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This thesis presents investigations of diversity at three genes (class I, DQA and DQB) of the Major Histocompatibility Complex (MHC) in cetaceans. The MHC genes encode for proteins that are crucial for initiating an immune response by binding invading pathogens in vertebrates. It has been acknowledged that a high diversity at these genes results in the ability to recognise a wider range of pathogens, therefore functional diversity is important for the survival of a species. Furthermore this diversity has been created under the influence of selection, which can reveal interesting contrasts with neutral markers about the history of selection of populations and species. The diversity at two genes (DQA and DQB) in natural populations of two contrasting species of cetaceans has been investigated in more detail. The species selected included both sub-species of Hector’s dolphin, the Hector’s dolphin (Cephalorhynchus hectori hectori) and the Maui’s dolphin (Cephalorhynchus hectori maui), as well as the long-finned pilot whale (Globicephala melas). These species were chosen, because both Hector’s dolphin sub-species contrast with the pilot whale species in regards to their population size, abundance, population structure and life history. For example both sub-species of Hector’s dolphin have small population sizes and only inhabit coastal areas around New Zealand, whereas the pilot whale is an abundant, pelagic dolphin species. In Chapter 2 the expression of class II MHC genes (DQA and DQB) was demonstrated for the first time for a cetacean species, the Hector’s dolphin. Using available information from the bottlenose dolphin (Tursiops truncatus), I also designed primers to investigate class I MHC. Fragments of MHC genes were amplified from cDNA, which was derived from blood samples of two Hector’s dolphins. These dolphins were the subject of a temporary live capture, presenting a unique opportunity for blood collection. No evidence was found for duplication of both MHC class II loci, but cloning suggested a minimum of three copies of class I genes within the genomic DNA. However, the expression of all class I genes was uncertain, since only one allele could be isolated from cDNA. Functionality for all three genes (class I, DQA and DQB) was supported by the evidence for balancing selection having operated on these genes, indicated by a higher ratio of non-synonymous to synonymous substitutions. In Chapter 3, a combination of single-strand conformation polymorphism (SSCP) and direct sequencing was used to describe DQA and DQB diversity in the Hector’s and Maui’s dolphin. Genetic samples for the Hector’s dolphin were available from previously collected stranding and biopsy samples (n = 233), representing three populations from the South Coast of New Zealand and the sub-species on the West Coast of the North Island of New Zealand. For the Hector’s dolphin of the South Island, a surprisingly large number of alleles at both loci (DQA = 4, DQB = 6) were found, considering their small population size and compared to other cetacean populations with larger population sizes. The Maui’s dolphin has been classified as critically endangered with less than 100 dolphins, but showed a relatively high nucleotide diversity for DQB ( = 4.5%). This diversity was based on only three alleles that have been retained in the sub-species, representing the most divergent of all six alleles. All populations showed strong geographic differentiation at both loci (DQA: FST=0.252; DQB: FST=0.333), with the greatest differentiation between the South Island population and the North Island Maui’s dolphin. Comparison to mitochondrial and microsatellite diversity suggested influence of stochastic genetic drift, although the pressure of balancing selection acting on DQB over an evolutionary time period was also evident by a higher ratio of non-synonymous to synonymous substitutions (dN/dS=5.9) and by a pattern of trans-specific allele sharing within the family of Delphinidae. In Chapter 4 similar methods were used to describe DQA and DQB in pilot whales using genetic samples from the long-finned pilot whale that were available from five mass-strandings from around New Zealand (n = 237). A larger number of alleles than for the Hector’s dolphin were found at both loci (DQA= 8; DQB= 8), although their large population size and pelagic abundance raises the expectation of an even greater number of alleles. The overall differentiation between mass-strandings was low, but significant for both loci (DQA: FST =0.012, DQB: FST =0.014). The differentiation of all strandings was greatest for the Golden Bay mass-stranding at DQA, but deviation from Hardy-Weinberg equilibrium at DQB suggested either sub-structure within mass-strandings (Wahlund effect) or the presence of null alleles. As for the Hector’s dolphin and other mammalian species, the influence of balancing selection acting on DQB over a long evolutionary time period was evident by a higher ratio non-synonymous to synonymous substitutions (dN/dS=9.3) and by a pattern of trans-specific allele sharing within the family of Delphinidae. Overall, diversity is surprisingly similar between these two cetacean species despite different life history characteristic, but low compared to domesticated ungulate species, such as the cow. If low MHC diversity is a general feature of cetaceans, due to the marine environment as suggested previously or rather a side effect of short-term demographic forces remains speculative. A standardised nomenclature for the increasing number of MHC alleles from cetacean is proposed in this thesis to assist with future development of this research.

