bonferroni correction was tested in the São Paulo population (570 cases and 380 controls). To evaluate the functional effect of this marker was carried a study of the production of IL-10 and TNF cytokines after stimulation with the sonicated antigen of M. leprae in 21 healthy controls. In the Rondonópolis populations was performed the HLA-DPA1* and HLA-DPB1* allele typing by Sequence-Specific Oligonucleotide – Polymerase Chain Reaction (SSO- PCR) using the Labtype-SSO kit (One Lambda, CA, USA). The genotyping results showed association with leprosy per se for the SNP rs9277341 in the HLA-DPA1 gene and two markers in the HLA-DPB1 gene (rs1431402 and rs9277469).The protections associations for the multibacilar form the leprosy was found for the markers rs9277341 and rs2301220 in the HLA-DPA1 gene and the marker rs 31350221 in the HLA-DPB1 gene. The rs9277341 with significatif datas after the Bonferroni correction and tested in the São Paulo state showed no association with leprosy. The functional study showed significantly increased IL-10 and TNF levels in G carriers. HLA typing analysis the HLA-DPB1*03 allele was associated with leprosy per se and the HLA-DPB1*02/03 genotype was associated with leprosy per se and the protection with MB form. These data suggest an association of HLA-DPA1 and HLA-DPB1 genes with leprosy.

<p>This Master Thesis report explains the project of trying to connect the humancognition architecture Soar (an AI architecture) to the simulation architecture HLA (High Level Architecture). Descriptions of both Soar and HLA are presented.</p><p>A connection between the two, whose main objective is the transfer of relevant data between Soar and HLA, is not a completely straightforward task. The task involves many different aspects of the HLA architecture, including threading in HLA interfaces, dealing with the HLA data representation, time management, transfer of ownership of HLA objects, HLA interactions, etc.</p><p>Analysis of the different aspects and their effects on a Soar component has been conducted and is presented. Different possible approaches to solutions to problems and their respective advantages are briefly described.</p><p>An implementation of a system including the most vital functions has been created. The system itself, some problems that came up during the implementation of the system and the actions taken to circumvent them, are described together with satisfactory results.</p><p>The paper ends with a discussion of different problems that includes some ideas of how to move forward.</p><p>The report concludes that creating a system that can deal with most of the aspects (inherent in the HLA architecture) in a general manner is probably too complex. With a more specified behaviour that a user will have to be confined to, a reliable system could be created. The report also concludes that in order to create a reusable HLA-connected system that not only works with Soar but with other AI architectures as well, the design of the system will probably have to be created with reusability plans in mind from the very beginning of the design process.</p>

This report embarks on an investigation of the ethnic diversity, using alloantisera, of three groups of HLA class I antigens, namely A2, A11, and C-locus antigens, and to define antigens that might be restricted to certain ethnic populations. As transplantation therapy has been widely accepted as the choice of treatment for patients with severe haematological disorders, such as acute leukemia, it is essential to match MHC antigens of unrelated donor and recipient pairs to avoid graft rejection or graft-versus-host disease and to reduce administration of immune suppressants. Results obtained from this study indicate that HLA-A2 consists of at least four variants as measured by isoelectric focusing assay. However, only three serological variants are demonstrated by alloantisera. Results further confirmed that two variants of HLA-A11 exist, as measured by serology and isoelectric focusing. The more frequent variant, A11.1, is found in donors of all ethnic backgrounds. The less frequent variant, A11.2, is found only in Oriental populations. This study also reveals two "new" serologically defined HLA-C locus antigens: HLA-Cw Hunt and HLA-Cw Grace. These two "new" antigens are found in Black, Caucasian, East Indian and Oriental ethnic groups. Identification of these two new C-locus antigens reveals that approximately 11% of the population was falsely typed and possess only one or no C-locus antigen. This study also identifies for the first time an alloantiserum that defines an ethnic-specific human minor histocompatibility antigen, hmH$\Phi,$ found only in the Oriental population with a frequency of approximately 5.5%. It was not found in 1,000 Caucasian, 200 East Indian, and 60 Black blood donors. (Abstract shortened by UMI.)

Cette étude a été concentrée sur L'influence de polymorphismes de gène de Non-HLA (Cytokines) et non classiques HLA (HLA-E) sur les résultats de transplantation de moelle. 91 patients indiens avec la bêta-thalassémie major et leurs donateurs ont été analysés. Des études de polymorphisme de gène ont été réalisées sur des groupes d'ADN de 91 patient et de leurs donateurs respectifs. Génotypes de distributeur/réceptifs pour le TNF-α308, IL-6-174, IL-10-1082-, -819,-592, IFN-γ-874 and TGF-β1+869, +-915 polymorphismes ont été analysés par PCR-SSP. HLA-E genoryping a été exécuté par SSP. Association significative d'exposition d'analyse entre le patient avec le bas phénotype de IL-6 et l'incidence de GVHD aigu, alors qu'il n'y avait aucune association significative entre le reste de polymorpbisnies de cytokines et l'incidence de GVHD aigu ou chronique. L'analyse n'a pas indiqué n'importe quelle association significative entre les allèles de HLA-E ou les génotypes et les événements d'infection. Cependant, nous avons trouvé une tendance vers l'association entre le homozygosity de HLA-E*0101/E*0101, dans le donateur et le destinataire, et l'incidence de la première infection aussi bien que la maladie de cytomégalovirus (CMV). Nous avons conclu de ce travail que le polymorphisme de gène de IL-6-174C/C a un rôle essentiel sur l'incidence de GVHD aigu et il a double rôle comme pro-inflammatoire et anti-inflammatoire, également nous pouvons présumer l'effet de l'état homozygous de l'allèle HLA-E*0101 comme facteur de risque pour infections graves<br>This study was focused on the non MHC and non classical MHC genetic polymorphisms and their influence on the BMT outcomes. 91 Indian patients with beta-thalassemia major and their donors were analyzed. Gene polymorphism studies were performed on DNA samples of 91 recipients and their respective donors. Donor/recipient genotypes for the TNF-α308, IL-6-174, IL-10-1082-, -819,-592, IFN-γ-874 and TGF-β1+869, +-915 polymorphisms were analyzed by polymerase chain reaction with sequence specific primers (PCR-SSP). HLA-E genotyping was performed by using sequence-specific primer (SSP) strategy. Analysis show significant association between recipient with Low IL-6 phenotype and the incidence of acute GVHD, while there was no significant association between the rest of cytokines polymorphisms and the incidence of either acute or chronic GVHD. Analysis failed to reveal any significant association between HLA-E alleles or genotypes and infection events. However, we found a trend toward association between HLA-E*0101/E*0101 homozygosity, both in donor and recipient, and overall incidence of first infection as well as cytomegalovirus (CMV) disease. We concluded from this work the vital role of rL-6~174C/C gene polymorphism in the incidence of acute GVHD and its double role as pro-inflammatory and anti-inflammatory agent, also we can hypothesize the effect of the homozygous state HLA-E*0101 allele as a risk factor for early severe infections

