Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-10-04T22:20:39Z No. of bitstreams: 1 Tese Soniane Costa.pdf: 5470162 bytes, checksum: c446c8cec1a4893eca8260700896103a (MD5) / Made available in DSpace on 2017-10-04T22:20:39Z (GMT). No. of bitstreams: 1 Tese Soniane Costa.pdf: 5470162 bytes, checksum: c446c8cec1a4893eca8260700896103a (MD5) Previous issue date: 2017-03-24 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / The decline of guava is a complex disease, which causes progressive galls in guava roots parasitized by Meloidogyne enterolobii. The objective of the present study is to study the genetic divergence in guava and ara?azeiros accessions by means of SNPs markers developed for Eucalyptus to serve as a subsidy for activities of genetic resources and improvement of guava, such as the broad genomic association and to estimate the number of genes involved in resistance to M. enterolobii, using F2 population of Psidium guajava and P. guineense hybrid.In the evaluation of the genetic diversity of Psidium accessions, presented in chapter I, the transferability of Eucalyptus SNPs to Psidium for application in analyzes of genetic divergence and other applications, including studies of genome association. The segregation results and broad sense heritability estimates in population F2, presented in chapter II, support dominance model controlled by two genes, with epistatic effects, and the presence of only one dominant allele conditions the resistance of the hybrid of P. guajava x P. guineense for M. enterolobii. New hybrids of P. guajava with other accessions of wild Psidium should be developed and evaluated in order to increase the sources of resistance to the pathogen, allowing effective control of the same in commercial areas of guava. Seven markers associated with resistance to Meloidogyne enterolobii in Psidium species presented in Chapter III were identified.The results also suggest the existence of different sources of resistance to the nematode, since several SNPs were identified on chromosome 3 of Eucalyptus. / O decl?nio da goiabeira ? uma doen?a complexa, que causa a podrid?o progressiva das ra?zes das goiabeiras parasitadas por Meloidogyneenterolobii. O objetivo do presente estudo ? estudar a diverg?ncia gen?tica em acessos de goiabeira e ara?azeiros por meio de marcadores SNPs (Polimorfismo de nucleot?deo ?nico) desenvolvidos para Eucalyptus, que servir?o de subs?dio as atividades de recursos gen?ticos e melhoramento da goiabeira, bem como a associa??o gen?mica ampla (GWAS), e estimar o n?mero de genes envolvidos na resist?ncia a M. enterolobii, tendo como refer?ncia a segrega??o em popula??o F2 de h?brido de Psidium guajava x P. guineense. Na avalia??o da diversidade gen?tica dos acessos de Psidium, apresentado no cap?tulo I, relata-se a transferibilidade de SNPs de Eucalyptus para Psidium, estes podem ser utilizados em an?lises de diverg?ncia gen?tica e outras aplica??es, incluindo estudos de associa??o do genoma. Os resultados de segrega??oe as estimativas de herdabilidade no sentido amplo em popula??o F2, apresentado no cap?tulo II, suportam modelo de resist?ncia dominante controlada por dois genes, com efeitos epist?ticos, sendo que, a presen?a de apenas um alelo dominante condiciona a resist?ncia do h?brido de P. guajava x P. guineensepara M.enterolobii. Novos h?bridos de P. guajava com outros acessos de Psidium silvestres devem ser desenvolvidos e avaliados de forma a aumentar as fontes de resist?ncia ao pat?geno, possibilitando efetivo controle do mesmo em ?reas comerciais de goiabeira. Foram identificados sete marcadores associados ? resist?ncia a Meloidogyne enterolobii em esp?cies de Psidium apresentado no cap?tulo III. Os resultados sugerem ainda a exist?ncia de diferentes fontes de resist?ncia ao nematoide, pois foram identificados v?rios SNPs no cromossomo 3 de Eucalyptus.