This thesis presents investigations of diversity at three genes (class I, DQA and DQB) of the Major Histocompatibility Complex (MHC) in cetaceans. The MHC genes encode for proteins that are crucial for initiating an immune response by binding invading pathogens in vertebrates. It has been acknowledged that a high diversity at these genes results in the ability to recognise a wider range of pathogens, therefore functional diversity is important for the survival of a species. Furthermore this diversity has been created under the influence of selection, which can reveal interesting contrasts with neutral markers about the history of selection of populations and species. The diversity at two genes (DQA and DQB) in natural populations of two contrasting species of cetaceans has been investigated in more detail. The species selected included both sub-species of Hector’s dolphin, the Hector’s dolphin (Cephalorhynchus hectori hectori) and the Maui’s dolphin (Cephalorhynchus hectori maui), as well as the long-finned pilot whale (Globicephala melas). These species were chosen, because both Hector’s dolphin sub-species contrast with the pilot whale species in regards to their population size, abundance, population structure and life history. For example both sub-species of Hector’s dolphin have small population sizes and only inhabit coastal areas around New Zealand, whereas the pilot whale is an abundant, pelagic dolphin species. In Chapter 2 the expression of class II MHC genes (DQA and DQB) was demonstrated for the first time for a cetacean species, the Hector’s dolphin. Using available information from the bottlenose dolphin (Tursiops truncatus), I also designed primers to investigate class I MHC. Fragments of MHC genes were amplified from cDNA, which was derived from blood samples of two Hector’s dolphins. These dolphins were the subject of a temporary live capture, presenting a unique opportunity for blood collection. No evidence was found for duplication of both MHC class II loci, but cloning suggested a minimum of three copies of class I genes within the genomic DNA. However, the expression of all class I genes was uncertain, since only one allele could be isolated from cDNA. Functionality for all three genes (class I, DQA and DQB) was supported by the evidence for balancing selection having operated on these genes, indicated by a higher ratio of non-synonymous to synonymous substitutions. In Chapter 3, a combination of single-strand conformation polymorphism (SSCP) and direct sequencing was used to describe DQA and DQB diversity in the Hector’s and Maui’s dolphin. Genetic samples for the Hector’s dolphin were available from previously collected stranding and biopsy samples (n = 233), representing three populations from the South Coast of New Zealand and the sub-species on the West Coast of the North Island of New Zealand. For the Hector’s dolphin of the South Island, a surprisingly large number of alleles at both loci (DQA = 4, DQB = 6) were found, considering their small population size and compared to other cetacean populations with larger population sizes. The Maui’s dolphin has been classified as critically endangered with less than 100 dolphins, but showed a relatively high nucleotide diversity for DQB ( = 4.5%). This diversity was based on only three alleles that have been retained in the sub-species, representing the most divergent of all six alleles. All populations showed strong geographic differentiation at both loci (DQA: FST=0.252; DQB: FST=0.333), with the greatest differentiation between the South Island population and the North Island Maui’s dolphin. Comparison to mitochondrial and microsatellite diversity suggested influence of stochastic genetic drift, although the pressure of balancing selection acting on DQB over an evolutionary time period was also evident by a higher ratio of non-synonymous to synonymous substitutions (dN/dS=5.9) and by a pattern of trans-specific allele sharing within the family of Delphinidae. In Chapter 4 similar methods were used to describe DQA and DQB in pilot whales using genetic samples from the long-finned pilot whale that were available from five mass-strandings from around New Zealand (n = 237). A larger number of alleles than for the Hector’s dolphin were found at both loci (DQA= 8; DQB= 8), although their large population size and pelagic abundance raises the expectation of an even greater number of alleles. The overall differentiation between mass-strandings was low, but significant for both loci (DQA: FST =0.012, DQB: FST =0.014). The differentiation of all strandings was greatest for the Golden Bay mass-stranding at DQA, but deviation from Hardy-Weinberg equilibrium at DQB suggested either sub-structure within mass-strandings (Wahlund effect) or the presence of null alleles. As for the Hector’s dolphin and other mammalian species, the influence of balancing selection acting on DQB over a long evolutionary time period was evident by a higher ratio non-synonymous to synonymous substitutions (dN/dS=9.3) and by a pattern of trans-specific allele sharing within the family of Delphinidae. Overall, diversity is surprisingly similar between these two cetacean species despite different life history characteristic, but low compared to domesticated ungulate species, such as the cow. If low MHC diversity is a general feature of cetaceans, due to the marine environment as suggested previously or rather a side effect of short-term demographic forces remains speculative. A standardised nomenclature for the increasing number of MHC alleles from cetacean is proposed in this thesis to assist with future development of this research.