Les molecules du complexe majeur d'histocompatibilite humain (hla) ont pour fonction de presenter des peptides aux lymphocytes t charges de la surveillance immunitaire. Certaines molecules hla predisposent au developpement de pathologies auto-immunes, en particulier les molecules hla-a29 et hla-b27. Nous avons etudie leurs caracteristiques de presentation peptidique grace a une approche biochimique d'elution de peptides. Les molecules hla-a29 conferent une susceptibilite aux retinopathies de type birdshot et les antigenes retiniens ag-s et irbp dont de potentiels autoantigenes. Pour determiner l'existence au sein de l'ag-s de peptides susceptibles d'etre presentes par la molecule hla-a29, nous avons tout d'abord caracterise le motif d'ancrage peptidique a la molecule : glu en position 2 et tyr, leu en position c-terminale. En integrant ces donnees, nous avons trouve 2 peptides capables de se lier a a29 parmi les peptides candidats issus de l'ag-s. Les 11 variants hla-b27 decrits ne predisposent pas de facon equivalente aux spondylarthropathies (sa). Comme ils different par un nombre restreint de substitutions, nous avons compare leurs caracteristiques de liaison. Nous confirmons le role des positions 77 et 116 de la poche f dans le choix du residu c-terminal du peptide et montrons l'absence de correlation entre le motif c-terminal d'ancrage aux molecules hla-b27 et la susceptibilite aux sa. La presence d'un peptide singulier arthritogenique a ete recherchee par analyse comparative des peptides presentes par un allele de forte susceptibilite, b*2705 et un allele decrit initialement comme moins associe, b*2703. Nous confirmons que la molecule b*2703 presente un sous-ensemble des peptides presentes par b*2705 et montrons que b*2705 presente un peptide de 11 acides amines qui n'est ni naturellement presente, ni fixe par b*2703. Ce peptide est interessant pour les homologies decrites avec des proteines bacteriennes. Certaines infections bacteriennes favoriseraient, en effet, le declenchement des sa chez les individus susceptibles. C'est pourquoi, nous avons analyse l'influence de l'infection par une bacterie candidate, shigella flexneri, sur la presentation antigenique par hla-b27. Nos resultats suggerent que l'infection entraine une modification de la nature des peptides presentes par b27.

Orientador: Manoel Barros Bertolo<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-26T03:37:39Z (GMT). No. of bitstreams: 1 Pires_CatarinaFernandes_D.pdf: 2601322 bytes, checksum: 1acb7f604b763cb1fd45c71455e387bf (MD5) Previous issue date: 2014<br>Resumo: O presente estudo de caso-controle avaliou 74 crianças piauienses com Artrite Idiopática Juvenil (AIJ) em suas diversas formas: Oligoarticular, Sistêmica, Poliarticular Fator Reumatoide Positivo (FR+), Poliarticular Fator Reumatoide Negativo (FR-), Artrite relacionada a Entesite (ERA), Artrite Psoriásica e Artrite indeterminada, classificadas de acordo com os Critérios da Liga Internacional de Associações de Reumatologia (ILAR) e cento e um controles saudáveis pareados de acordo com idade, sexo, procedência e etnia. Os objetivos foram identificar e determinar os alelos HLA-DR e HLA-DQ em uma amostra da população infanto-juvenil piauiense com AIJ nas formas oligoarticular, sistêmica, poliarticular FR+, poliarticular FR-, artrite psoriásica, artrite relacionada a entesite e artrite indeterminada e compará-las com as frequências observadas no grupo de controle saudáveis; conhecer os alelos HLA-DR e HLA-DQ que estão associados a maior susceptibilidade e os que conferem maior proteção na população de crianças com AIJ em suas diversas formas. As tipagens dos alelos HLA-DR e HLA-DQ foram realizadas por meio da técnica de amplificação pela Reação de Cadeia da Polimerase (PCR), utilizando cadeias específicas de primers DR e DQ. O resultado da análise demostrou que, entre todas as formas de apresentação da AIJ, houve associação estatística significativa para a susceptibilidade da doença com o HLA-DRB1*10 OR 8,8 (IC 1,1 a 74,9) e HLA-DRB1*16 OR 2,8 (IC 1,1 a 7.5). Na forma oligoarticular a associação estatística significativa para a susceptibilidade ocorreu com o alelo HLA-DRB1*08 OR 4,6 (IC 1,4 a 14,6). Na forma sistêmica, essa associação aconteceu com o alelo HLA-DRB1*10 OR 22,2 (IC 1,8 a 269,5).Na forma Poliarticular FR+ a associação estatística significativa para a susceptibilidade ocorreu com os alelos DRB1*09 OR 12,1 (IC 2,2 a 66,9) e DRB1*10 OR 28,5 (IC 2,3 a 355,0). Na forma Poliarticular FR-, a associação estatística significativa para a susceptibilidade foi com o alelo DRB1*16 OR 13,4 (IC 1,6 a 111,2). A Artrite Psoriásica não apresentou nenhuma associação estatística significativa com os HLA pesquisados. O resultado da análise demostrou que, entre todas as formas de apresentação da AIJ, houve associação estatística significativa para a proteção da doença com o HLA-DRB1*03 OR 0,7 (IC 0,5 a 0,9) e, na forma sistêmica, essa associação aconteceu igualmente com DRB1*03 OR 0,7 (IC 0,6 a 0,8). As outras formas de apresentação da doença não demonstraram associação estatística significativa para a proteção com nenhum tipo da HLA pesquisado. A população estudada não apresentou nenhum caso de ERA e de Artrite indeterminada. O estudo encontrou ainda, associação para o risco entre o HLA-DQB1*03 OR = 6,06 (IC 1,3 a 27,2) e uveíte em pacientes com a forma oligoarticular da AIJ. Desta forma, concluímos que os nossos resultados apresentam tanto semelhanças em relação a susceptibilidade, quanto diferenças, principalmente quanto a proteção, nas associações tipicamente encontradas na literatura<br>Abstract: This case-controle valuated 74 children from Piauí with Juvenile Idiopathic Arthritis (AIJ) in its various forms: Oligoarticular, Systemic, Polyarticular Rheumatoid Factor Positive (FR +), Polyarticular Rheumatoid Factor Negative (FR-), Enthesitis-related Arthritis (ERA), Arthritis Psoriatic and Arthritis indeterminate, classified according to the Criteria of the International League of Associations for Rheumatology(ILAR), and one hundred and one healthy control smatched according to age ,sex, origin and ethnicity. The objectives were to identify and determine the HLA-DR and HLA-DQ in a sample of Piauí juvenile population subtypes JIA in oligoarticular, systemic, polyarticular RF + polyarticular RF -, psoriatic arthritis, enthesitis-related arthritis and arthritis indeterminate and compares them with the observed frequencies in the group of healthy control; know the HLA-DR and HLA-DQ alleles are associated with increased susceptibility and conferring greater protection in the population with JIA in its various forms and identify a possible relationship between the alleles HLA-DR and HLA-DQ and the most frequent and most aggressive form of the disease in the population studied. Polymerase Chain Reaction (PCR) using primers specific chains of DR and DQ performed the typing of HLA-DR and HLA-DQ using the technique of amplification. The result of the analysis demonstrate damong all cases of JIA was statistically significant association for disease susceptibility with HLA-DRB1*10 OR 8.8 (CI 1.1 to 74.9) and HLA-DRB1*16 OR 2.8 (CI 1.1 to 7.5). In oligoarticular JIA significant statistical association to susceptibility occurred with HLA-DRB1*08 allele OR 4.6 (CI 1.4 to 14.6), association systemic form happened to HLA-DRB1*10 OR 22.2 (CI 1.8 to 269.5) allele in polyarticular RF + the statistically significant association to susceptibility occurred with the DRB1*09 alleles OR 12.1(CI 2.2 to 66.9) and DRB1*10 OR 28.5(CI 2.3 to 355.0) in polyarticular RF- significant statistical association to susceptibility was with DRB1*16 allele OR 13.4 (CI 1.6 to 111.2). The Psoriatic Arthritis showed no statistically significant association with HLA surveyed. The result of the analysis demonstrate damong all cases of JIA was statistically significant association for disease protection with HLA-DRB1*03 OR 0.7(CI 0.5 to 0.9) and the systemic form this association also happened DRB1*03 OR with 0.7 (CI 0.6 to 0.8). The other forms of the disease showed no statistically significant association for protection on any type of HLA searched. The study population did not show any cases of ERA and indeterminate Arthritis. The study also found, statistically significant difference between the HLA-DQB1*03 OR 6.0 (CI 1.3 to 27.2), and uveitis in patients with oligoarticular form of JIA. Thus, we conclude that our results show both similarities in terms of susceptibility, and differences, especially regarding the protection, associations typically found in the literature<br>Doutorado<br>Medicina Interna<br>Doutora em Ciências Médicas

