This thesis is comprised of studies of proteins involved in class I and class II major histocompatibility complex (MHC) antigen procressing. In class I MHC processing, structural and functional studies were conducted of an aminopeptidase, ERAP1, that mediates the final step in antigen processing to understand how it is particularly suitable for cleavage of antigenic peptides for class I MHC presentation. In the class II MHC antigen presentation pathway, structural studies were conducted to characterize a fluorogenic peptide that can be used to understand peptide loading events in vivo and in real time. Also structural studies of class II MHC and peptide complexes were conducted to understand the nature of an unique C-terminal secondary structure element exhibited by an HIV derived peptide in the peptide binding groove of class II MHC. The studies discussed in this thesis provide insights into the proteins involved in the class I and class II MHC antigen presentation pathway. The endoplasmic reticulum (ER) aminopeptidase, ERAP1, is a 941 amino acid member of the M1 family of zinc metalloaminopeptidases. Unlike other aminopeptidases, ERAP1 has a length and C-terminal preference for its substrates. Interestingly, ERAP1 has been shown to trim antigenic peptides to lengths of 8 or 9 amino acids long. This length matches the length required to bind into the peptide binding groove of class I MHC molecules. In addition, ERAP1 is upregulated in the ER of cells treated with interferon gamma (IFN-γ). Knock-down of ERAP1 by siRNA results in less overall antigenic presentation during IFN-γ treatment, although the knock-down does not affect all class I MHC epitopes equally. Knock-out studies show that ERAP1 effects the antigen repertoire at the cell surface. These and other data implicate ERAP1 as an important player in class I MHC antigen presentation. A chapter of this thesis will describe the crystallographic work describing the structures of ERAP1 with an aminopeptidase inhibitor, bestatin, and ERAP1 without an inhibitor that suggest possible peptide binding site in ERAP1 that will allow it to generate suitable substrates for a subset of class I MHC alleles. Class II MHC plays a key role in the immune response by presenting antigenic peptides on CD4+ cytotoxic cell surfaces for T-cell response. The binding of peptides onto the MHC is an important step in creating an immune response. Structures of peptide bound MHC class II show conserved side chain binding pockets within the overall peptide-binding groove. In HLA-DR1, a common human class II MHC, the P1 pocket shows a preference for large hydrophobic side chains. Development of environmentally sensitive peptide analogs, that can bind into the class II MHC the same way as native peptides, can assist in visualizing the antigen binding process. A chapter in this thesis describes the crystallographic work showing that (4-DAPA)-HA can be used to study antigen-presenting processes in a cell by visualizing the changes in fluorescence of the synthesized peptide upon antigen loading. Crystallographic analysis of MHC class II, HLA-DR1, in complex with HIV gag-derived peptide, GagP16(PEVIPMFSALSEGATP), and superantigen, SEC3- 3B2, reveals the conventional polyproline conformation up to MHC binding pocket residue, P9, while the C-terminus of GagP16 adopts an unusual β- hairpin loop structure. Additionally, interactions between the leucine at P8 (LeuP8) and other residues on the loop such as ThrP16 and AlaP14 of the hairpin loop, was observed. Importantly, GagP16 requires the last 4 amino acids (P13-P16), which is part of the hairpin loop, for T-cell recognition. Understanding what dictates the C-terminal hairpin loop and the interaction motif of HLA-DR1/GagP16 complex with its TCR will provide insights on why it is important for T cell activation. A chapter in this thesis discusses the structural investigation conducted to understand the determinants of the loop at the C-terminus of GagP16 using designed peptides. It will also discuss work involving HLA-DR1 with the T cell receptor, AC25, that was cloned from T cells that are specific to HLA-DR1 in complex with the GagP16 peptide.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2009. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

No description available.

