Submitted by admin tede (tede@pucgoias.edu.br) on 2018-06-19T18:06:59Z No. of bitstreams: 1 SAMARA SOCORRO SILVA PEREIRA.pdf: 764936 bytes, checksum: 434a8cd13e6701c05b6ea08edda95150 (MD5) / Made available in DSpace on 2018-06-19T18:06:59Z (GMT). No. of bitstreams: 1 SAMARA SOCORRO SILVA PEREIRA.pdf: 764936 bytes, checksum: 434a8cd13e6701c05b6ea08edda95150 (MD5) Previous issue date: 2018-03-14 / Copy Number Variation (CNV) has been associated with intelectual disability (ID) and this condition occur in approximately 2% of world population. Chromosomal Microarray Analysis (CMA) is being indicated as first-tier test for individuals with ID and has also helped to understand the mechanisms of CNV formation, classification of these rearrangements, type of recurrence, and its origin. The aim of this study was to infer the parental chromosome origin of de novo pathogenic CNV in patients with ID and their mechanisms of formation. Patients with clinical indications of ID were referred to Replicon Research Group/LaGene for G-band karyotyping. CMA approach was done for patients without numerical and/or structural rearrangements results in karyotype. After performing CMA and classification of CNVs, the parental origin of pathogenic CNVs was done using Mendelian error check based on SNPs markers available by ChAs software. In addition, the UCSC Genome Browser website was used to detect Low Copy Repeats (LCR) surrounding the CNVs to infer the mechanisms of their formation. In the period from 2013 to 2015 was performed G-band karyotyping in 290 patients with clinical indication of ID and a total of 193/290 (66.5%) were diagnosed by Karyotype. The group of patients who were not diagnosed using the karyotype, only 76/97 (78.3%) agreed to continue the investigation by CMA’s approach. After performing CMA, a total of 15 de novo pathogenic CNVs were observed, 10 CNV of loss and 5 CNV of gain, in 13/76 (17.1%) patients. The analysis of the parental origin showed 60% of CNVs are of maternal origin and 40% of paternal origin. It was not possible to detect the influence of parental age in the formation of CNVs. After analyzing the presence of surrounding LCRs, it was observed that 46.7% are recurrent CNVs and the mechanism of formation was Non- Allelic Homologous Recombination (NAHR), and 71.4% of these recurrent CNVs are of maternal origin. These data are in agreement with studies that affirm that the majority of CNVs of paternal origin are nonrecurrent due to germ cells replicate many times their genetic material in the pre-meiotic phase, being possible to infer the mechanism of formation of CNV that may have been by Microhomology-mediated break-induced replication (MMBIR) or Non-homologous end joining (NHEJ). / A variação no número de cópias (CNV) no genoma é um dos fatores etiológicos que pode desencadear a condição da deficiência intelectual (DI), sendo que esta condição atinge cerca de 2% da população mundial. A metodologia de análise cromossômica por microarranjo (CMA) além de ser indicada como teste de primeira escolha para pacientes com DI, tem ajudado também na compreensão da formação de CNVs e classificação destes rearranjos, quanto à patogenicidade, o tipo de recorrência e sua origem. E este estudo objetivou inferir a origem cromossômica parental das CNVs de novo patogênicas em pacientes com DI e seu mecanismo de formação. Os pacientes com indicação clínica de DI foram encaminhados ao Núcleo de Pesquisas Replicon/LaGene para realização do cariótipo com bandeamento GTG, e subsequentemente, os que não tiveram alteração numérica e/ou estrutural no cariótipo foram convidados a continuar a investigação em nível genômico, pela metodologia de CMA. Após realização do CMA e classificação das CNVs, foram realizadas a análise da origem parental das CNVs de novo patogênicas pela análise do erro mendeliano usando os marcadores de SNPs disponibilizado pelo software ChAS. Adicionalmente, foi usado o UCSC Genome Browser para detectar Repetições De Poucas Cópias (LCR) circundantes as CNVs para inferir o mecanismo de formação das mesmas. Foi realizado o cariótipo em 290 pacientes com indicação clínica de DI entre os anos de 2013 a 2015 e em 193/290 (66,5%) foram diagnosticados pelo cariótipo. Do conjunto de pacientes que não foram diagnosticados usando o cariótipo, apenas 76/97 (78,3%) aceitaram continuar a investigação pelo CMA. Após realizar o CMA, foi observado 15 CNVs de novo patogênicas, 10 CNVs de perda e 5 CNVs de ganho, em 13/76 (17,1%) pacientes. Na análise da origem parental, observou-se que 60% das CNVs são de origem materna e 40% de origem paterna. Não foi possível detectar a influência da idade parental na formação das CNVs. Ao analisar a presença de LCRs circundantes, observou-se que 46,7% das CNVs de novo patogênicas são recorrentes e o mecanismo de formação foi a Recombinação Homologa Não Alélica (NAHR), e 71,4% dessas CNVs recorrentes são de origem materna. Esses dados corroboram com os estudos que afirmam que a maioria das CNVs de origem paterna são não recorrentes devido às células germinativas replicarem inúmeras vezes o seu material genético na fase pré-meiótica, sendo possível inferir sobre o mecanismo de formação que pode ter sido por Replicação Induzida por Quebra e Mediada por Microhomologia (MMBIR) ou Junção de Extremidade Não Alélica (NHEJ).