Le developpement d'un modele de souris transgeniques pour des molecules d'histocompatibilite de classe i humaines dans le but d'etudier les reponses cytotoxiques chez l'homme se heurte au probleme de la cooperation heterospecifique entre molecules d'histocompatibilite humaines (hla) et complexe de reconnaissance t murin (recepteur t et molecules accessoires). Le repertoire t cytotoxique de ces animaux reste majoritairement restreint par les molecules d'histocompatibilite de souris et l'utilisation des molecules hla de classe i demeure marginale

Made available in DSpace on 2016-08-29T15:34:36Z (GMT). No. of bitstreams: 1 tese_9536_Dissertac&#807;a&#771;o completa versa&#771;o final.pdf: 3098397 bytes, checksum: bed15892e2bc7fb6e3d38393c123bc36 (MD5) Previous issue date: 2015-10-27<br>A Síndrome de Parry-Romberg (SPR) ou Atrofia hemifacial progressiva (HFA) é uma doença rara (1:250.000 a 1:700.000), de causa desconhecida, caracterizada por atrofia unilateral da face, acometendo pele, tecidos moles, músculos e tecidos ósseos subjacentes de forma lenta, progressiva e autolimitada. A patogênese da SPR é heterogênea, parece ser sobreposta à da esclerodermia linear, mas ainda não é totalmente compreendida. Infecções virais, traumas, atividade eurológica e autoimunidade têm sido propostos para explicar a etiologia da SPR. Tendo por base o mecanismo inflamatório e autoimune proposto como uma das causas para SPR e do gene HLA-G estar envolvido em algumas doenças inflamatórias e autoimunes, o objetivo deste trabalho é verificar se os níveis de expressão das isoformas HLA-G e HLA-G5 diferem entre pacientes com SPR e controles. Células tronco mesenquimais derivadas de tecido adiposo (ASCs) foram isoladas de gordura obtida por lipoaspiração tanto dos pacientes (n=9), antes do uso para lipoenxertia autóloga com fins terapêuticos, quanto dos controles (n=9), nesse caso para fins estéticos. O RNA total foi isolado das ASCs e a técnica de PCR quantitativa em Tempo Real foi utilizada para avaliar a expressão das isoformas HLA-G (membranar) e HLA-G5 (solúvel) do gene HLA-G. A análise estatística foi feita através do teste não paramétrico de Mann-Withney. Foi observado que as ASCs dos pacientes com SPR apresentaram menor expressão relativa de HLA-G - isoforma de membrana (p = 0,000), mas não de HLA-G5 isoforma solúvel (p = 0,387). O HLA-G é uma molécula imunomoduladora associada à doenças inflamatórias e autoimunes, portanto esse trabalho mostra, pela primeira vez, que baixos níveis de expressão de HLA-G em pacientes com SPR podem estar relacionados à supressão da resposta imunológica, levando a uma autoagressão. Estudos adicionais verificando a reprodutibilidade desse resultado são necessários para melhor compreensão da importância do HLA-G e dos mecanismos moleculares envolvidos na etiologia da SPR, para melhorar estratégias de diagnóstico precoce e abrir perspectivas terapêuticas visando o aumento da atividade imunossupressora por meio do estímulo ao aumento da expressão de HLA-G nesses pacientes. Palavras-chave: Síndrome de Parry-Romberg. Atrofia hemifacial progressiva. HLA-G. HLA-G5.

A successful kidney transplant is the best treatment for established renal failure, yet around 300 patients per annum are denied transplants because they have antibodies, most notably directed against donor HLA or ABO in their blood, which have the potential to cause acute and chronic rejection of the transplant. Such antibodies are present in 25% (roughly 1750 of the 7000 on the kidney transplant waiting list) of the patients listed for a deceased donor transplant. Programmes to remove antibody and transplant patients across HLA antibody barriers have been developed, but are limited by a high rate of acute rejection. This thesis explores the factors which may impact upon the pathogenicity of HLA-specific antibodies and also aims to enhance the understanding of the techniques used in the laboratory to define these antibodies. A range of studies were carried out examining factors such as the IgG subclass composition of the anti-HLA response. Assay variations were designed to enable a higher definition of antibody specificity to be achieved, and for the first time in the literature the direct quantification of HLA-specific antibodies in patient sera was performed. In addition, proof of principle design and testing was carried out on a novel prototype therapeutic device for the selective depletion of HLA-specific antibodies directly from patient plasma and sera. The antibodies isolated from this approach were also used in studies to examine the factors which determine serum cytotoxicity.

Cette thèse s’inscrit dans le contexte de HLA, une architecture de simulation distribuée dont l'objectif est de permettre l'interopérabilité et la réutilisation d’applications de simulation. L'ONERA a développé une plate-forme de simulation distribuée compatible HLA (appelée CERTI). L'objectif de cette thèse est d'étendre le travail entrepris sur CERTI en se basant sur l'introduction de la notion classique de domaine. Nous étudions différentes techniques impliquant cette notion et dont les objectifs concernent l'interopérabilité, la sécurité, la réutilisation ainsi que l'amélioration des performances. Notre contribution tient d’une part dans la réalisation de simulateurs-ponts permettant l'interconnexion de plusieurs simulations et d’autre part dans le développement de services de CERTI destinés à optimiser les échanges de données entre domaines.