This thesis presents investigations of diversity at three genes (class I, DQA and DQB) of the Major Histocompatibility Complex (MHC) in cetaceans. The MHC genes encode for proteins that are crucial for initiating an immune response by binding invading pathogens in vertebrates. It has been acknowledged that a high diversity at these genes results in the ability to recognise a wider range of pathogens, therefore functional diversity is important for the survival of a species. Furthermore this diversity has been created under the influence of selection, which can reveal interesting contrasts with neutral markers about the history of selection of populations and species. The diversity at two genes (DQA and DQB) in natural populations of two contrasting species of cetaceans has been investigated in more detail. The species selected included both sub-species of Hector’s dolphin, the Hector’s dolphin (Cephalorhynchus hectori hectori) and the Maui’s dolphin (Cephalorhynchus hectori maui), as well as the long-finned pilot whale (Globicephala melas). These species were chosen, because both Hector’s dolphin sub-species contrast with the pilot whale species in regards to their population size, abundance, population structure and life history. For example both sub-species of Hector’s dolphin have small population sizes and only inhabit coastal areas around New Zealand, whereas the pilot whale is an abundant, pelagic dolphin species. In Chapter 2 the expression of class II MHC genes (DQA and DQB) was demonstrated for the first time for a cetacean species, the Hector’s dolphin. Using available information from the bottlenose dolphin (Tursiops truncatus), I also designed primers to investigate class I MHC. Fragments of MHC genes were amplified from cDNA, which was derived from blood samples of two Hector’s dolphins. These dolphins were the subject of a temporary live capture, presenting a unique opportunity for blood collection. No evidence was found for duplication of both MHC class II loci, but cloning suggested a minimum of three copies of class I genes within the genomic DNA. However, the expression of all class I genes was uncertain, since only one allele could be isolated from cDNA. Functionality for all three genes (class I, DQA and DQB) was supported by the evidence for balancing selection having operated on these genes, indicated by a higher ratio of non-synonymous to synonymous substitutions. In Chapter 3, a combination of single-strand conformation polymorphism (SSCP) and direct sequencing was used to describe DQA and DQB diversity in the Hector’s and Maui’s dolphin. Genetic samples for the Hector’s dolphin were available from previously collected stranding and biopsy samples (n = 233), representing three populations from the South Coast of New Zealand and the sub-species on the West Coast of the North Island of New Zealand. For the Hector’s dolphin of the South Island, a surprisingly large number of alleles at both loci (DQA = 4, DQB = 6) were found, considering their small population size and compared to other cetacean populations with larger population sizes. The Maui’s dolphin has been classified as critically endangered with less than 100 dolphins, but showed a relatively high nucleotide diversity for DQB ( = 4.5%). This diversity was based on only three alleles that have been retained in the sub-species, representing the most divergent of all six alleles. All populations showed strong geographic differentiation at both loci (DQA: FST=0.252; DQB: FST=0.333), with the greatest differentiation between the South Island population and the North Island Maui’s dolphin. Comparison to mitochondrial and microsatellite diversity suggested influence of stochastic genetic drift, although the pressure of balancing selection acting on DQB over an evolutionary time period was also evident by a higher ratio of non-synonymous to synonymous substitutions (dN/dS=5.9) and by a pattern of trans-specific allele sharing within the family of Delphinidae. In Chapter 4 similar methods were used to describe DQA and DQB in pilot whales using genetic samples from the long-finned pilot whale that were available from five mass-strandings from around New Zealand (n = 237). A larger number of alleles than for the Hector’s dolphin were found at both loci (DQA= 8; DQB= 8), although their large population size and pelagic abundance raises the expectation of an even greater number of alleles. The overall differentiation between mass-strandings was low, but significant for both loci (DQA: FST =0.012, DQB: FST =0.014). The differentiation of all strandings was greatest for the Golden Bay mass-stranding at DQA, but deviation from Hardy-Weinberg equilibrium at DQB suggested either sub-structure within mass-strandings (Wahlund effect) or the presence of null alleles. As for the Hector’s dolphin and other mammalian species, the influence of balancing selection acting on DQB over a long evolutionary time period was evident by a higher ratio non-synonymous to synonymous substitutions (dN/dS=9.3) and by a pattern of trans-specific allele sharing within the family of Delphinidae. Overall, diversity is surprisingly similar between these two cetacean species despite different life history characteristic, but low compared to domesticated ungulate species, such as the cow. If low MHC diversity is a general feature of cetaceans, due to the marine environment as suggested previously or rather a side effect of short-term demographic forces remains speculative. A standardised nomenclature for the increasing number of MHC alleles from cetacean is proposed in this thesis to assist with future development of this research.

