Peu d’hommes sont touchés par les maladies auto-immunes (MAI), maladies où la réponse immune est très forte et attaque l’hôte. La polyarthrite rhumatoïde (PR), une maladie inflammatoire chronique, suit cette règle avec 3 femmes pour 1 homme atteint. Dans cette thèse, nous analysons les différences en fonction du sexe et les raisons d’un tel biais. D’après des observations chez l’animal, nous nous sommes demandés si les rares hommes atteints de PR ont une augmentation du nombre de copies d’un gène impliqué dans la réponse immune et porté par le chromosome (Chr) X. Contrairement aux femmes, les hommes n’ont qu’un Chr X et de ce fait qu’une copie de ce gène. Cependant, nous avons montré par différentes techniques, que ces patients avaient 10% de cellules portant 2 copies de ce gène, et que cette augmentation venait de cellules ayant 2 Chr X. Nos recherches soulignent l’importance du Chr X dans l’auto-immunité et ouvrent un nouveau champ d'investigation pour les hommes atteints de MAI. / Few men are affected by autoimmune diseases (AID), diseases where the immune response is very strong and attacks the host. Rheumatoid arthritis (RA), a chronic inflammatory disease, follows this rule with 3 women affected for 1 man. In this thesis, we analyse gender differences and the reasons for such bias. Based on observations in animals, we wondered whether the rare men with RA have an increased copy number of a gene involved in the immune response and carried by the X chromosome (Chr). Unlike women, men have only one X Chr and one copy of this gene. However, we showed by different techniques that these patients had 10% of cells carrying 2 copies of this gene, and that this increase came from cells with 2 X Chr. Our research emphasizes the importance of the X Chr in autoimmunity and opens up a new field of investigation for men with AID.

Orientadora: Regina Maria Barreto Cicarelli / Banca: Raquel Mantuaneli Scarel Caminaga / Banca: João Aristeu da Rosa / Banca: Leonor Gusmão / Banca: Rodrigo Rodenbusch / Resumo: A análise dos polimorfismos do DNA é a melhor ferramenta encontrada para resolução de casos de identificação humana, sendo os marcadores STRs (short tandem repeat) localizados em regiões autossômicas os principais e mais utilizados para esta finalidade. Apesar da indiscutível reprodutibilidade destes marcadores, quando são analisados em amostras degradadas podem não apresentar bons resultados, o que dificulta a resolução dos casos. Assim, há a necessidade de uma alternativa e, atualmente, a mais indicada é a utilização dos polimorfismos bialélicos. A análise do cromossomo sexual X também vem ganhando importância significativa no contexto forense, devido ao seu padrão de transmissão entre os genitores. Neste trabalho, caracterizamos as populações brasileiras do estado do Mato Grosso e do Distrito Federal pela análise de 32 polimorfismos de inserção/deleção no cromossomo X (X-InDels), tendo em vista que os dados destes polimorfismos nestas populações são ainda inéditos, e para que esses marcadores também possam no futuro auxiliar a resolução de casos forenses em nosso país. Para identificar a diversidade genética foram analisados os perfis genotípicos de 303 indivíduos não aparentados nascidos no estado do Mato Grosso e 179 indivíduos não aparentados e residentes em Brasília. Os resultados indicam que o painel dos 32 X-Indels é bastante eficiente para a sua finalidade, sendo que praticamente todos os marcadores se mostraram altamente informativos para as populações estudadas. O... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The analysis of DNA polymorphisms is the best tool found for solving cases of human identification, and the Short Tandem Repeat (STR) markers used in the autosomal regions are the main and most marker used for this purpose. Despite the undeniable reproducibility of these markers, when analyzed in degraded samples they may not present good results, which makes it difficult to solve the cases. Because of this, there is a need for an alternative tool and currently the most indicated is the use of the biallelic polymorphisms. In this way, the analysis of the sex chromosome X has gained significant importance in the forensic context, due to its pattern of transmission between the parents. In this work, we characterize the Brazilian populations of the state of Mato Grosso and the Federal District by the analysis of 32 insertion/deletion polymorphisms on the X chromosome (X-InDels), considering that the data of this polymorphism in these populations are still unpublished, and for that these markers may also in the future help the resolution of forensic cases in our country. To identify the genetic diversity, the genotypic profiles of 303 unrelated individuals born in the state of Mato Grosso and 179 unrelated individuals living in Brasília were analyzed. The results shows that the 32 X-Indels panel is very efficient to its purpose, being almost all markers highly informative for the studied populations. The Hardy-Weinberg equilibrium test was performed on the female samples from bot... (Complete abstract click electronic access below) / Doutor