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-08-14T21:36:36Z No. of bitstreams: 1 WilliamFariasPortoTese2017.pdf: 13965916 bytes, checksum: 627497be290e274ade4f2d64a0b0d119 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-08-14T21:37:32Z (GMT) No. of bitstreams: 1 WilliamFariasPortoTese2017.pdf: 13965916 bytes, checksum: 627497be290e274ade4f2d64a0b0d119 (MD5) / Made available in DSpace on 2018-08-14T21:37:33Z (GMT). No. of bitstreams: 1 WilliamFariasPortoTese2017.pdf: 13965916 bytes, checksum: 627497be290e274ade4f2d64a0b0d119 (MD5) Previous issue date: 2017-08-08 / Antimicrobial peptides (AMPs) are part of the innate immune system. Genetic modifications can lead to the imbalance in the production of AMPs, which in turn, can lead to several inflammatory and/or infectious conditions. In this context, the identification and characterization of AMPs variants caused by point mutations are important to medical monitoring of bearers of such mutations; mainly due to the fact that the effectiveness of conventional antimicrobial agents has been reduced due to the development of resistance by bacteria, a breakthrough of new drugs is made. In this context, synthetic AMPs, generated through several rational design methods, have been proposed as an alternative. Thus, aiming at solutions to this scenario, the present work presents two new approaches, which consist of a model for the prediction of activities of variants of human defensins; and the computer-aided design of plant peptides. In the first approach, it was elaborated a system of median lethal dose prediction, correlating previously published data and the solvation potential energy of the variants. This model was applied to human defenses, HD5 and HBD1, which in turn showed that several variants may be less potent and consequently their carriers may be more susceptible to bacterial infections. In this way, in the second approach, the guava peptide, Pg-AMP1, was used as a model for the development of new synthetic peptides, the guavanins. Structural analyzes of Pg- AMP1 indicated an extremely flexible and variable structure. Thus, a genetic algorithm for computer-aided rational design was applied to obtain a more stable structure. The prototype, guavanin 2, presented α-helix structuring in hydrophobic environments, and showed 100% efficacy against Gram-negative bacteria at low concentrations through the rupture of the bacterial membrane and causing hyperpolarization of the same. In sum, the methodologies and the molecules developed here bring new perspectives for the treatment of infections. / Os peptídeos antimicrobianos (PAMs) fazem parte do sistema imune inato. Alterações genéticas podem levar ao desequilíbrio em sua produção, podendo gerar diversos quadros inflamatórios e/ou infecciosos. Neste contexto, a identificação e caracterização de variantes de PAMs geradas por mutações pontuais em seus respectivos genes são importantes para o acompanhamento médico dos portadores destas mutações; principalmente pelo fato de que a eficácia dos antimicrobianos convencionais está sendo reduzida devido ao desenvolvimento de resistência por parte das bactérias, tornando necessário o desenvolvimento de novos fármacos. Desta forma, PAMs sintéticos, gerados por meio de métodos de desenho racional, têm sido propostos como uma alternativa. Assim, visando desenvolver soluções para este cenário, o presente trabalho apresenta duas novas abordagens, que consistem em um modelo para predição de atividade de variantes de defensinas humanas; e o desenho assistido por computador de peptídeos de planta. Na primeira abordagem foi elaborado um sistema de predição de dose letal mediana correlacionando dados previamente publicados e a energia potencial de solvatação das variantes. Este modelo foi aplicado a defensinas humanas, HD5 e HBD1, indicando que diversas variantes são menos potentes e consequentemente seus portadores podem ser mais susceptíveis às infecções bacterianas. Neste sentido, na segunda abordagem, o peptídeo de goiaba, Pg-AMP1, foi utilizado como modelo para o desenvolvimento de novos peptídeos sintéticos, as guavaninas. As análises estruturais do Pg-AMP1 indicaram uma estrutura extremamente flexível e variável. Desse modo, um algoritmo genético para o desenho racional assistido por computador foi aplicado para a obtenção de uma estrutura mais estável. O protótipo, guavanina 2, apresentou estruturação em α-hélice em ambientes hidrofóbicos, e mostrou eficácia de 100% contra bactérias Gram-negativas em baixas concentrações através do rompimento da membrana bacteriana e causando hiperpolarização da mesma. Em suma, as metodologias e as moléculas desenvolvidas aqui trazem novas perspectivas para o tratamento de infecções.