Orientador: Noeme Sousa Rocha / Resumo: O tumor venéreo transmissível canino (TVTC) ocorre naturalmente em cães, sem predileção por raça ou sexo sendo transmitido durante o coito ou hábitos sociais. É também um tumor transplantável experimentalmente e tem sido utilizado como modelo para o estudo da relação entre tumor e hospedeiro. Apesar da maior infiltração inflamatória intratumoral e da expressão de moléculas do complexo principal de histocompatibilidade (MHC) estar associada à regressão do tumor, o papel central das células imunes do hospedeiro na evolução clínica do TVTC ainda não está claro. Neste estudo nós buscamos analisar a interação entre TVTC natural e hospedeiro, especialmente sob o ponto de vista da imunidade celular tumoral. Aqui nós identificamos e quantificamos por citometria de fluxo células T (CD3+, CD4+ e CD8+), células NK, células B, macrófagos, em amostras de sangue e de tumor, além da expressão imunoistoquímica de moléculas do MHC de classe I e II, sobretudo nas diferentes fases clínicas do tumor, assim como classificamos os subtipos citológicos do tumor e avaliamos o comportamento tumoral frente ao tratamento quimioterápico com sulfato de vincristina em uma amostra de 22 cães com TVTC natural. A quimioterapia foi efetiva no tratamento da maioria dos casos. Encontramos predomínio de TVTC linfocitóide e que metástases e resistência quimioterápica ocorreram apenas nos tumores de fenótipo linfocitóide e misto. Identificamos aumento significativo na expressão de moléculas de MHC classe I e II na ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The transmissible venereal canine tumor (CTVT) occurs naturally in dogs, without predilection for race or gender being transmitted during intercourse or social habits. It is also an experimentally transplantable tumor and it has been used as a pattern for the study about the relationship between the tumor and the host. Despite the greater intratumoral inflammatory infiltration and the expression of major histocompatibility complex molecules (MHC) is associated with tumor regression, the central role of host immune cells in the CTVT clinical evolution is not clear yet. In this study we sought to analyze the interaction between natural and host CTVT, especially from the point of view of tumor cell immunity. Here we identify and quantify by flow cytometry cells T (CD3+, CD4+ e CD8+), cells NK, cells B, macrophages, in blood and tumor samples, besides the immune histochemical expression of MHC class I and II molecules, specially in the different clinical phases of the tumor as well as classifying the cytological subtypes of the tumor and evaluating the tumor behavior against the chemotherapy treatment with vincristine sulfate in a sample of 22 dogs with natural CTVT. Chemotherapy was effective in the treatment of most cases. We found a predominance of lymphocytoid CTVT and that metastases and chemotherapeutic resistance occurred only in tumors of lymphocytoid and mixed phenotype. We identified a significant increase in the expression of MHC class I and II molecules in the regress... (Complete abstract click electronic access below) / Mestre

O sucesso da gestação, e nascimento da prole saudável, depende da adequada formação, desenvolvimento e funcionamento da placenta. Entretanto, são observadas altas perdas durante o primeiro terço da gestação de embriões bovinos produzidos por transferência de núcleo de célula somática (TNCS), principalmente causadas por alterações placentárias, decorrentes da incompleta reprogramação epigenética no desenvolvimento embrionário. Normalmente, após a fecundação, o DNA paterno é desmetilado durante as primeiras horas do desenvolvimento, enquanto o DNA materno é desmetilado de forma passiva. Entretanto nos embriões TNCS a desmetilação do DNA é tardia e incompleta, resultando numa alteração do padrão epigenético, principalmente nos níveis de metilação (5mC) e hidroximetilação (5hmC) do DNA e nas histonas. Além disso, na gestação TNCS há expressão precoce das moléculas do complexo de histocompatibilidade principal de classe I (MHC-I), associada ao aumento de infiltrados inflamatórios na placenta e à rejeição