A compensação da dosagem gênica entre fêmeas XX e machos XY em mamíferos é adquirida através de um complexo mecanismo epigenético que resulta na inativação de grande parte de um dos cromossomos X nas células femininas. O processo de inativação do cromossomo X (XCI) se inicia cedo durante a embriogênese, concomitantemente à diferenciação celular, e envolve a aquisição de modificações epigenéticas características do cromossomo X inativo (Xi). Uma estabelecido, o padrão de inativação é estavelmente mantido através de todas as mitoses celulares subsequentes e por toda a vida do organismo (exceto para células germinativas que sofrem reativação do Xi). Os mecanismos envolvidos na iniciação e estabelecimento da XCI foram extensivamente estudados, especialmente em camundongos. Embora algumas características epigenéticas associadas à manutenção da XCI tenham sido descritas, a identidade e modo específico de ação de fatores envolvidos durante essa fase da XCI são aspectos ainda não bem compreendidos. Além disso, o processo de XCI apresenta diferenças importantes entre humanos e camundongos e estudos direcionados para a identificação de novos componentes envolvidos na manutenção da XCI em humanos tornam-se de fundamental importância. Triagens funcionais genômicas por bibliotecas de shRNAs constituem uma ferramenta poderosa para a identificação de genes envolvidos em diferentes mecanismos celulares e vias bioquímicas. Sendo assim, utilizamos essa ferramenta para triar genes envolvidos na manutenção da XCI em humanos. Células somáticas femininas primárias HPRT+/-/HPRT- foram transduzidas com uma biblioteca lentiviral de shRNAs e posteriormente tratadas em meio de cultura contendo a droga HAT para seleção de células HPRT+ nas quais esperava-se que o cromossomo Xi presente tivesse sofrido reativação em decorrência do knockdown de genes envolvidos na manutenção da XCI. Essa estratégia nos permitiu identificar 20 novos genes candidatos a estarem envolvidos na manutenção da XCI. Esses candidatos deverão ser avaliados individualmente para confirmar seu papel no processo de controle epigenético do cromossomo X / Transcriptional dosage compensation between mammalian XX females and XY males is acquired through a complex epigenetic mechanism that leads to the inactivation of most part of one of the X chromosomes in the female cells. The X chromosome inactivation (XCI) process takes place early during embryogenesis and involves the acquisition of epigenetic modifications that are characteristic of the inactive X chromosome (Xi). Once silencing is established, the inactivation pattern is maintained in through all the subsequent mitosis and the same X chromosome remains stably silenced in all the descendant cells and throughout the life of the organism (except for the germ line cells that undergo X chromosome reactivation). The initiation of XCI has been studied extensively, especially in mice. Although some epigenetic features associated with the maintenance of XCI have already been described, the identity and specific mode of action of the factors involved in this phase of XCI are largely unknown. Moreover, the XCI process presents important differences between mice and humans, and studies directed to the identification of new players involved in the maintenance of human XCI are fundamentally important. Functional genome-wide screens using multiplex shRNA libraries are a powerful tool for the identification of genes involved in different cellular mechanisms and biochemical pathways. In order to screen for genes involved in the maintenance of XCI in humans, a population of HPRT+/-/HPRT- primary somatic female cells were transduced with a multiplex lentiviral shRNA library and subsequently treated in HAT medium to select for HPRT+ cells in which we expected that the Xi would undergo reactivation as a result of the knockdown of genes involved in the maintenance of XCI. As a result, we identified 20 new candidate genes that could potentially be involved in the maintenance of XCI. These candidates should be individually evaluated in order to confirm their role in the epigenetic control of the X chromosome