Le complexe majeur d'histocompatibilité (CMH) est une région génomique complexe des Vertébrés, encore imparfaitement connue chez le poulet, qui présente à certains locus une très grande variabilité génétique et qui joue un rôle central dans l'organisation de la réponse immunitaire d'un animal aux pathologies infectieuses. Le CMH est également une région de choix pour étudier le déterminisme génétique de l'adaptation aux agents pathogènes dans un contexte évolutif. De plus une meilleure connaissance du déterminisme génétique de la réponse immunitaire contre les agents pathogènes est un atout important pour développer une stratégie globale de lutte contre les maladies infectieuses. Nous avons donc entrepris d'utiliser les récents outils de la génomique, notamment des marqueurs génétiques de type SNP (Single Nucleotide Polymorphism) afin de caractériser la région B du CMH du poulet. En premier lieu, la diversité génétique a été évaluée dans plus de 80 races ou populations en utilisant le marqueur LEI0258. Ensuite, afin de couvrir l'ensemble de la région à l'aide de marqueurs SNPs, nous avons choisi d'identifier ces polymorphismes par reséquençage de 11 gènes d'intérêt et par comparaison des séquences obtenues entre elles et avec la séquence du génome et les séquences de référence disponibles dans les bases de données. En parallèle, ces travaux ont également permis d'améliorer la connaissance de certains gènes, notamment trois gènes de classe II non classiques de type DM. Une puce de 96 SNPs dédiée à la région B du CMH du poulet a été produite et devrait rapidement permettre d'exploiter des études d'infections expérimentales réalisées à l'INRA.

<p>Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. </p><p>A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. </p><p>Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations.</p><p>Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family.</p><p>The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.</p>

Contexte. Les phénotypes ABO et Rh(D) des donneurs de sang ainsi que des patients transfusés sont analysés de façon routinière pour assurer une complète compatibilité. Ces analyses sont accomplies par agglutination suite à une réaction anticorps-antigènes. Cependant, pour des questions de coûts et de temps d’analyses faramineux, les dons de sang ne sont pas testés sur une base routinière pour les antigènes mineurs du sang. Cette lacune peut résulter à une allo-immunisation des patients receveurs contre un ou plusieurs antigènes mineurs et ainsi amener des sévères complications pour de futures transfusions. Plan d’étude et Méthodes. Pour ainsi aborder le problème, nous avons produit un panel génétique basé sur la technologie « GenomeLab _SNPstream» de Beckman Coulter, dans l’optique d’analyser simultanément 22 antigènes mineurs du sang. La source d’ADN provient des globules blancs des patients préalablement isolés sur papiers FTA. Résultats. Les résultats démontrent que le taux de discordance des génotypes, mesuré par la corrélation des résultats de génotypage venant des deux directions de l’ADN, ainsi que le taux d’échec de génotypage sont très bas (0,1%). Également, la corrélation entre les résultats de phénotypes prédit par génotypage et les phénotypes réels obtenus par sérologie des globules rouges et plaquettes sanguines, varient entre 97% et 100%. Les erreurs expérimentales ou encore de traitement des bases de données ainsi que de rares polymorphismes influençant la conformation des antigènes, pourraient expliquer les différences de résultats. Cependant, compte tenu du fait que les résultats de phénotypages obtenus par génotypes seront toujours co-vérifiés avant toute transfusion sanguine par les technologies standards approuvés par les instances gouvernementales, les taux de corrélation obtenus sont de loin supérieurs aux critères de succès attendus pour le projet. Conclusion. Le profilage génétique des antigènes mineurs du sang permettra de créer une banque informatique centralisée des phénotypes des donneurs, permettant ainsi aux banques de sang de rapidement retrouver les profiles compatibles entre les donneurs et les receveurs. / Background. ABO and Rh(D) phenotyping of both blood donors and transfused patients is routinely performed by blood banks to ensure compatibility. These analyses are done by antibody-based agglutination assays. However, blood is not routinely tested for minor blood group antigens on a regular basis because of cost and time constraints. This can result in alloimmunization of the patient against one or more minor antigens and may complicate future transfusions. Study design and Methods. To address this problem, we have generated an assay on the GenomeLab SNPstream genotyping system (Beckman Coulter, Fullerton, CA) to simultaneously test polymorphisms linked to 22 different blood antigens using donor’s DNA isolated from minute amounts of white blood cells. Results. The results showed that both the error rate of the assay, as measured by the strand concordance rate, and the no-call rate were very low (0.1%). The concordance rate with the actual red blood cell and platelet serology data varied from 97 to 100%. Experimental or database errors as well as rare polymorphisms contributing to antigen conformation could explain the observed differences. However, these rates are well above requirements since phenotyping and cross-matching will always be performed prior to transfusion. Conclusion. Molecular profiling of blood donors for minor red blood cell and platelet antigens will give blood banks instant access to many different compatible donors through the set-up of a centralized data storage system.