imunológica ao feto. O objetivo desse trabalho foi de verificar, na placenta bovina TNCS e controle, os níveis globais de 5mC e 5hmC, e de algumas modificações de histonas importantes na formação do trofectoderma ou por serem modificações clássicas, além de imunolocalizar moléculas de MHC-I. Para tanto foram utilizadas amostras de placentônio TNCS no primeiro (n = 5) e terceiro (n = 6) terço gestacional; e como controle, placentônios com controle no primeiro (n = 6) e terceiro (n = 6). Foram realizadas reações de imunohistoquímica para modificações no DNA (5mC, 5hmC) e nas histonas (H3K4me3, H3K27me3, H3K9ac, H3K9me2/3) e para MHC-I (Qa-2 e IL-A88); além de reações de PCR quantitativo para enzimas responsáveis pelas modificações no DNA (DNMT1, TET1, TET3), para alguns genes relacionados com o desenvolvimento da placenta (PAG9, PHDLA2, SNRPN e TSSC4) e para isoformas de MHC-I (NC1-4 e JSP-1). Houve aumento dos níveis globais de metilação, e diminuição de hidroximetilação, na placenta TNCS durante o primeiro trimestre gestacional. Os níveis de H3K4me3 foram estáveis na placenta controle e crescentes na placenta TNCS, enquanto que a H3K27me3 decresceu na placenta controle e foi estável na placenta TNCS. Na placenta TNCS, aos 60 dias, foram observados os menores níveis globais H3K9ac, porém H3K9me2/3 não diferiram entre as idades e tipos gestacionais estudados. Foi identificado MHC-I no trofoblasto do placentônio bovino nas idades analisadas, com variações de intensidade dependendo da isoforma detectada, além de que a placenta controle e a TNCS apresentam padrões diferentes na expressão de MHC-I. De modo geral, o menores níveis de metilação do DNA encontrados na placenta TNCS aos 60 dias de gestação, indica ser um mecanismo compensatório para ativar a expressão gênica. Visto que a as modificações de histona levam a um estado repressivo da expressão gênica, já que a bivalência entre H3K4me3 e H3K27me3 e os níveis de H3K9ac estão diminuídos. Ainda na placenta TNCS aos 60 dias, há uma alta expressão de MHC-I, levando a resposta imune do sistema materno contra os tecidos fetais. Portanto vários eventos são presentes e parecem contribuir para instabilidade da gestação inicial em clones, coincidindo com a alta taxa de perdas gestacionais nessa fase / Pregnancy success depends of adequate placental formation, development and function. Therefore, the highest loses rates are found during first trimester pregnancies of bovine embryos produced by somatic cell nuclear transfer (NT), majorly by placental alterations, due to incomplete epigenetic reprogramming during embrionary development. Normally, after fecundation, paternal DNA is actively demethylated at first hours of development; where as maternal DNA is passively demethylated. However in NT embryos the DNA demethylation is late and incomplete, resulting in changes of epigenetic patterns, majorly in methylation (5mC), hydroxymethylation (5hmC) and histone levels. Furthermore, in NT pregnancy there is premature expression of class I major histocompatibility complex (MHC-I), associated with inflammatory infiltrates increases and immunological rejection against fetus. The aim of this work was to evaluate global levels of 5mC, 5hmC and some posttranslational histone modifications and MHC-I molecules expression of bovine placenta in NT and control models. For this, NT and control bovine placentome were collected at first (n = 6) and third (n = 6) trimester of pregnancy. Immunohistochemistry reactions were performed to assay DNA methylation (5mC) and (5hmC) and posttranslational histone modifications (H3K4me3, H3K27me3, H3K9ac, H3K9me2/3) and MHC-I molecules (Qa-2 e IL-A88). Quantitative PCR reactions were performed to evaluate the expression of DNA modifications related enzymes (DNMT1, TET1 and TET2), MHC-I classical (JSP-1) and non-classical (NC1-4) isoforms, and imprinted genes expression (SNRPN, TSSC4 and PHDLA2). In the NT placenta there was an increase in the global methylation and decreased hydromethylation level at first trimester. Levels of H3K4me3 were constant at control placenta while increased in NT placenta. For H3K27me3, the levels decreased in control