In dieser Arbeit wurde die Überlebenszeit von Nierentransplantaten untersucht und deren Abhängigkeit von zwei Faktoren: der HLA-Kompatibilität und der Antikörperdynamik. Hierzu konnten Daten von 327 Patienten gesammelt werden, die zwischen 1991 und 1996 eine postmortale Spenderniere erhielten. Eine konventionelle Gewebetypisierung erfolgte mittels serologischer und molekularbiologischer Untersuchungen. Eine neue Matchingmethode wurde durchgeführt auf Ebene von Aminosäuren. Ein Antikörperscreening erfolgte vor und nach Transplantation mittels Lymphozytotoxtest und ELISA. Zur statistischen Bewertung benutzten wir die Kaplan-Meier-Methode zur Berechnung der Überlebenszeit und eine Cox Regression zur Berechnung des relativen Risikos. Bezüglich der Gewebeübereinstimmung konnten wir beim konventionellen Matching eine Tendenz feststellen, daß Patienten mit einer guten Übereinstimmung eine längere Transplantatüberlebenszeit zeigten, als Patienten mit einer schlechten Übereinstimmung. Beim Matching auf Aminosäureebene konnten keine Unterschiede in der Transplantatfunktion nachgewiesen werden. Bei Betrachtung des Antikörperverhaltens der Empfänger konnten wir signifikante Unterschiede nachweisen dahingehend, daß Nierentransplantierte mit einer Antikörperbildung eine schlechtere Transplantatüberlebenszeit besaßen als Patienten ohne Antikörpernachweis. Außerdem konnte gezeigt werden, daß Patienten mit vielen Transfusionen vor Transplantation eine signifikant kürzere Transplantatüberlebenszeit zeigten, als Patienten mit wenigen Transfusionen. Anhand unserer Ergebnisse empfehlen wir ein konventionelles Matching als Grundlage der Nierentransplantation. Ein Matching auf Ebene von Aminosäuren könnte zukünftig das konventionelle Match ergänzen oder ablösen. Außerdem empfehlen wir ein generelles Antikörperscreening der Empfänger vor und nach Transplantation, da Aussagen möglich werden zum Verlauf nach Transplantation und die immunsuppressive Therapie angepaßt werden kann.<br>In this study we examined the survival of kidney transplants and the influence of two factors: the hla-compatibility and the dynamics of antibodies. For this we collected full data of 327 Patients, who were transplanted with a postmortal kidney transplant between the years 1991-1996. A conventional tissue typing was done with serological and molecular biological tests. A new matching method was done at the level of amino acids. A screening for antibodies was done before and after transplantation using lymphocytotoxtest and ELISA. For statistical valuation we used the Kaplan-Meier-method for the calculation of the transplant survival time and a cox regression for the calculation of the relative risk. Regarding the tissue similarities at the conventional match we saw the trend of a longer transplant survival time at patients with a good match compared to patients with more missmatches. At matching at amino acid-level we couldn´t show any differences in the transplant survival time. By observing the dynamics of antibodies of the receiver we could show a significant difference: kidney transplant receivers developing antibodies show a shorter transplant survival time than patients, who didn´t develop antibodies. Additionally we could show that patients with many transfusions before transplantation have a significantly worser transplant function than patients with less transfusions. Resulting from our examinations we recommend a conventional matching as a basic for kidney transplantation. In future a matching at amino acid-level could supplement or replace the conventional match. Additionally we recommend an antibodyscreening of the transplant receivers before and after transplantation. A prediction for the posttransplant course will be possible and an individual adjustment of the immunsuppressive therapy.

Orientadores: Lilian Tereza Lavras Costallat, Fernando Ferreira Costa<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas<br>Made available in DSpace on 2018-07-21T16:00:30Z (GMT). No. of bitstreams: 1 Bertolo_ManoelBarros_D.pdf: 5744580 bytes, checksum: f7167e9a18551a891129b8899097c781 (MD5) Previous issue date: 1996<br>Resumo: A associação de antígenos de histocompatibilidade com a artrite reumatóide (AR) vem sendo demonstrada em inúmeros estudos. A principal associação em população caucasóide é com o HLA-DR4, contudo, também vem sendo observada com HLA-DRl e outros antígenos. Resultados de vários trabalhos sugerem que, o HLA-DR4 está mais associado com a gravidade do que com suscetibilidade. Com a introdução das técnicas de biologia molecular foi possível determinar que, os subtipos do HLA-DR4, relacionados com AR, são os alelos HLA-DRBl *0401, *0404 e *0405, que estão mais associados à gravidade da doença do que com a suscetibilidade. Em alguns estudos verificou-se, também, que os subtipos do HLA-DRl associados com a doença são os alelos HLA DRBl *0101 e *0102. Os propósitos deste estudo foram os de analisar a freqüência dos antígenos HLA-DR, identificar os alelos específicos do HLA-DRB 1, determinar sua freqüência, correlacionar estes alelos com as manifestações clínicas e laboratoriais e caracterizar aqueles que podem predizer o padrão evolutivo da AR em pacientes caucasóides. Foram avaliados 65 pacientes caucasóides, com AR, diagnosticados pelos critérios. da "American College of Rheumatism" (ACR). Todos foram avaliados clinicamente quanto ao envolvimento articular e extra-articular. O quadro funcional foi analisado usando-se a classificação funcional de Steinbrocker (CFS). Fator reumatóide (FR) e exame radiográfico foram realizados em todos os pacientes. Na determinação dos antígenos de classe I e TI utilizou-se a reação da microlinfocitotoxicidade. O DNA genômico dos pacientes positivos para HLA-DRl e DR4 foi amplificado pela reação da cadeia da polimerase (PCR), e a identificação dos alelos efetuada através da hibridização de sondas de oligonucleotídeos com seqüências específicas (SSO). Nesta casuística, o HLA mais freqüente foi o HLA-DRl (26,2%), quando comparado com o grupo controle (11,5%) (p<0,05). HLA-DR4 ocorreu em 24,6% dos pacientes e em 18% dos controles (p>0,05). Os pacientes com HLA-DRl não mostraram associação com nenhuma das variáveis clínicas e laboratoriais estudadas. Aqueles com DR4 positivo, quando comparados com DR4 negativo, apresentaram maiores títulos de FR, erosão óssea e um pior grau na CFS (p<0,05). Os alelos HLA-DRBI *0401 e *0404 apresentaram associação com um quadro funcional mais incapacitante, FR em altos títulos e erosão óssea, não se evidenciando predomínio significativo de um sobre o outro, com exceção do Fator Reumatóide, que estava em maiores títulos nos pacientes com o alelo *0404. Os alelos HLA-DRBI *0101 e *0102, embora mais freqüentes, não apresentaram associação com as variáveis clínicas e laboratoriais. Os pacientes com HLA-DRBI *0401, *0404 e *0101, que possuem seqüências semelhantes de aminoácidos na região 70-74 (QKRRA - QRRAA), mostraram maior freqüência de erosões. A partir dos dados obtidos, concluímos que os alelos HLA-DRBI *0101 e *0102 estavam relacionados com a suscetibilidade na AR, enquanto que os alelos HLA-DRB 1 *0401 e *0404 estavam associados com doença mais agressiva<br>Abstract: Several reports have shown that rheumatoid arthritis (RA) is associated with HLA-DR4, however association of RA and HLA-DRI has been found also in caucasian population. Subsets of HLA-DR4 were recognized using molecular biology technics. It has been mentioned that DR4 aneles (HLA-DRB 1 *0401, *0404, *0405) are more associated with severity than susceptibility to the disease. Our goals were to determine the HLA-DR frequency, to identify the specifics alleles of HLA-DRBI, to correlate these aneles with the clinical and laboratorial manifestations and to determine the aneles that can identify patients with more aggressive disease in caucasian with rheumatoid arthritis. Sixty five caucasian patients with RA (ACR's criteria) were analyzed with clinical, serological, radiographic data and functional status according to the Steinbrocker scale. To determine Class I and 11 antigens the microlinfocitotoxicity reaction was employed. Genomic DNA from HLA-DRI and DR4 positive patients was amplified by the polymerase chain reaction (PCR). Dot blotting and hybridization with sequence specific oligonucleotide (SSO) probes were performed. The phenotypic frequency of HLA-DRI in RA patients (26,2%) was significantly greater than controls (11,5%) (p<O,05). HLA-DR4 occurred in 24,6% of the patients and in the 18% of controls (p>0,05). The HLA-DRI positive patients did,'not show ditTerences when compared to HLA-DRI negative. Positive DR4 patients had significantly higher tittles of rheumatoid factor, bone erosions and worst functional status (p<0,05). The alleles HLA-DRB 1 *0401 e *0404 were associated with worst functional status, bone erosions and rheumatoid factor. HLA-DRBI *0101 and *0102 were the most frequent aneles, however not associated with clinical and laboratory characteristics. Those with HLA-DRBI *0101, *0401 e *0404 aneles, who presented de shared epitope (QKRAA - QRRAA) at position 70-74, showed erosion more frequently. We can conclude that the HLA-DRBI *0101 and *0102 aneles are associated with susceptibility to RA while *0401 and *0404 with more aggressive disease<br>Doutorado<br>Clinica Medica<br>Doutor em Medicina