Background: Parkinson’s disease (PD) is a complex disease with a multi-factorial aetiology, comprising both genetic and environmental risk factors. The disease pathology is progressive and neurodegenerative where dopaminergic nerve cell death occurs predominantly in the substantia nigra pars compacta (SNpc) with the subsequent loss of the dopamine neurotransmitter in the basal ganglia. The most significant risk factors for PD include an advancing age and a family history of the disease, while environmental and lifestyle risk factors such as pesticide exposure and smoking are widely accepted as risk altering exposures. Currently up to 10% of PD is attributed to Mendelian inherited PD at one of 13 PARK loci in 9 genes. The pursuit of common susceptibility alleles for idiopathic PD has proven challenging with only a few loci reproducibility associated with an altered risk. The aim of this thesis is to study, using a candidate gene case-control design, the potential role of genetic variants in PD. The APOE candidate gene was hypothesized to modify the risk of PD as it is a proven modifier of Alzheimer’s disease (AD). The common pathological finding in PD of elevated levels of iron within the SNpc is proposed to increase the oxidative state of the nerve cells and predispose the dopaminergic neurons to apoptosis. Therefore, susceptibility alleles within the candidate genes that regulate iron metabolism and homeostasis are hypothesized to alter iron metabolism and predispose to iron-induced neurodegeneration in PD. Missense variants and common “tagging” SNPs with the HFE, Transferrin and Transferrin Receptor genes are investigated extensively in this thesis. Finally, autosomal recessively inherited PD can result from mutations in the parkin gene at the PARK2 locus. The final hypothesis explored in this thesis suggests that non-deleterious missense variants in the parkin gene modify the risk for developing sporadic PD. Further genetic variation in the parkin gene such as exon rearrangements is a frequently reported mutation where heterozygosity for these rearrangements may increase the risk of PD. Heterozygous deletions or duplications of exons in the parkin gene provide technical challenges for their detection. In this thesis a novel assay for the detection of these mutations is investigated. Methods: Genotyping was performed using PCR-RFLP for genetic variants in the APOE (E2 and E4 alleles), HFE (C282Y, H63D and S65C), Transferrin receptor (TfR; S142G), Transferrin (Tfn; P570S and G258S), IREB2 genes (L159V) and the parkin gene (S167N, R366W and V380L) in a cohort of 425 PD cases and 387 controls recruited from throughout Queensland, Australia. A tagged SNP high-throughput genotyping approach was then employed to try to replicate single SNP associations in 6 iron-related genes using a cohort of 1034 PD cases and 774 controls. These genetic variants were analysed for direct association with PD risk, age of onset effects as well as potential gene x gene (GxG) and gene x environment (GxE) interactions. Additionally, a quantitative PCR assay was developed to detect heterozygous deletions and duplications within the parkin gene and utilised to screen 43 YOPD cases for these mutations. Results: The initial study of the HFE C282Y variant revealed a significant protective association with PD in the two independent cohorts studied. Further study did not reveal significant associations with PD for the other HFE variants or missense variants within the Tfn and TfR genes. When analysed for GxE interactions, the C282Y, P589S and G277S variants showed evidence for an increased risk of PD in synergy with pesticide and herbicide exposure. Carriers of the risk variant and with toxin exposure were at two-fold increased risk of PD, although the number of individuals in this category was small. A further investigation of the role of common genetic polymorphisms in iron genes revealed only one of the 20 SNPs genotyped using high-throughput multiplex methods, remained significantly associated with PD after correction for age and sex. The rs198855 SNP is downstream of the HFE gene and further implicates a role for HFE in PD. The APOE E4 allele demonstrated modifying effects for the age of PD onset, restricted to the female cases. Analysis of the parkin missense variants also demonstrated a modifying effect on the age of PD onset in carriers of the S167N variant, with putative interactions between the APOE E4 allele, a family history of PD and toxin exposure that further reduced the age of onset. Twenty individuals of the 43 YOPD cases screened demonstrated heterozygous parkin exon rearrangements using the novel qPCR method. Conclusions: Non-synonymous variants within iron-related genes or the parkin gene putatively interact with herbicide and pesticide exposure to increase the risk of PD or modify the phenotype, highlighting the need for future studies to address the multi-factorial aetiology of PD in their study design and analysis. This thesis provides evidence for the association between genetic variation within the HFE locus and PD and for the APOE E4 allele as a modifier of PD.