Le génotypage d’une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d’identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l’implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d’identité. Dans une première étude, une délétion de 200Kb englobant l’ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d’implication de DPY19L2 s’élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène. / Performing a genome wide scan by SNP microarray on a Jordanian consanguineous family where five brothers were diagnosed with complete globozoospermia, we show in a first study that the four out of five analysed infertile brothers carried a homozygous deletion of 200 kb on chromosome 12 encompassing only DPY19L2. The gene encodes for a transmembrane protein and is surrounded by two low copy repeats (LCRs). Very similar deletions were found in three additional unrelated patients. Later, we have pursued our patient screen by recruiting a largest cohort of patients. Out of a total of 54 patients analysed, 36 (66.7%) showed a mutation in DPY19L2. Out of 36 mutated patients, 20 are homozygous deleted, 7 heterozygous composite and 4 showed a homozygous point mutation. We characterized a total of nine breakpoints that clustered in two recombination hotspots, both containing direct repeat elements. These findings confirm that the deletion is due to a nonallelic homologous recombination (NAHR) between the two LCRs. Thus, Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene.

BACKGROUND:Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes.RESULTS:Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome.CONCLUSIONS:We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in the most recent duplication are the main contributions of our study.

This thesis is an ethnography of Palestinian political factions in Lebanon through an immersion in the daily life of homes. It explores the nature of factions and faction membership from the vantage point of those who form their very basis. It asks how did Palestinian political factions, which are clearly made of people, come to be seen as autonomous bodies that are studied as a whole and spoken of in the singular (‘Fatah did this’ and ‘Hamas declared that’). Through a detailed account of the everyday practices of Palestinian refugees I problematise the underlying conceptualization of factions in the academic literature as bounded structures defined by their respective ideologies. I explore how factions appear in the daily life of Palestinian refugees in Lebanon; how Palestinians join factions; how their relationship evolves over time; how they demand, and at times obtain, aid; how and whether they participate in events organized by factions; and how factionalism affects their understandings of what factions are. This ethnographic approach reveals that what binds Palestinian refugees to factions is not the ideology or regional or international alliances of the factions. For example, young Palestinians do not join a faction based on whether it is Islamic, Marxist, or nationalist; rather they do so based on where they have friends or family, and sometimes depending on which faction has the closest youth centre to their home. In fact, it is those personal relationships, including those developed with other faction members that keep Palestinians affiliated to factions. Factions appear as a loose network of people held together by different degrees of trust and cohesion. Yet my work does not dismiss the fact that factions also appear as structures, as coherent entities. On the contrary, in the second part of this thesis, I trace another set of practices, that of aid distribution, criticism, physical representation, and factionalism, to show how factions metamorphose from loose networks based on interpersonal relations into impersonal structures defined by ideology. An examination of the everyday practices and representations of Palestinian political factions reveals how those structures come into being, how that operation creates and maintains a certain configuration of power in Palestinian society, and how factions remain the center of political life in the face of widespread condemnation.

The human ribosomal RNA genes are critically important for cell metabolism and viability. They code for the catalytic RNAs which, encased in a housing of more than 80 ribosomal proteins, link together amino acids by peptide bonds to generate all cellular proteins. Because the RNAs are not repeatedly translated, as is the case with messenger RNAs, multiple copies are required. The genes which code for the human ribosomal RNAs (rRNAs) are arranged as clusters of tandemly repeated sequences. Three of four catalytic RNAs are spliced from a single transcript. The genes are located on the short arms of the five acrocentric chromosomes (13, 14, 15, 21, and 22). The genes for the fourth rRNA are on chromosome 1q42, also arranged as a cluster of tandem repeats. The repeats are extremely similar in sequence, which makes them ideal for misalignment, non‐allelic homologous recombination (NAHR), and genomic destabilization during meiosis , replication, and damage repair. In this dissertation, I have used pulse‐field gel electrophoresis and in‐blot Southern hybridization to explore the physical structure of the human rRNA genes and determine their stability and heritability in normal, healthy individuals. I have also compared their structure in solid tumors compared to normal, healthy tissue from the same patient to determine whether dysregulated homologous recombination is an important means of genomic destabilization in cancer progression. Finally, I used the NCI‐60 panel of human cancer cell lines to compare the results from the pulsed‐field analysis, now called the gene cluster instability (GCI) assay, to two other indicators of homologous‐recombination-mediated genomic instability: sister chromatid exchange, and 5‐hydroxymethyl‐2’deoxyuridine sensitivity.