La sequence qkraa de la troisieme region hypervariable de hla-drb1#*0401 porte la susceptibilite a developper une polyarthrite rhumatoide. Le role de la sequence qkraa n'est pas encore connu. Il implique sans doute une interaction avec un ou plusieurs ligands. Nous avons recherche si la sequence qkraa de la troisieme region hypervariable de hla-drb1#*0401 interagit avec des proteines susceptibles de jouer un role dans la polyarthrite rhumatoide. Nous avons montre que : 1- le peptide de troisieme region hypervariable de hla-drb1#*0401 contenant qkraa fixe specifiquement la proteine de choc thermique bacterienne, dnak. Le motif qkraa suffit a fixer dnak. 2- le motif qkraa est implique dans l'interaction de dnak avec la proteine de choc thermique, dnaj, qui porte aussi la sequence qkraa. 3- dans les lignees lymphoblastoides humaines, hla-drb1#*0401 interagit specifiquement avec la proteine de choc thermique constitutive hsp73. Hsp73 transporte directement hla-drb1#*0401 du reticulum endoplasmique vers les lysosomes.

This study proposes a metamodel, named Federation Architecture Metamodel (FAMM), for describing the architecture of a High Level Architecture (HLA) compliant federation. The metamodel provides a domain specific language and a formal representation for the federation adopting Domain Specific Metamodeling approach to HLA-compliant federations. The metamodel supports the definitions of transformations both as source and as target. Specifically, it supports federate base code generation from a described federate behavior, and it supports transformations from a simulation conceptual model. A salient feature of FAMM is the behavioral description of federates based on live sequence charts (LSCs). It is formulated in metaGME, the meta-metamodel for the Generic Modeling Environment (GME). This thesis discusses specifically the following points: the approach to building the metamodel, metamodel extension from Message Sequence Chart (MSC) to LSC, support for model-based code generation, and action model and domain-specific data model integration. Lastly, this thesis presents, through a series of modeling case studies, the Federation Architecture Modeling Environment (FAME), which is a domain-specific model-building environment provided by GME once FAMM is invoked as the base paradigm.

There has been a recent interest in the model-based development approach in the modeling and simulation community. The Model-Driven Architecture (MDA) of OMG envisions a fully model-based development process where models are created for capturing not only requirements, but also designs and implementations. Domain-specific metamodels and model transformations constitute the cornerstones of this approach. We have developed transformations from the data part of Field Artillery (FA) domain models to High Level Architecture (HLA) Object Model Template (OMT) models, honoring the MDA philosophy. In the MDA terminology, the former corresponds to the CIM (Computation-Independent Model) or, arguably, PIM (Platform-Independent Model), and the latter corresponds to the PSM (Platform-Specific Model), where the platform is HLA. As a case study for the source metamodel, we have developed a metamodel for the data model part of the (observed) fire techniques of the FA domain. All of the entities in the metamodel are derived from the NATO&rsquo<br>s Command and Control Information Exchange Data Model (C2IEDM) elements.

This thesis puts forth a process for ontology driven distributed simulation through a case study. Ontology is regarded as a domain model, including objects, attributes, methods and object relations. The case study involves trajectory simulation. A trajectory simulation is a piece of software that calculates the flight path and other parameters of a munition, such as its orientation and angular rates, from launch to impact. Formal specification of trajectory simulation domain is available as a domain model in the form of an ontology, called Trajectory Simulation ONTology (TSONT). Ontology driven federation development process proposed in this thesis is executed in three steps. The first step is to analyze the TSONT and to create instances of individuals guided by the requirements of the targeted simulation application, called Puma Trajectory Simulation. Puma is the simulation of a ficticious air-to-ground guided bomb. The second step is to create the High Level Architecture(HLA) Federation Object Model (FOM) using Puma Simulation individuals. FOM will include the required object and interaction definitions to enable information exchange among federation members, including the Puma federate and the Exercise Manager federate. Transformation from the ontology to FOM is realized in two ways: manually, and by using a tool called OWL2OMT. The third step is to implement the Trajectory Simulation federation based on the constructed FOM. Thus, the applicability of developing HLA federates and the federation under the guidance of ontology is demonstrated.

The High Level Architecture (HLA) is IEEE's 1516 standard [1] which defines a software architecture for the development and execution of Distributed Interactive Simulation (DIS). It also provides a general-purpose network communication mechanism for DIS through its implementation---Run-Time Infrastructure (RTI). However, HLA does not address two critical features: stream (audio/video) and real-time transmission. This thesis focuses on solving these two issues. The first part of this thesis reviews the basic concepts of DIS and its evolving history. Then it illustrates how the HLA supports the requirements of the DIS via its service groups, using a virtual shopping mall application as an example. The second part introduces an HLA-compliant solution for stream transmission and control. An audio playback application is illustrated to show how to transfer stream data, using HLA defined APIs and control transfer schedule, using the operating system interval timer. Then, a presentational audio-video continuous-media retrieval application is illustrated to show how to control stream retrieval In the third part, a real-time extension proposal to HLA and an architecture of real-time RTI is presented. The Internet is moving to Quality of Service (QoS) age, which will provide delay and jitter bounded services. With the IP QoS and real-time operating systems involved, it is shown that HLA can support real-time DIS by taking advantages of these new technologies. After analyzing the limitation of current HLA and RTI, a proposal of real-time extension to HLA and the architecture of real-time RTI is illustrated. In the fourth part, our experiences of building QoS networks and a performance analysis, including end-to-end RSVP for unicast and multicast, end-to-end DiffServ, RSVP over differentiated service network, etc., are presented. We hope that the thesis' contributions would create a discussion in the DIS community on the topic of Real-Time DIS (RT-DIS). With new real-time technologies in operating systems and networks blooming, RT-DIS has the possibility of coming true, while its final realization depends on the effort of the whole DIS community.