A identificação de regiões cromossômicas, ou blocos de dependência dentro do genoma humano, que são transmitidas em conjunto para seus descendentes (haplótipos) tem sido um desafio e alvo de várias iniciativas de pesquisa, muitas delas utilizando dados de plataformas de marcadores moleculares do tipo SNP (Single Nucleotide Polymorphisms - SNPs), com alta densidade dentro do DNA humano. Este trabalho faz uso de uma modelagem estocástica de campos Markovianos de alcance variável, em uma amostra estratificada de diferentes populações, para encontrar blocos de SNPs, independentes entre si, estruturando assim o genoma em regiões ilhadas de dependência. Foram utilizados dados públicos de SNPs de diferentes populações mundiais (projeto HapMap), além de uma amostra da população brasileira. As regiões de dependência configuram janelas de influência as quais foram usadas para caracterizar as diferentes populações de acordo com sua ancestralidade e os resultados obtidos mostraram que as janelas da população brasileira têm, em média, tamanho maior, evidenciando a sua história recente de miscigenação. É também proposta uma otimização da função de verossimilhança do problema para obter as janelas de consenso maximais de todas as populações. Dada uma determinada janela de consenso, uma medida de distância apropriada para variáveis categóricas, é adotada para medir sua homogeneidade/heterogeneidade. Janelas homogêneas foram identificadas na região HLA (Human Leukocyte Antigen) do genoma, a qual está associada à resposta imunológica. O tamanho médio dessas janelas foi maior do que a média encontrada no restante do cromossomo, confirmando a alta dependência existente nesta região, considerada como bastante conservada na evolução humana. Finalmente, considerando a distribuição dos SNPs entre as populações nas janelas mais heterogêneas, a Análise de Correspondência foi aplicada na construção de um classificador capaz de determinar o percentual relativo de ancestralidade de um indivíduo, o qual, submetido à validação, obteve uma eficiência de 90% de acerto da população originária. / The identification of chromosome regions, or dependency blocks in the human genome, that are transmitted together to offspring (haploids) has been a challenge and object of several research initiatives, many of them using platforms of molecular markers such as SNP (Single Nucleotide Polymorphisms), with high density inside the human DNA. This work makes use of a stochastic modeling of Markov random fields, in a stratified sample of different populations, to find SNPs blocks, independent of each other, thus structuring the genome in stranded regions of dependency. Public data from different worldwide populations were used (HapMap project), beyond a Brazilian population. The dependence regions constitute windows of influence which were used to characterize the different populations according of their ancestry and the results showed that the Brazilian populations windows have, on average, a bigger size, showing their recent history of admixture. It is also proposed an optimization of likelihood function of the problem for the maximal windows of consensus from all populations. Given a particular window of consensus, a distance measure appropriated to categorical variables, it is adopted to evaluate its homogeneity/heterogeneity. Homogeneous windows were identified within region of genome called HLA (Human Leukocyte Antigen), which is associated with the immune response. The average size of these windows was bigger than the average found in the rest of the chromosome, confirming the high dependence verified in this region, considered highly conserved in the human evolution. Finally, considering the distribution of the SNPs among the populations in the most heterogeneous windows, the Correspondence Analysis was applied to build a classifier able to determine, for a given individual, the ancestry proportion from each population considered, which, submitted to a validation, obtained a 90% accuracy of the original population.