Estudos relacionando a síndrome de Turner com o estado nutricional relativo aos micronutrientes, em especial o zinco e selênio, são praticamente inexistentes. Portanto, o presente estudo teve como objetivo avaliar o estado nutricional relativo ao zinco e ao selênio dessa população, considerando as diferentes fases de vida. A avaliação antropométrica das crianças mostrou que 55,6% estavam eutróficas e 44,4% com sobrepeso, de acordo com o índice de peso/estatura. As adolescentes foram classificadas segundo o percentil de IMC ajustado para a idade, onde 73,7% estavam eutróficas e 26,3% com sobrepeso. Em relação ao grupo das adultas, 42,9% estavam com sobrepeso e 14,3% com obesidade, considerando o IMC (kg/(m)2). A avaliação do consumo alimentar foi realizada por meio do software Nutwin, mostrando que a ingestão de zinco dietético de 35,7% das participantes do estudo estava abaixo da EAR. Em relação à ingestão de selênio, observou-se que 100% das pacientes consumiram quantidades deste mineral acima da EAR. Na avaliação da concentração de zinco plasmático foi demonstrado que 22,2%, 68,4% e 14,3% das crianças, adolescentes e adultas estavam deficientes em zinco. A concentração de zinco eritrocitário apresentou-se deficiente em 66,7% das crianças, 57,9% das adolescentes e 28,6% das adultas. Na avaliação da excreção urinária de zinco observou-se que mais de 50% da população estudada estavam eliminando baixas concentrações desse mineral. Em relação ao estado nutricional de selênio, 77,8% das crianças, 78,9% das adolescentes e 85,7% das adultas encontravam-se deficientes em selênio plasmático e 55,6%, 52,6% e 57,1% das crianças, adolescentes e adultas, respectivamente, estavam deficientes em selênio eritrocitário. Opercentual de crianças, adolescentes e adultas com baixas concentrações de selênio na urina foi de 100%, 94,7% e 100%, respectivamente. A determinação da concentração de selênio nas unhas mostrou que 100% das crianças, 93,8% das adolescentes e 66,7% das adultas se encontravam com valores reduzidos neste compartimento. Os resultados da atividade da glutationa peroxidase se mostraram dentro dos limites de normalidade nas três fases de desenvolvimento. Assim, pode se concluir que o estado nutricional relativo ao zinco e ao selênio está deficiente para grande parte das pacientes, visto que as concentrações desses micronutrientes encontram-se reduzidos para a maioria dos parâmetros utilizados. / Studies relating to Turner Syndrome with the nutritional status on micronutrients, in particular zinc and selenium are practically non-existent. This study aimed to assess the nutritional status on zinc and selenium in this population, considering the different stages of life. Anthropometric evaluation of the children showed that 55.6% were eutrophic and 44.4% with overweight, according to the rate of weight/height. The adolescents were c1assified according to the percentile of BMI adjusted for age, where 73.7% were eutrophic and 26.3% with overweight. Regarding the group of adults, 42.9% were overweight and 14.3% with obesity, according BMI (kg/m2). The assessment of food consumption was made through the software Nutwin, demonstrating that the intake of dietary zinc, 35.7% of participants in the study were below the EAR. Regarding the intake of selenium, it was observed that 100% of patients consumed quantities of this mineral above the EAR. In assessing the plasma concentration of zinc has been shown that 22.2%, 68.4% e 14.3% of children, adolescents and adults were deficient in zinco The concentrations of zinc in erythrocyte were deficient 66.7% of children, 57.9% of adolescents and 28.6% of adults. In assessing the urinary excretion of zinc observed that 55.6%,57.9% and 66.7% of children, adolescents and adults, respectively, eliminate low concentrations of this mineral. The nutritional status of selenium, 77.8% of children, 78.9% of adolescents and 85.7% of adults were found deficient in plasmatic selenium and 55.6%, 52.6% and 57.1% of children, adolescents and adults, respectively, were deficient for selenium in erythrocyte. The percentage of children, adolescents and adults with low concentrations of selenium in urine was 100%, 94.7% and 100% respectively. The determination of the concentration of selenium Nail showed that 100% of children, adolescents and 93.8% from 66.7% of adults with values were reduced in this compartment. The results of the activity of glutathione peroxidase were within the limits of normality in the three stages of development. Thus, it can be concluded that the nutritional status on the zinc and selenium is deficient for most patients, because the concentrations of these micronutrients, are reduced for most of the parameters used.

No description available.