A endometriose é uma doença inflamatória crônica, estrógeno-dependente e de etiologia multifatorial, caracterizada pela implantação e crescimento de tecido endometrial fora da cavidade uterina e associada à dor pélvica e infertilidade. A doença é classificada de acordo com os estádios e sítios de acometimento nos órgãos pélvicos. Variantes genéticas, endócrinas e ambientais podem contribuir para a geração de uma deficiência na resposta imune local permitindo a implantação das células ectópicas na cavidade pélvica. Alterações constatadas no padrão de citocinas presentes no microambiente pélvico poderiam promover um ambiente imunossupressor, justificando a diminuição da resposta imune efetora verificada na endometriose. Dentre os possíveis fatores imunomoduladores, está o antígeno leucocitário humano-G (HLA-G), cuja expressão se dá intensamente nas células trofoblásticas, sendo reconhecido por induzir a tolerância materno-fetal. A proteína HLA-G pode ser expressa na membrana celular ou ser secretada na forma solúvel. HLA-G encontra receptores inibitórios nas células do sistema imune inato e adaptativo e tem sua expressão induzida sob condições não fisiológicas, como em transplantes alogênicos, doenças inflamatórias ou neoplasias malignas. Assim, a hipótese deste estudo é a de que a proteína HLA-G seria produzida em níveis superiores nas mulheres com endometriose, o que poderia contribuir para a imunossupressão no microambiente da doença. Para testar esta hipótese, a proteína solúvel foi mensurada no soro e no fluido peritoneal de mulheres com e sem endometriose, por ensaio de imunoabsorção enzimática (ELISA). Além disto, a expressão gênica de HLA-G foi avaliada nos tecidos de endométrio, por qRT-PCR, bem como a expressão da proteína, avaliada por imunohistoquímica, nos tecidos de endométrio e de lesão de endometriose, em mulheres com e sem a doença. Como resultados, verificaram-se maiores níveis da proteína solúvel no soro de mulheres que apresentavam endometriose em estádios avançados, especialmente naquelas com endometriose ovariana. Entretanto, na comparação entre os fluidos peritoneais, não houve diferença significativa entre os grupos com e sem endometriose. A expressão do transcrito gênico (mRNA) se mostrou maior no endométrio de mulheres sem a doença, mas a presença da proteína foi semelhante entre os endométrios de mulheres com e sem endometriose. Por outro lado, a expressão da proteína HLA-G nos tecidos de lesão de endometriose avançada se mostrou superior à do endométrio de mulheres sem a doença, indicando que a expressão de HLA-G seria induzida ectopicamente, no microambiente pélvico da doença. Portanto, os resultados apontam para um aumento da expressão de HLA-G em endometriose avançada<br>Endometriosis is a chronic inflammatory, estrogen-dependent disease of multifactorial etiology characterized by implantation and growth of endometrial tissue outside the uterine cavity, and associated with pelvic pain and infertility. Endometriosis is classified according to the stages and sites of the disease. Genetic, endocrine and environmental factors may contribute to the deficit on local immune response, allowing ectopic implantation of endometrial cells into the pelvic cavity. Changes in cytokines pattern in the pelvic microenvironment might promote an immune suppressor environment and explain the decreased immune effector cells response verified in endometriosis. Among possible immunomodulatory factors is the human leucocytary antigen-G (HLA-G) which is intensively expressed in trophoblasts and recognized by inducing maternal-fetal tolerance. HLA-G protein is expressed in both membrane-bound and soluble forms. HLA-G binds inhibitory receptors on innate and adaptive immune cells surface and its expression is induced in non-physiological conditions, such as allogeneic transplants, inflammatory diseases or neoplastic malignancies. Thus, this study hypothesizes that the HLA-G protein would be overexpressed in women with endometriosis, and could contribute to the immunosuppression in the disease microenvironment. To test this hypothesis soluble HLA-G protein was measured in serum and peritoneal fluid of women with and without endometriosis. Moreover, HLA-G gene expression were evaluated on endometrial tissue using RT-qPCR, and HLA-G protein expression were evaluated in matched ectopic and eutopic endometrium of women with and without endometriosis. As results, higher levels of soluble HLA-G were found in serum of women with advanced endometriosis, especially in those with ovarian endometriosis. However, soluble HLA-G levels in peritoneal fluid did not show significant differences between women with and without endometriosis. HLA-G mRNA expression were higher in eutopic endometrium of women without endometriosis, but the HLA-G protein expression were similar in eutopic endometrium of women with and without endometriosis. On the other hand, HLA-G protein expression in ectopic endometrium of women with advanced endometriosis was higher than in eutopic endometrium of women without endometriosis, suggesting that HLA-G expression was induced ectopically, in the pelvic microenvironment of the disease. In conclusion, the results point to an upregulation of HLA-G expression in advanced endometriosis