A identificação de regiões cromossômicas, ou blocos de dependência dentro do genoma humano, que são transmitidas em conjunto para seus descendentes (haplótipos) tem sido um desafio e alvo de várias iniciativas de pesquisa, muitas delas utilizando dados de plataformas de marcadores moleculares do tipo SNP (Single Nucleotide Polymorphisms - SNPs), com alta densidade dentro do DNA humano. Este trabalho faz uso de uma modelagem estocástica de campos Markovianos de alcance variável, em uma amostra estratificada de diferentes populações, para encontrar blocos de SNPs, independentes entre si, estruturando assim o genoma em regiões ilhadas de dependência. Foram utilizados dados públicos de SNPs de diferentes populações mundiais (projeto HapMap), além de uma amostra da população brasileira. As regiões de dependência configuram janelas de influência as quais foram usadas para caracterizar as diferentes populações de acordo com sua ancestralidade e os resultados obtidos mostraram que as janelas da população brasileira têm, em média, tamanho maior, evidenciando a sua história recente de miscigenação. É também proposta uma otimização da função de verossimilhança do problema para obter as janelas de consenso maximais de todas as populações. Dada uma determinada janela de consenso, uma medida de distância apropriada para variáveis categóricas, é adotada para medir sua homogeneidade/heterogeneidade. Janelas homogêneas foram identificadas na região HLA (Human Leukocyte Antigen) do genoma, a qual está associada à resposta imunológica. O tamanho médio dessas janelas foi maior do que a média encontrada no restante do cromossomo, confirmando a alta dependência existente nesta região, considerada como bastante conservada na evolução humana. Finalmente, considerando a distribuição dos SNPs entre as populações nas janelas mais heterogêneas, a Análise de Correspondência foi aplicada na construção de um classificador capaz de determinar o percentual relativo de ancestralidade de um indivíduo, o qual, submetido à validação, obteve uma eficiência de 90% de acerto da população originária. / The identification of chromosome regions, or dependency blocks in the human genome, that are transmitted together to offspring (haploids) has been a challenge and object of several research initiatives, many of them using platforms of molecular markers such as SNP (Single Nucleotide Polymorphisms), with high density inside the human DNA. This work makes use of a stochastic modeling of Markov random fields, in a stratified sample of different populations, to find SNPs blocks, independent of each other, thus structuring the genome in stranded regions of dependency. Public data from different worldwide populations were used (HapMap project), beyond a Brazilian population. The dependence regions constitute windows of influence which were used to characterize the different populations according of their ancestry and the results showed that the Brazilian populations windows have, on average, a bigger size, showing their recent history of admixture. It is also proposed an optimization of likelihood function of the problem for the maximal windows of consensus from all populations. Given a particular window of consensus, a distance measure appropriated to categorical variables, it is adopted to evaluate its homogeneity/heterogeneity. Homogeneous windows were identified within region of genome called HLA (Human Leukocyte Antigen), which is associated with the immune response. The average size of these windows was bigger than the average found in the rest of the chromosome, confirming the high dependence verified in this region, considered highly conserved in the human evolution. Finally, considering the distribution of the SNPs among the populations in the most heterogeneous windows, the Correspondence Analysis was applied to build a classifier able to determine, for a given individual, the ancestry proportion from each population considered, which, submitted to a validation, obtained a 90% accuracy of the original population.

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...