The nematode Auanema rhodensis has recently been established as a new model organism. A. rhodensis is characterized by the simultaneous existence of three phenotypical sexes (males, females and hermaphrodites; called trioecy), skewed sex ratios which do not follow Mendel's laws, and variant segregation patterns according to sex and type of gametogenesis. Recently, A. rhodensis has been used to study the possible mechanisms for sex determination in three-sexed species and the variability of basic processes during sexual reproduction including meiotic divisions. During male meiosis, a diploid primary spermatocyte undergoes two consecutive divisions to form four haploid spermatids. Surprisingly, male meiosis in A. rhodensis results in two functional and two nonfunctional spermatids depending on the presence of an X-chromatid. A. rhodensis males exhibit a set of paired autosomes and one single X-chromosome, like males of the wellestablished nematode model organism Caenorhabditis elegans. In contrast to C. elegans, however, the X-chromosome in A. rhodensis divides precociously into its sister chromatids during the first meiotic division followed by a lagging X-chromatid and its uneven distribution during meiosis II. Additionally, the second meiotic divi-sion within this species is characterized by an asymmetric organelle distribution and a spindle structure reminiscent of a monopolar spindle. In this study, serial section electron tomography was used to analyse the ultrastructure of the microtubule skeleton in spermatocytes of A. rhodensis. The analysis of spermiogenesis using electron tomography posed some key advantages compared to standard transmission electron microscopy. First, the microtubule (MT) network could be studied in detail including spindle formation, organization of spindle poles, rearrangement of MTs, and inter-action between MTs and chromosomes. Second, the number and shape of chromosomes could be visualized. And third, the morphology of organelles could be observed at high resolution, and different organelles as well as their distribution pattern could be distinguished and quantified. This study provides highresolution 3D information about male meiosis in A. rhodensis. The results of this thesis confirm the complexity of the male meiotic program and the promi-nent position of the X-chromatid in meiosis II in this organism. Like previous light microscopic studies, electron tomography supports the hypothesis of an X-chromatid-dependent distribution of cellular organelles such as fibrousbody membranousorganelles (FB-MOs) and mitochondria. Furthermore, the formation of an asymmetric spindle could be observed with progressing anaphase II and might be associated with the X-chromatid distribution. Additionally, the analysis of the number of chromosome-associating MTs and their association character gives new insights into possible chromosome segregation mechanisms. Finally, significant differences to the male meiotic program in C. elegans have been identified. For the first time, the MT network in A rhodensis spermatocytes of different division stages has been observed in detail, and several different analyses could be done, including an analysis of the length distribution of MTs in the spindles. Because this ultrastructural analysis is based on fixed samples, live-cell imaging should be performed in the future to gain further information on the chromosome dynamics in this species. / Der Fadenwurm Auanema rhodensis hat sich in den letzten Jahren mehr und mehr zu einem neuen Modellorganismus entwickelt. Typisch für A. rhodensis ist das zeitgleiche Vor-kommen dreier phänotypischer Geschlechter (Männchen, Weibchen und Hermaphroditen; die sogenannte Triözie) und deren zahlenmäßig ungleiches Verhältnis zueinander, welches sich nicht durch Mendelsche Regeln erklären lässt. Ebenfalls auffällig sind nach Geschlecht und Gametogenese (Oogenese oder Spermatogenese) abweichende chromosomale Segregationsmuster. Unlängst hat A. rhodensis zu Erkenntnissen über die Geschlechtsdeterminierung in dreigeschlechtlichen Arten und die Varianz grundlegender Prozesse in der Meiose beigetragen. Während der männlichen Meiose (Spermatogenese) teilt sich eine diploide primäre Vorläuferzelle (primäre Spermatozyte) in zwei aufeinanderfolgenden Teilungen in insgesamt vier haploide Spermatiden. Bei A. rhodensis führt die Spermatogenese ungewöhnlicher-weise zu zwei funktionalen und zwei nicht-funktionalen Spermatiden, wobei