published_or_final_version / Pathology / Master / Master of Philosophy

Understanding the genetic basis of fitness differences has been a major goal for evolutionary biologists over the last two decades. Although there are many studies investigating how natural selection can promote local adaptation, few have succeeded to find the link between genotype and fitness of the phenotype. Polymorphic genes of the major histocompatibility complex (Mhc) are excellent candidates for such associations as they are a central component of the vertebrate immune system, playing an important role in parasite resistance, and hence can have direct effects on survival of their bearers. Although associations between Mhc and disease resistance are frequently documented, the epidemiological basis of the host-parasite interaction is often lacking and few studies have investigated the role that Mhc genes play in individual variation in fitness; thus comparatively little is known about the fitness consequences of Mhc in wild populations. Furthermore, the majority of work to date has involved testing associations between Mhc genotypes and disease. However, the mechanism by which any direct selection on the Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. The aim of this thesis was to characterize Mhc alleles in terms of their predicted functional properties and to investigate whether and how selection operates on Mhc class I functional variation using the great tit (Parus major) population at Wytham Woods as a model host species. Through a comprehensive characterization effort and the use of 454 pyrosequencing platform, I performed a detailed analysis of genetic variation at Mhc class I exon 3 and grouped alleles with similar antigen-binding affinities into supertypes to classify functionally distinct Mhc types. There was extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. A total of 862 alleles were detected from 857 individuals; the highest number yet characterized in a wild bird species. The functional alleles were clustered into 17 supertypes; there was clear evidence that functional alleles were under strong balancing selection. To understand the role of Mhc in disease resistance, I examined the linkage between Mhc supertypes, Plasmodium infection and great tit survival, and showed that certain functional variants of Mhc confer resistance to two divergent Plasmodium parasite species that are common in the environment. I further investigated the fitness consequences of functional variation at Mhc, using mark-recapture methods and long-term breeding data; and tested the hypotheses that selection: (i) maximizes Mhc diversity; (ii) optimizes Mhc diversity, or (iii) favours specific functional variants. I found that the presence of three different supertypes was associated with three different components of individual fitness: adult survival, annual recruitment probabilities and lifetime reproductive success. In contrast, there was no evidence for a selective advantage of Mhc functional diversity, either in terms of maximal or optimal supertype diversity. Finally, I explored the role that Mhc plays in female mate choice decisions and examined the reproductive fitness consequences of Mhc-dependent mating patterns. There was little evidence to suggest that functional dissimilarity at Mhc has any influence on female mate choice decisions or that dissimilarity at Mhc affects the reproductive output of the social pair. Overall, this thesis provides strong support for the suggestion that selection favours specific functional variants of Mhc, possibly as a result of supertype-specific resistance or susceptibility to parasites that exert strong selective pressures on their hosts; whereas there is no support for selection favouring maximal or optimal Mhc diversity. More importantly it demonstrates that functional variants of Mhc class I loci are an important determinant of individual fitness in natural populations.

The Human Leukocyte Antigen (HLA)-B27 is a Major Histocompability Complex (MHC) class I antigen that is strongly associated with development of a group of closely related arthritic diseases, collectively known as the spondyloarthropathies (SpA). However, the mechanism by which HLA-B27 confers this susceptibility is unclear. Studies have shown that HLA-B27 heavy chains can form classical heterotrimers associated with peptide and β2-microglobulin (B27HT), and also non-classical heavy chain homodimers (B27₂). B27₂ assemble intracellularly during maturation and are also expressed at the cell surface following endosomal recycling of B27HT. A pathogenic role for B27₂ has been proposed in two of the current theories of pathogenesis: the B27 homodimer theory and the B27 misfolding and UPR theory. Yet, determinations of the extent, distribution, and triggers of B27₂ expression, as well as the functional consequences of its receptor interactions in AS pathogenesis, have been hampered by the lack of a specific detection reagent. Therefore, to investigate the role of B27₂ in AS, we generated a novel antibody to B27₂ – HD6 – using phage display technology, which binds to in vitro refolded B27₂ but not B27HT complexes by ELISA. This thesis provides evidence that HD6-reactive molecules, which include B27₂, are expressed at the cell surface in both cell lines and in the context of a disease setting. Recognition is B27-specific and strongly correlated with the magnitude of B27 expression, which could account for the lack of staining in some cell subsets. Moreover, staining was comparable in cell lines expressing the disease-associated B*27:05 and the less disease-associated subtype B*27:09. In addition, I have shown cells expressing physiologic levels of B27, including EBV-transformed BCLs and AS patient PBMCs, are capable of expressing the HD6 epitope upon low pH treatment. Interestingly, these ‘acid-inducible HD6’ molecules were absent from cells lacking a functional PLC. Finally, I have shown that HD6-reactive molecules can derive from pre-existing folding B27 molecules at the cell surface, which may be inhibited by the addition of exogenous B27-binding peptides. These findings are consistent with a mechanism of pathogenesis involving the surface expression and recognition of B27₂ and/or other aberrantly folded forms of B27, as proposed in the homodimer theory. HD6 will be a powerful tool to address the potential pathogenic role of B27₂ in SpA and may additionally have therapeutic potential.

The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.

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.