Os genes HLA (Antígenos leucocitários humanos) estão localizados no Complexo Principal de Histocompatibilidade humano (o MHC), e possuem os maiores níveis de variação do genoma, com milhares de alelos, altas taxas de heterozigose e diversidade nucleotídica. No presente estudo, nosso objetivo foi a identificação dos principais alvos da seleção natural nos genes HLA. Para isso, propusemos duas abordagens que resultaram na redação de dois manuscritos. Na primeira abordagem, nós testamos a hipótese de que os principais alvos da atuação da seleção natural nas moléculas HLA seriam os aminoácidos que compões os sítios que ancoram os peptídeos antigênicos (os bolsões B e F da região de ligação de peptídeos (PBR)). Para isso, utilizamos um conjunto de dados de 6.435 e 6.409 indivíduos genotipados para os genes HLA-A e -B respectivamente, gerados para o 13º Workshop Internacional de Histocompatibilidade (IHW) e pertencentes a 55 populações espalhadas por todos os continentes. Nós estimamos a diversidade nucleotídica (&PI;) das sequências que codificam para os bolsões B e F e comparamos esses dados com os obtidos de outros bolsões da PBR. Concomitantemente, utilizamos a classificação de alelos dos locos HLA-A e -B em supertipos, que são agrupamentos alélicos com similaridades nos perfis de ligação de peptídeos, devido a semelhanças em aminoácidos específicos dos bolsões B e F. Nós descrevemos os padrões observados de variação dos supertipos e desenvolvemos um teste de hipótese onde comparamos os estimadores observados de diferenciação populacional (Gst) e diversidade genética (taxa de heterozigose (He) e número de alelos (k)) com os obtidos a partir de 10.000 réplicas constituídas por agrupamentos aleatórios de alelos. O bolsão B foi a região que apresentou os maiores níveis de diversidade no gene HLA-B (p <0,00001, teste de soma de ranques de Mann-Whitney) e boa parte de sua variação está estruturada entre os supertipos desse mesmo loco. Além disso, os padrões observados de variação nos supertipos de HLA-B não foram reproduzidos pelos agrupamentos aleatórios de alelos (com 98 % das simulações apresentando valores de Gst menores do que os observados, utilizando as amostras africanas, europeias e asiáticas). Esse resultado indicou que os supertipos e consequentemente as especificidades do bolsão B estão significativamente estruturas entre as populações, um indicativo de adaptações locais aos patógenos específicos de diferentes regiões geográficas. Esses mesmos padrões não foram reproduzidos na análise do loco HLA-A, pois boa parte da variação no PBR desse gene não está localizada nos bolsões B e F. Além disso, as simulações envolvendo os supertipos de HLA-A reproduziram mais frequentemente os padrões observados de variação, indicando que os bolsões B e F não são os principais alvos da seleção nesse gene, ou que os níveis de seleção em HLA-A sejam menores dos atuantes em HLA-B. Na segunda abordagem, o nosso principal objetivo foi a identificação de genes que contribuíram para a adaptação local das populações nativas das Américas, que teria ocorrido durante o recente processo de colonização desse continente. Nós sequenciamos os exons 2 e 3 dos loci de classe I HLA-B e -C e o exon 2 do loco de classe II -DRB1 em 635, 524 e 568 indivíduos, respectivamente, pertencentes a 32 populações nativas do continente Americano. Os dados de sequência foram utilizados na estimativa das frequências alélicas, taxa de heterozigose (He), grau de compartilhamento de alelos entre populações (medido pela distância de Prevosti) e desvios de neutralidade utilizando o teste D de Tajima. Nós também comparamos os padrões de variação das taxas de heterozigose obtidas a partir dos loci HLA ao longo do continente com os obtidos a partir de um conjunto de 61 microssatélites espalhados ao longo do genoma, permitindo-nos a diferenciação dos padrões provavelmente gerados pela história demográfica ou seleção natural. O loco HLA-B apresentou o maior número de pares de populações em que não observamos compartilhamento de alelos (44 pares contra 4 e 6 para os loci HLA-C e -DRB1, respectivamente) sendo que a região leste da América do Sul (SAE) foi a que apresentou os menores níveis de compartilhamento de alelos com outras regiões das Américas (39 dos 44 pares de populações continham uma população SAE). Essa maior diferenciação do gene HLA-B nas populações SAE é uma consequência da presença de alelos exclusivos dessa região, originados por eventos de conversão genica e/ou recombinação envolvendo alelos presentes em outras regiões do continente. As populações SAE também apresentaram níveis elevados de variação para o gene HLA-B, resultado evidenciado pela falta de correlação entre a diminuição da taxa de heterozigose e o aumento da distância em relação ao Estreito de Bering (r2 = -0,1117, p > 0,05), o que contrasta com a tendência geral observada nos microssatélites e genes HLA -C e -DRB1 (r2 = -0,1957, -0,2261 e -0,2637, respectivamente (p < 0,05)). Finalizando, as populações SAE apresentaram valores de D de Tajima maiores (p <0,001, teste de soma de ranques de Mann-Whitney) e mais significativos (p < 0,0000005, aplicando um teste binomial exato) no loco HLA-B, quando comparadas às populações das outras regiões. Essas diferenças entre regiões geográficas não foram observadas nos genes HLA-C e -DRB1, corroborando a explicação seletiva para o aumento da frequência dos alelos de HLA-B originados por conversão gênica/recombinação em resposta aos novos desafios ambientais das regiões tropicais na América do Sul. As conclusões obtidas a partir de ambas as abordagens do presente trabalho apontam o gene HLA-B como o principal alvo da seleção natural, uma vez que esse loco concentra as maiores evidências de atuação de seleção natural recente quando comparado aos demais genes HLA analisados. Nós também demonstramos com as análises intragênicas que o bolsão B do PBR de HLA-B concentra por boa parte das diferenças observadas entre as populações, implicando em diferenças nos perfis de apresentação de peptídeos entre essas mesmas populações, o que pode ser interpretado como um indicativo de adaptações locais aos conjuntos de patógenos presentes em distintas regiões geográficas<br>The Classical HLA genes (Human Leucocyte Antigens) are located in the human Major Histocompatibility Complex (the MHC) and present the highest levels of variation on the Human genome, with thousands of alleles associated with high levels of heterozygosis and nucleotide diversity. In the present study, our goal was the identification of the main targets of natural selection on the HLA genes. We proposed two different approaches to address this issue resulting in two manuscripts. At the first approach, we performed an intragenic analysis, verifying if the amino acids at the peptide-binding region (PBR) anchor positions (the B and F pockets) exhibit higher evidences of evolution under natural selection when compared with the remaining regions of the HLA molecules. To do so, we used a dataset generated for the 13th International Histocompatibility Workshop (IHW), composed by 6,435 and 6,409 individuals genotyped for HLA-A and -B respectively, belonging to 55 populations scattered along all the continents. We measured the levels of nucleotide diversity (&pi;) of the sequences coding for the B and F pockets and compared them with the remaining PBR pockets. Concomitantly, we applied the supertype classification which consists in groups of HLA-A and -B alleles which bind overlapping sets of peptides, as a consequence of sharing specific amino acids at B and F pockets and described the patterns of supertype variation in the observed data. Next, we developed a hypothesis test in which the observed patterns of population differentiation (Gst) and variability (heterozygosity (He) and number of alleles (k)), using the supertype definition, were compared with 10,000 replicates of random assigned groups of alleles. At the HLA-B locus, the B pocket presented the highest levels of variation (p < 0.00001, Wilcoxon rank sum test) and concentrated most of the differences between supertypes. Our simulations results revealed that the reassignment of alleles into random groups could not reproduce the observed patterns of population differentiation (with 98% of the simulations presenting Gst values smaller than the observed, using the African, European and Asiatic samples), indicating that supertypes and more specifically the B pocket specificities are significantly structured among populations, which could be an indicative of adaptations to local pathogens. We did not observe the same patterns at the HLA-A locus which presented relative lower levels of variation at B and F pockets when compared with the remaining PBR regions, and simulated values of Gst and He which often reproduced the observed data. At the second approach, our main objective was the identification of genes that contributed for local adaptation on Native American Populations because of the relatively recent colonization of the new American environments. We sequenced the exons 2 and 3 of the HLA-B and -C class I loci and the exon 2 of the -DRB1 class II locus in 635, 524 and 568 individuals, respectively, belonging to 32 different Native American Populations scattered along all the Americas. We estimated the allele frequencies, expected heterozygosity (He), degree of allelic sharing between populations (measured by the Prevost\'s Distance) and departure from neutral expectation using the Ewens-Watterson (EW) and Tajima\'s D test. Concomitantly, we used a dataset of 61 microsatellites scattered along the genome as a demographic control, comparing the degree of variation of the heterozygosity along the continent. The HLA-B locus showed the highest number of pairs of populations in which we did not observe any sharing of alleles (44 pairs against 4 and 6 for HLA-C and -DRB1 loci, respectively) and the Eastern South American (SAE) region was the one presenting the smallest levels of allelic sharing with other American regions at this locus (39 out the 44 pair of populations contained a SAE population). The presence of exclusive gene conversion and/or recombination alleles accounts for the higher differentiation of SAE populations at the -B locus. The -B locus also exhibited a higher level of variation at SAE populations which was evidenced

Imputation of human leukocyte antigen (HLA) alleles from SNP-level data is attractive due to importance of HLA alleles in human disease, widespread availability of genome-wide association study (GWAS) data, and expertise required for HLA sequencing. However, comprehensive evaluations of HLA imputations programs are limited. We compared HLA imputation results of HIBAG, SNP2HLA, and HLA* IMP: 02 to sequenced HLA alleles in 3,265 samples from BioVU, a de-identified electronic health record database coupled to a DNA biorepository. We performed four-digit HLA sequencing for HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1 using long-read 454 FLX sequencing. All samples were genotyped using both the Illumina HumanExome BeadChip platform and a GWAS platform. Call rates and concordance rates were compared by platform, frequency of allele, and race/ethnicity. Overall concordance rates were similar between programs in European Americans (EA) (0.975 [SNP2HLA]; 0.939 [HLA* IMP: 02]; 0.976 [HIBAG]). SNP2HLA provided a significant advantage in terms of call rate and the number of alleles imputed. Concordance rates were lower overall for African Americans (AAs). These observations were consistent when accuracy was compared across HLA loci. All imputation programs performed similarly for low frequency HLA alleles. Higher concordance rates were observed when HLA alleles were imputed from GWAS platforms versus the HumanExome BeadChip, suggesting that high genomic coverage is preferred as input for HLA allelic imputation. These findings provide guidance on the best use of HLA imputation methods and elucidate their limitations.