Le génotypage d'une famille jordanienne consanguine constituée de 5 frères globozoospermiques et de 3 frères fertiles sur puce Affymetrix, a permis d'identifier un nouveau gène responsable de la globozoospermie situé dans un intervalle de 6.4Mb en 12q14.2. Au regard de son expression prédominante dans le testicule et l'implication de son orthologue, chez C. elegans, dans la polarisation cellulaire, le gène DPY19L2 est un gène candidat parfait. Le gène, codant pour une protéine transmembranaire, est flanqué par deux séquences répétées (LCRs) qui partagent 96,5% d'identité. Dans une première étude, une délétion de 200Kb englobant l'ensemble du gène a été mise en évidence chez les 4 frères infertiles de cette famille jordanienne ainsi que chez 3 autres patients non apparentés. Nous avons ensuite recruté une plus grande cohorte de 54 patients. Parmi ces patients, 20 sont homozygotes pour la délétion de DPY19L2 et 7 sont hétérozygotes composites associant la délétion hétérozygote et une mutation ponctuelle. En outre, nous avons identifié, 4 patients avec des mutations ponctuelles homozygotes. Par conséquent, la fréquence d'implication de DPY19L2 s'élève à 66.7%. En tout, 9 points de cassures, regroupés en deux hotspots au sein des LCRs, ont pu être mis en évidence. Ceci confirme que le mécanisme sous-jacent de la délétion est une recombinaison homologue non allélique (NAHR) entre les LCRs. En conclusion, nous confirmons que DPY19L2 est le principal gène de la globozoospermie et nous élargissons le spectre des mutations possible dans ce gène.

Les duplicacions segmentàries (DSs), o també anomenades duplicons o Low copy Repeats (LCRs), són regions de coma mínim 1 kb amb un alt nivell d'identitat (>90%), que estan presents almenys dues vegades en el genoma. La regió 8p23.1 consta de 6.5 Mb a la part distal del braç curt del cromosoma 8 i està flanquejada per duplicacions segmentàries. Degut a la seva arquitectura genòmica aquesta regió és susceptible a patir reordenaments mediats per recombinació homòloga no al·lèlica entre les DSs, com per exemple la inversió polimòrfica de 8p23.1 [inv(8)(p23)], present en un de cada quatre individus de la població general europea i japonesa, així com d'altres reorganitzacions menys corrents.El treball realitzat en aquesta tesi doctoral pretén aprofundir en la caracterització de la complexa arquitectura genòmica d'aquesta regió. En la nostra primera aproximació a l'estudi de les DSs que flanquegen la regió cromosòmica 8p23.1, es va identificar una nova família gènica específica de primats, la família gènica FAM90A.Així, bona part d'aquesta tesi doctoral està centrada en l'anàlisi de l'origen, formació, evolució i expansió de FAM90A en els homínids. Per altra banda també s'ha analitzant en detall la variabilitat de FAM90A com a variant en número de còpia (CNV) en diferents poblacions humanes.Finalment, s'ha establert la freqüència de la inversió que afecta a 8p23.1 en població espanyola. També s'ha procedit a genotipar diversos individus homozigots per la inversió i s'ha predit l' estatus de la inversió en 150 individus del projecte HapMap i s'ha analitzat l'efecte que té aquesta reorganització sobre els nivells d'expressió dels gens de la regió. / Segmental duplications (SDs), also known as duplicons or Low Copy Repeats (LCRs), are regions of a minimum of 1 kb with a high sequence identity level (>90%), which are present at least two times in the genome. The 8p23.1 region extends 6.5 Mb at the distal part of the short arm of chromosome 8 and it is flanked by segmental duplications. Due to its genomic architecture the region is prone to suffer rearrangements mediated by non-allelic homologous recombination between these SDs, such as the polymorphic inversion of 8p23.1 [inv(8)(p23)], which is present in one out of every four of European and Japanese general population individuals, as well as other less frequent rearrangements.The aim of the work presented in this doctoral thesis is to get insights in the characterization of the genomic architecture of this complex region. Our first approach to study the SDs flanking 8p23.1 region resulted in the identification of a novel gene family which is primate specific, the FAM90A gene family. Thus, this doctoral thesis is mainly focused on the analysis of the origins, formation, evolution and expansion of FAM90A in hominoids. It has also been analyzed in detail the variability of FAM90A as a copy number variant (CNV) in different human populations.Finally, it has been established the frequency of the inversion affecting 8p23.1 region in the Spanish population. Several homozygous inverted individuals have been genotyped and the status for the inversion has been predicted for 150 HapMap individuals, as well as the effect of this rearrangement on the gene expression levels of the genes contained in the region.