die Funktionsfähigkeit vom Auftreten eines X-Chromatids abhängt. Männchen von A. rhodensis besitzen, ähnlich wie im Modellorganismus Caenorhabditis elegans, eine Reihe gepaarter Autosomen sowie ein einzelnes X-Chromosom. Im Gegensatz zu C. elegans teilt sich das ungepaarte X-Chromosom in A. rhodensis vorzeitig schon während der ersten meiotischen Teilung in seine Chromatiden, wodurch es zu einer verzögerten und ungleichen Verteilung des X-Chromatids während der zweiten meiotischen Teilung kommt. Diese zweite meiotische Teilung bei A. rhodensis ist außerdem durch eine asymmetrische Verteilung der Organellen und Mikrotubuli gekennzeichnet, letztere ähneln einer monopolaren Spindel. In dieser Arbeit wurde die Methode der seriellen Elektronentomographie genutzt, um die Ultrastruktur der Mikrotubuli in meiotischen Spindeln in Spermatozyten von A. rhodensis zu untersuchen. Zum einen wurden mittels Elektronentomographie das Netzwerk der Mikrotubuli und die Spindelorganisation, die Struktur der Spindelpole sowie die Interaktion zwischen Mikrotubli und Chromosomen drei-dimensional (3D) analysiert. Zum anderen wurde die Form der Chromosomen und die Morphologie und Verteilung der verschiedenen Organellen quantitativ erfasst. Somit stellt diese Studie hochauflösende 3D-Information über den Ablauf der männlichen Meiose in A. rhodensis zur Verfügung und bestätigt damit die Komplexität der männlichen Meiose und die zentrale Rolle des X-Chromatids während der zweiten meiotischen Teilung in diesem Organismus. Basierend auf vorangegangenen lichtmikroskopischen Experimenten an fixierten Proben unterstützt die Elektronentomographie die Hypothese einer vom X-Chromatid abhängigen Verteilung zellulärer Organellen wie spermienspezifischer FB-MOs oder Mitochondrien während der zweiten meiotischen Teilung. Außerdem konnte die Ausbildung einer asymmetrischen Spindel beobachtet werden, welche ebenfalls mit der ungleichen Verteilung des X-Chromatids in Zusammenhang stehen könnte. Eine zusätzliche Analyse chromosomenassoziierter Mikrotubuli brachte erste Erkenntnisse über mögliche zugrundeliegende Mechanismen der Chromosomensegregation. Die Ergebnisse dieser Arbeit konnten mit ähnlichen Untersuchungen in C. elegans verglichen und Unterschiede herausgearbeitet werden. Zum ersten Mal wurden hier meiotische Spindeln unterschiedlicher Teilungsstadien in 3D untersucht und unterschiedliche quantitative Analysen zur Längenverteilung der Mikrotubuli durchgeführt. Da alle hier gewonnenen Ultrastrukturdaten auf fixierten Proben basieren, sollte eine Betrachtung einer transgenen Wurmlinie mit Fluoreszenzmarkern mittels live-cell imaging auf diese Ultrastrukturanalyse folgen.

Einige biologische Prozesse sind dosisabhängig, wobei nicht nur die Anwesenheit oder Abwesenheit bestimmter Genprodukte, sondern auch deren spezifische Mengen wichtig sind. Ein Beispiel ist die Dosis-Kompensation für Geschlechtschromosomen bei Säugetieren, die durch X-Chromosomen-Inaktivierung erreicht wird. Dieser Mechanismus ist auf Frauen beschränkt, da sie zwei X-Chromosomen besitzen, im Gegensatz zu Männern mit nur einem X-Chromosom. Dosisabhängigkeit spielt auch bei der Differenzierung pluripotenter Stammzellen eine Rolle. Geringe Schwankungen in der Menge des Pluripotenzfaktors OCT4 (POU5F1) können bestimmen, ob Maus-Embryonale Stammzellen (mESCs) sich in das Trophektoderm oder in meso-endodermale Linien differenzieren. Ebenso ist die Menge des Pluripotenzfaktors NANOG entscheidend für die Steuerung der naiven und vorbereiteten pluripotenten Zustände. Das Verständnis der dosisabhängigen Regulation biologischer Prozesse ist entscheidend, jedoch technisch anspruchsvoll, da es erfordert, die Proteinmenge quantitativ zu modulieren. Hier wurde ein auf Degron- und CRISPR/Cas-basiertes Toolkit, CasTuner, entwickelt, um die endogene Genexpression analog zu steuern. CasTuner basiert auf Cas-abgeleiteten Repressoren, die an eine Degron-Domäne fusioniert sind und durch die Titration der Konzentration eines Liganden gesteuert werden können. CasTuner ermöglicht eine homogene (analoge) Steuerung der Genexpression, im Gegensatz zum KRAB-basierten CRISPRi-System, das eine bimodale (digitale) Repression zeigt. Mit CasTuner wurden die Dosis-Wirkungs-Beziehungen von NANOG und OCT4 mit ihren Zielgenen und dem zellulären Phänotyp gemessen. Schließlich wurde CasTuner eingesetzt, um die dosisabhängige Rolle des X-gebundenen Xist-Aktivators RNF12 und des neu entdeckten Faktors ZIC3 zu