placenta and were stable at NT counterparts. At 60 days of pregnancy in NT placenta, were observed low levels of global H3K9ac, but no differences of H3K9me2/3 levels were found between pregnancy ages or type. We identified MHC-I at bovine placentomal trophoblast in all ages analyzed, with intensity variation of different isoforms. In general, low levels of DNA methylation at day 60 of pregnancy of TNCS placenta, indicates a compensatory mechanism to active genic expression. Since histone modifications, in this period, leads to repressive status, because bivalence of H3K4me3 and H3K27me3 and levels of H3K9ac were diminished. Also at day 60 of pregnancy of TNCS placenta, MHC-I is highly expressed and leads to maternal immune response against fetus tissue. In conclusion, several events are present and apparently contribute to early clone pregnancy instability, coinciding with high pregnancy losses in that phase

Realizamos neste trabalho a automatização da nova metodologia MHCalc, desenvolvida anteriormente em nosso laboratório, que teve como resultado o programa homônimo escrito em linguagem C para ambiente GNU/Linux, o qual avalia quantitativamente a seletividade de uma determinada proteína MHC. Esta avaliação quantitativa possibilita o estabelecimento de uma escala de preferência dos resíduos de ocorrência natural para cada uma das posições de interação da fenda de apresentação do MHC; por permutação destes resíduos, podem-se derivar regras de composição para peptídeos reconhecíveis em cada alelo estudado, inclusive na ausência de dados experimentais. A metodologia desenvolvida em nosso laboratório se baseia na avaliação da seletividade de cada bolsão independentemente, através da energia de interação do mesmo com cada aminoácido de ocorrência natural. O programa MHCalc utiliza o pacote de programas THORMM [Moret, 1998] de modelagem molecular para otimização geométrica das estruturas descritas, e tem como entrada unicamente um arquivo de coordenadas em formato POB contendo as coordenadas de um complexo MHC/peptídeo, tendo como saída uma tabela de dados contendo os resíduos de ocorrência natural em ordem de preferência para cada bolsão. Testamos o programa MHCalc para o complexo formado pela molécula HLA-DR1 (DRA DRB1 *01 01) e o peptídeo de Hemaglutinina, obtido no Brookhaven Protein Data Bank com a entrada 10LH, sendo este sistema escolhido por ser altamente estudado e com abundância de dados experimentais e teóricos para comparação de resultados. Até o presente momento obtivemos os dados referentes ao bolsão 1, os quais estão em pleno acordo com a literatura. / In this work we automated the new methodology MHCalc, developed previously in our laboratory, which resulted in the computational program with the same name, written in C language to GNU/Linux environment. The program MHCalc evaluates quantitatively the selectivity of a given MHC protein. This quantitative evaluation allows the establishment of a preference scale of the naturally occurring residues for each pocket of a MHC molecule. From such a study it may be derived composition rules to peptides recognizable in each allele studied, even in the absence of experimental data. The methodology developed in our laboratory is based on the selectivity evaluation of each pocket independently, through its interaction energy with each naturally occurring amino acid. The program MHCalc uses the molecular modeling package THORMM [Moret, 1998] to optimize the geometry of the structures described below, and needs as only entry the PDS like file with the coordinates of the MHC/peptide complex. The program finishes printing out a data file containing the naturally occurring residues in preference order for each pocket and the data used to order these residues. We tested the program MHCalc to the complex molecule HLA-DR1 (DRA DRB1 *01 01) with the Hemaglutinin peptide, obtained at the BrookHaven Prote in Data Bank (1 DLH entry). This system was chosen because it has been highly studied and so offers abundant experimental and theoretical data to compare our results. We obtained data referring to pocket 1 so far, which is in full agreement with the literature.