Alloreactivity is the major barrier to solid organ and hematopoietic stem cell transplantation (HSCT), determining important clinical events such as graft rejection and graft versus host disease. This biological phenomenon is due to the immunogenicity of allogeneic Human Leukocyte Antigens (HLA) expressed in transplanted tissues and recognized by T cell receptor (TCR) on the surface of alloreactive T cells. In the past, the hosting laboratory identified an immunogenic T cell epitope (TCE) encoded by a subset of HLA-DPB1 alleles, thereby defining permissive and non-permissive DPB1 mismatches between donor and recipient associated with different clinical outcomes after HSCT. Aim of this thesis is to elucidate the molecular basis underlying the different clinical impact of DPB1 mismatches in order to improve our understanding of HLA immunogenicity in the context of transplantation. To achieve this aim, we investigated if the clinical effect of DPB1 mismatches was reflected by the strength of their in vitro alloreactive T cell response and, subsequently, we characterized the alloreactive response to one of the most immunogenic DPB1 alleles, HLA-DPB1*09:01 (HLA-DP9), at the molecular level by homology modeling driven site directed mutagenesis. The strength of the alloreactive T cell response to HLA-DP was assessed in Mixed Lymphocyte Reactions between healthy responder/stimulator pairs mismatched only at the DPB1 locus, followed by quantitative assessment of responder T cells upregulating the cell surface activation marker CD137. We observed significantly higher frequencies of activated T cells against non-permissive mismatches (n=9; mean 10.13% ± 7.51%) compared to permissive mismatches (n=15; mean 2.34% ± 2.82% p<0.05). SDM of HLA-DP9 was performed on 6 polymorphic amino acid residues predicted to be crucial for interaction with bound peptide (positions 9, 35, 55, 69, 76 and 84), and on 2 amino acid residues putatively involved in direct interaction with the TCR (positions 56 and 57). These residues were mutated into amino acids naturally occurring in other HLA-DP variants. A lentiviral vector expression platform was used to express the wild type or mutant HLA-DP in 2 HLA homozygous reporter B lymphoblastoid Cell lines. A panel of 8 alloreactive T cell effectors, specific for wild type HLA-DPB1*09:01 (n=6) or for DPB1*10:01 and DPB1*1701, respectively, but crossreactive to DPB1*09:01 (n=2), was used to evaluate the impact of mutagenesis on T cell recognition by gIFN ELISpot or CD107a degranulation assay. For T cells specifically alloreactive to HLA-DPB1*09:01, recognition was influenced by a complex pattern of residues, which was different for each of the 6 effectors studied. In contrast, for the 2 T cells cross-reactive to HLA-DPB1*09:01, only 2 amino acids (positions 69 and 76) had an influence on allorecognition, suggesting the presence of a more restricted set of T cell epitopes in the context of cross-reactivity rather than nominal specificity. These results are consistent with the peptide involvement in alloreactivity to HLA-DPB1*09:01, and underline the complexity of allorecognition which may be one of the mechanisms underlying the immunogenicity of this molecule. Finally, an additional collaborative study with the North Italian Program for Solid Organ Transplantation was aimed at predicting the functional role of amino acid point mutations in newly described naturally occurring variants of HLA-A3 and A32, respectively, by homology modeling. These studies confirmed data from the literature suggesting a relevant putative role of amino acid substitutions at positions 114 and 116, present in a variant of HLA-A3, on the molecular shape and charge of the peptide binding groove. In contrast, point mutations at position 151 and 258 found in two variants of HLA-A32 and A3, respectively, were not predicted to have important functional impacts, due to limited structural or biochemical changes of the groove. In conclusion, our results shed new light on the molecular basis of T cell alloreactivity. In particular, we showed that the stronger immunogenicity of non-permissive DPB1 mismatches is reflected by a divergent and complex T cell epitope repertoire with a putative involvement of the peptide repertoire presented to the alloreactive TCRs. Identification of specific allopeptides associated with these responses will allow to further dissect their molecular basis.

Orientador: Manoel Barros Bértolo<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-16T11:59:40Z (GMT). No. of bitstreams: 1 Nishimura_WesterEidi_M.pdf: 1381412 bytes, checksum: a01c05ab2c7b9409c11bde6b5d507e4f (MD5) Previous issue date: 2010<br>Resumo: O objetivo do presente estudo foi avaliar a freqüência e a associação clínica do HLA classe I e II em pacientes brasileiros com vasculite reumatóide (VR). Nós avaliamos 57 pacientes com artrite reumatóide (AR) estabelecida pelos critérios do Colégio Americano de Reumatologia (ACR) - 1987. Dezessete apresentavam VR de acordo com os critérios de Scott e Bacon - 1984. Foram avaliados nestes pacientes dados demográficos, fator reumatóide (FR), anticorpo anti-peptídio citrulinado cíclico (anti-CCP), tempo de diagnóstico da AR e a atividade da doença pelo escore de atividade da doença (DAS 28). Os alelos HLA foram tipados usando a reação em cadeia da polimerase hibridizado com seqüências específicas de "primers" de baixa resolução. Quanto à atividade da doença nos pacientes sem VR observou-se freqüência aumentada do HLA-B*15 (p=0.033) e HLA-DRB1*01 (p=0.014) com média de DAS 28 >3.2 _ 5.1 e o HLA-Cw*16 (p=0.027) e HLA-B*07 (p=0.027) com média de DAS 28>5.1. Não houve significância estatística de qualquer classe do HLA com o DAS 28 nos pacientes com VR. A comparação entre os 2 grupos mostrou diferença estatística (p=0.001) para o DAS 28 com rank médio = 39.94 para os pacientes com VR. O HLADQB1* 05 (p=0.035) esteve presente em 5 pacientes com VR com média de tempo de diagnóstico de AR de 17 anos e ausente em 12 pacientes com VR com média de tempo de diagnóstico AR de 11.45 anos. Os pacientes com VR tiveram freqüência aumentada do HLA-B*14 (p=0.006) e HLA-Cw*08 (p=0.006). Uma freqüência aumentada do HLA-DRB5*01 (p=0.048) foi encontrada em pacientes sem VR. Nossos resultados mostram na amostra estudada que a VR está associada ao sexo feminino, raça branca, FR e anti-CCP positivos. O HLA-B*15 e HLA-DRB1*01 podem estar envolvidos na atividade moderada da AR sem VR e o HLA-Cw*16 e HLA-B*07 podem estar envolvidos na atividade intensa da AR sem VR. Não houve diferença estatística das classes do HLA com o DAS 28 para VR, porém a doença foi mais ativa em pacientes com VR quando comparados com pacientes sem VR. O HLA-DQB1*05 pode estar envolvido nos casos tardios de AR para a manifestação da VR. O HLA-B*14 e HLACw* 08 podem estar envolvidos na suscetibilidade para VR. O HLA-DRB5*01 pode conferir proteção contra esta manifestação extra-articular da AR<br>Abstract: Our purpose was to evaluate the frequency and clinical association of HLA class I and class II in Brazilian patients with rheumatoid vasculitis (RV). We evaluated 57 patients with rheumatoid arthritis (RA) (American College of Rheumatology -ACR, 1987 criteria). Seventeen had RV according to Scott and Bacon's criteria - 1984. Demographic data, time of RA diagnosis, disease activity by the Disease Activity Score (DAS 28), rheumatoid factor (RF) and cyclic citrullinated peptide (anti-CCP) were analyzed. HLA alleles were typed using polymerase chain reaction-amplified DNA hybridized with low-resolution sequence-specific primers. HLA-B*15 (p=0.033) and HLA-DRB1*01 (p=0.014) were associated with moderate activity of RA without RV, and HLA-B*07 (p=0.027) and HLA-Cw*16 (p=0.027) with intense activity of RA without RV; no statistical significance of HLA class and DAS 28 was observed in RV. HLA-DQB1*05 (p=0.035) was related to RV in patients with late RA. The comparison between the groups showed an increased frequency of HLA-B*14 (p = 0.006) and HLA-Cw*08 (p = 0.006) in patients with RV, and an increased frequency of HLADRB5* 01 (p = 0.048) in patients without RV. In conclusion, the HLA-B*14 and HLACw* 08 may be involved in susceptibility to RV and HLA-DRB5*01 may confer protection against this extra articular manifestation of RA<br>Mestrado<br>Clinica Medica<br>Mestre em Clinica Medica