Este trabalho teve como objetivo principal descrever a diversidade genética e identificar a estrutura populacional de populações de tamanduá-mirim distribuídas ao longo dos biomas brasileiros através do uso de ferramentas de genética de populações e do acesso a regiões neutras e adaptativas do genoma da espécie. A amostragem de indivíduos de tamanduá-mirim é complicada pela difícil detecção do animal em trabalhos de campo, pela sua complicada captura e manipulação. Assim, fez-se necessário o uso de espécimes de museu e de amostras não-invasivas. A fim de validar o uso das mesmas para a genotipagem confiável de oito locos de microssatélites desenvolvidos para a espécie foi utilizado um método de padronização da qualidade das amostras (Índice de Qualidade, QI) e estimativa dos erros de genotipagem. Foi observada uma qualidade superior das amostras não invasivas (N=19) em relação às peles de museu (N=138). Foi possível também eliminar amostras com desempenho ruim (QI<0.7 e sucesso de amplificação maior que 75%), e garantir a confiabilidade dos resultados de microssatélites das amostras que permaneceram no estudo para análises posteriores. Devido à grande área de distribuição da espécie de forma contínua, se tornou complicado traçar populações pré-definidas. Assim, a abordagem da genética da paisagem foi a ferramenta mais apropriada para o estudo da estrutura de populações em T. tetradactyla (N=176). Comparativamente, duas abordagens foram usadas: agrupamento de indivíduos pelo critério de proximidade geográfica, designado aqui como a priori (20 populações), e análises baseadas no indivíduo, sem informação prévia de agrupamento populacional, referida no capítulo como a posteriori (quatro transectos testados). Foram encontrados níveis de diversidade moderados (média de 11.38 alelos por lóco) e poucas evidências de estruturação entre populações das localidades amostradas (K=2 na maioria dos testes). Os indivíduos que mostraram maior diferenciação foram originados da Floresta Amazônica. Esta região também demonstrou maior diversidade genética (riqueza alélica e heterozigosidade esperada) que as outras. Por outro lado, populações distribuídas ao longo da Mata Atlântica e regiões adjacentes demonstraram um padrão de isolamento por distância. Populações do Brasil central (Cerrado e Pantanal) não demonstraram diferenciação em relação às demais. Finalmente, foi estudada a variação genética adaptativa da espécie através da diversidade do gene DRB do complexo MHC. Esta família gênica codifica proteínas envolvidas no reconhecimento de antígeno e ativação da resposta imune adaptativa, e são regulados por seleção natural, especialmente por pressão seletiva dirigida por patógenos. É esperado que a diversidade de patógenos seja distinta nos biomas brasileiros, sendo os ambientes florestais mais biodiversos neste quesito do que ambientes da Diagonal Seca, o que representa pressões seletivas diferentes. Assim, foi investigada a diversidade do gene DRB éxon 2 em indivíduos (N=65) dos diferentes biomas brasileiros através de sequenciamento de nova geração (454 GS Junior), e os resultados de distribuição dos alelos foram comparados com os microssatélites. Foi encontrada uma alta diversidade (60 alelos no nível de aminoácido e 70 alelos no nível de nucleotídeos) e assinaturas claras de seleção positiva no gene (dN/dS=2.94). Maior riqueza alélica e proporção de alelos privados foram encontradas em biomas florestados, especialmente na Floresta Amazônica. Além disso, os marcadores neutros (microssatélites), demonstraram padrões similares ao DRB, revelando a força de eventos demográficos e deriva genética que também moldaram os padrões de diversidade deste gene do MHC / This work aimed to describe the genetic diversity and population structure of populations of the lesser anteater distributed along Brazilian biomes through population genetic tools, accessing neutral and adaptive genomic regions of the species. Sampling lesser anteater individuals is complicated due to infrequent detection of the animal in field works, its complicated capture and manipulation. Thus, it was necessary to use museum specimens and noninvasive samples. To validate these samples for the reliable genotyping of eight microsatellite loci developed for the species, a standardization method of the quality of samples (Quality Index, QI) and genotyping errors was used. A superior quality of noninvasive samples (N=19) compared to study skins (N=138) was observed. It was also possible to eliminate samples with a bad performance (QI<0.7 and amplification success higher than 75%), and thus guarantee the reliability of microsatellites results for samples kept in further analysis. Due to the great and continuous distribution area of the species, it becomes difficult to delineate predefined populations. Thereby, landscape genetics approach was a better suited tool for studying the population structure of T. tetradactyla individuals (N=176). Comparatively, two approaches were used: grouping of individuals by a geographical proximity criterion, designated here as a priori (20 populations), and analysis based on individuals, without previous information of population grouping, referred here as a posteriori (four transects tested). Moderate levels of diversity were found (average of 11.38 alleles per locus), and few evidences for structuring between populations of the sampled localities (K=2 in most tests). The individuals showing the major differentiation were originated from the Amazon Forest. This region also demonstrated higher genetic diversity (allelic richness and expected heterozygosity) than others. By the other hand, populations distributed in Atlantic Forest and adjacent regions demonstrated a pattern of isolation by distance. Populations from central Brazil (Cerrado and Pantanal) did not show distinction from the others. Finally, the adaptive genetic variation of the species was studied through DRB gene diversity, from MHC. This gene family codes for proteins involved in the antigen recognition and activation of the adaptive immune response, and are regulated by natural selection, especially by selective pressure driven by pathogens. It is expected that the pathogen diversity is distinct in Brazilian biomes, being florested biomes more diverse than dry central Brazil environments, which represents different selective pressures. Therefore, the diversity of DRB exon 2 gene in individuals (N=65) from different Brazilian biomes was investigated through Next Generation Sequencing (454 GS Junior), and the results of allele distributions were compared with microsatellites. A high diversity was found (60 alleles in amino acid level and 70 in nucleotide level) and clear signatures of positive selection in DRB (dN/dS=2.94). Greater allelic richness and private allele number were found in forested biomes, especially in the Amazon Florest. Besides, neutral markers (microsatellites) demonstrated similar patterns to DRB, revealing the strength of demographic events and genetic drift in shaping the diversity patterns in MHC