<p>U&nbsp; ovom&nbsp; radu&nbsp; sagledan&nbsp; je&nbsp; molekularni&nbsp; diverzitet,&nbsp; filogeografska&nbsp; struktura,<br />prostorna&nbsp; distribucija&nbsp; molekularnog&nbsp; diverziteta,&nbsp; kao&nbsp; i&nbsp; prisustvo&nbsp; selekcionih<br />signala i genetičkih signala lokalnih adaptacija kod 251 jedinke&nbsp; vrste&nbsp; Lepus<br />europaeus (Pallas,&nbsp; 1778)&nbsp; sa&nbsp; teritorije&nbsp; Evrope&nbsp; i&nbsp; Bliskog&nbsp; Istoka,&nbsp; a&nbsp; na&nbsp; osnovu<br />analize&nbsp; varijabilnosti&nbsp; sekvenci&nbsp; D&nbsp; petlje&nbsp; mtDNK, MT-ND2,&nbsp; MT-ND6,&nbsp; MHCDQA, MHC-DQB i TLR2 gena. Uočen je visok nivo parametara molekularnog<br />diverziteta&nbsp; za&nbsp; sve&nbsp; ispitivane&nbsp; molekularne&nbsp; markere.&nbsp; Utvrđeno&nbsp; je&nbsp; postojanje<br />filogeografske&nbsp; strukturiranosti&nbsp; vrste&nbsp; na&nbsp; osnovu&nbsp; mtDNK,&nbsp; kao&nbsp; i&nbsp; asimetričan<br />protok gena jedinki sa teritorije Anadolije na teritoriju Balkana&nbsp; na osnovu D<br />petlje mtDNK, MT-ND2 i MT-ND6 gena, dok je na osnovu sekvenci D petlje<br />mtDNK uočena gotovo tri puta veća stopa protoka gena sa Balkana u centralnu<br />i&nbsp; zapadnu&nbsp; Evropu.&nbsp; Utvrđeno&nbsp; je&nbsp; prisustvo&nbsp; signala&nbsp; poizivne&nbsp; selekcije&nbsp; u&nbsp; okviru<br />MT-ND6&nbsp; gena,&nbsp; kao&nbsp; i&nbsp; efekat&nbsp; klimatskih&nbsp; parametara&nbsp; (precipitacije)&nbsp; na<br />distribuciju&nbsp; proteinskih&nbsp; varijanti&nbsp; ND6C&nbsp; i&nbsp; ND6F,&nbsp; kao&nbsp; moguća&nbsp; posledica<br />regionalnih adaptacija na razlike u sredinskim uslovima. Pokazano je odsustvo<br />signala&nbsp; filogeografske&nbsp; strukturiranosti&nbsp; na&nbsp; osnovu MHC-DQA, MHC-DQB i<br />TLR2 gena.&nbsp; Uočeno&nbsp; je&nbsp; postojanje&nbsp; prostorne&nbsp; strukturiranosti&nbsp; na&nbsp; osnovu&nbsp; gena<br />imunskog&nbsp; sistema,&nbsp; i&nbsp; definisane&nbsp; su&nbsp; dve&nbsp; prostorne&nbsp; grupe,&nbsp; jedna&nbsp; koja&nbsp; je<br />obuhvatala&nbsp; jedinke&nbsp; sa&nbsp; teritorije&nbsp; Bliskog&nbsp; Istoka,&nbsp; i&nbsp; druga&nbsp; koja&nbsp; je&nbsp; obuhvatala<br />jedinke&nbsp; sa&nbsp; teritorije&nbsp; Evropi.&nbsp; Vi&scaron;e&nbsp; vrednosti&nbsp; parametara&nbsp; molekularnog<br />diverziteta uočene su u anadolijskoj grupi, u poređenju sa evropskom grupom.<br />Uočen je signal delovanja pozitivne i negativne selekcije u MHC-DQA i MHCDQB genima, kao i signal negativne selekcije u TLR2 genu. Pokazan je efekat<br />klimatskih&nbsp; parametara&nbsp; na&nbsp; distribuciju&nbsp; najzastupljenijih&nbsp; proteinskih&nbsp; varijanti<br />MHC-DQA&nbsp; i&nbsp; MHC-DQB&nbsp; gena kao&nbsp; indirektni&nbsp; pokazatelj&nbsp; imunogenetičkih<br />adaptacija&nbsp; na&nbsp; sredinski&nbsp; uslovljene&nbsp; pretpostavljene&nbsp; razlike&nbsp; u&nbsp; distribuciji<br />patogena.&nbsp; Mehanizam&nbsp; oblikovanja&nbsp; varijabilnosti&nbsp; MHC&nbsp; gena&nbsp; rezultat&nbsp; je<br />uzajamnog delovanja mutacija, rekombinacija i selekcije.</p> / <p>In&nbsp; this&nbsp; doctoral&nbsp; dissertation,&nbsp; molecular&nbsp; diversity,&nbsp; phylogeographic&nbsp; structure,<br />spatial&nbsp; distribution&nbsp; of&nbsp; molecular&nbsp; diversity,&nbsp; detection&nbsp; of&nbsp; possible&nbsp; selection<br />signals&nbsp; shaping&nbsp; the&nbsp; evolution of&nbsp; these&nbsp; genes,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; presence&nbsp; of<br />local/regional&nbsp; adaptations&nbsp; in&nbsp; correlation&nbsp; was&nbsp; examined&nbsp; in&nbsp; 251&nbsp; brown&nbsp; hares<br />from&nbsp; Europe&nbsp; and&nbsp; the&nbsp; Middle East&nbsp; based&nbsp; on&nbsp; the&nbsp; analyses&nbsp; of&nbsp; mitochondrial&nbsp; D<br />loop,&nbsp; mitochondrially&nbsp; Encoded&nbsp; NADH&nbsp; Dehydrogenase&nbsp; 2&nbsp; (MT-ND2),<br />mitochondrially&nbsp; Encoded&nbsp; NADH&nbsp; Dehydrogenase&nbsp; 6&nbsp; (MT-ND6),&nbsp; exon&nbsp; 2&nbsp; of<br />MHC Class II genes MHC-DQA,MHC-DQB and Toll Like Receptor 2 (TLR2)<br />gene sequences. A high level of molecular diversity was found based on the all<br />applied&nbsp; markers.&nbsp; Strong&nbsp; signal&nbsp; of&nbsp; phylogeographical&nbsp; and&nbsp; spatial&nbsp; structuring<br />was&nbsp; observed&nbsp; for&nbsp; mtDNA,&nbsp; most&nbsp; likely&nbsp; as&nbsp; a&nbsp; consequence&nbsp; of&nbsp; climatic<br />perturbations&nbsp; during&nbsp; the&nbsp; Pleistocene.&nbsp; The&nbsp; evolutionary&nbsp; development&nbsp; of&nbsp; hares<br />from&nbsp; Anatolia/Israel&nbsp; to&nbsp; the&nbsp; Balkans,&nbsp; and&nbsp; furthermore&nbsp; to&nbsp; central&nbsp; and&nbsp; western<br />Europe was suggested by several lines of evidences, which include dating the<br />population&nbsp; demography&nbsp; based&nbsp; on&nbsp; D-loop&nbsp; sequences,&nbsp; the&nbsp; observed&nbsp; migration<br />patterns,&nbsp; results&nbsp; of&nbsp; demographic&nbsp; tests,&nbsp; and&nbsp; apparent&nbsp; reduction&nbsp; in&nbsp; molecular<br />diversity&nbsp; indices&nbsp; along&nbsp; this&nbsp; trajectory.&nbsp; Positive&nbsp; selection&nbsp; acting&nbsp; on MT-ND6<br />gene&nbsp; was&nbsp; detected,&nbsp; together&nbsp; with&nbsp; significant&nbsp; climatic&nbsp; effect&nbsp; shaping&nbsp; the<br />distribution&nbsp; of&nbsp; the&nbsp; most&nbsp; prevalent&nbsp; protein&nbsp; variants&nbsp; found&nbsp; in&nbsp; this&nbsp; gene,<br />supposedly&nbsp; as&nbsp; a&nbsp; consequence&nbsp; to&nbsp; local/regional&nbsp; adaptations&nbsp; due&nbsp; to&nbsp; the<br />environmentally induced different energetic requirements and optimization of<br />OXPHOS&nbsp; genes.&nbsp; On&nbsp; the&nbsp; other&nbsp; side,&nbsp; less&nbsp; evident&nbsp; phylogeographic&nbsp; signal&nbsp; and<br />absence&nbsp; of&nbsp; strong&nbsp; structuring&nbsp; was&nbsp; revealed&nbsp; in&nbsp; MHC&nbsp; genes.&nbsp; High&nbsp; diversity&nbsp; at<br />MHC genes seems to be shaped by the interplay of recombination, selection<br />mechanisms&nbsp; and&nbsp; adaptations.&nbsp; Balancing&nbsp; selection&nbsp; seems&nbsp; to&nbsp; maintain&nbsp; a&nbsp; high<br />molecular&nbsp; diversity&nbsp; within&nbsp; these&nbsp; genes,&nbsp; while&nbsp; directional selection&nbsp; promotes<br />local/regional adaptations to pathogenic landscapes, as indirectly suggested by<br />a&nbsp; significant&nbsp; effect&nbsp; of&nbsp; climatic&nbsp; parameters&nbsp; on&nbsp; the&nbsp; distribution&nbsp; of&nbsp; protein<br />variants in both examined MHC genes.</p>

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Background: Arterial allografts are used as vascular conduits in the treatment of prosthetic graft infection. Immunosuppression decreases their rupture risk rate. However, immunosuppression can be unprofitable in florid infection. Previously, we confirmed inhibition of cell-mediated destruction of rat aortic grafts by delayed use of tacrolimus. In this work, we studied the influence of this protocol on the antibody-mediated rejection.