untersuchen. Dabei wurde ein modifiziertes Modell für die zufällige X-Chromosomen-Inaktivierung vorgeschlagen. / Certain biological processes are dose-dependent, depending not only on the presence or absence of given gene products but also on their specific. The importance of quantitative regulation of gene expression is illustrated by the need for dosage compensation for sex chromosomes and by the presence of genes whose decreased expression is linked to diseases. The mechanism by which mammals achieve X-dosage compensation, X-chromosome inactivation, is itself dose-dependent, being restricted to females through sensing the two-fold higher dose for X-linked genes in females compared to males. Dose-dependency has been described in the differentiation of pluripotent stem cells into different lineages: small variations in the quantity of the pluripotency factor OCT4 (POU5F1) can determine the differentiation of mouse embryonic stem cells (mESCs) into the trophectoderm or meso-endoderm lineages. Similarly, the amount of the pluripotency factor NANOG is critical for the control of naïve and primed pluripotent states. Understanding the principles underlying the dose-dependent regulation of biological processes is crucial, but also technically challenging, since it requires the ability to quantitatively modulate protein abundance. Here, I developed a degron- and CRISPR/Cas-based toolkit, CasTuner, for analogue tuning of endogenous gene expression. CasTuner relies on Cas-derived repressors fused to a degron domain, which can be tuned by titrating the concentration of a ligand. I demonstrate homogenous (analogue) tuning of gene expression across cells, as opposed to the KRAB-based CRISPRi system, which exhibits bimodal (digital) repression. I employ CasTuner to measure the dose-response relationships of NANOG and OCT4 with their target genes and the cellular phenotype. Finally, I apply CasTuner to study the dose-dependent role of the X-linked Xist activator RNF12 and the newly discovered factor ZIC3, and propose a modified model for random X-chromosome inactivation.

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No. of bitstreams: 1 polverari_fs_dr_araiq_int.pdf: 3019599 bytes, checksum: cac87174a4589a63a284c7075604c847 (MD5) Previous issue date: 2018-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A análise dos polimorfismos do DNA é a melhor ferramenta encontrada para resolução de casos de identificação humana, sendo os marcadores STRs (short tandem repeat) localizados em regiões autossômicas os principais e mais utilizados para esta finalidade. Apesar da indiscutível reprodutibilidade destes marcadores, quando são analisados em amostras degradadas podem não apresentar bons resultados, o que dificulta a resolução dos casos. Assim, há a necessidade de uma alternativa e, atualmente, a mais indicada é a utilização dos polimorfismos bialélicos. A análise do cromossomo sexual X também vem ganhando importância significativa no contexto forense, devido ao seu padrão de transmissão entre os genitores. Neste trabalho, caracterizamos as populações brasileiras do estado do Mato Grosso e do Distrito Federal pela análise de 32 polimorfismos de inserção/deleção no cromossomo X (X-InDels), tendo em vista que os dados destes polimorfismos nestas populações são ainda inéditos, e para que esses marcadores também possam no futuro auxiliar a resolução de casos forenses em nosso país. Para identificar a diversidade genética foram analisados os perfis genotípicos de 303 indivíduos não aparentados nascidos no estado do Mato Grosso e 179 indivíduos não aparentados e residentes em Brasília. Os resultados indicam que o painel dos 32 X-Indels é bastante eficiente para a sua finalidade, sendo que praticamente todos os marcadores se mostraram altamente informativos para as populações estudadas. O teste de equilíbrio de HardyWeinberg foi realizado nas amostras femininas de ambas as populações e não foram verificados desvios significativos. O painel demonstrou alta eficiência forense, confirmado pelo alto poder de discriminação para Mato Grosso (PDF=0,999999999997 e PDM=0,99999997) e para Brasília ((PDF=0,999999999998 e PDM=0,99999998), e pelo elevado poder de exclusão observado em trios: suposto pai/mãe/filha (0,999998/Mato Grosso e 0,999997/Brasília) e duos: suposto pai/filha (0,9996/Mato Grosso e 0,9995/Brasília). No estudo comparativo com outras populações, Mato Grosso e Brasília estão mais próximos ao estado de São Paulo, à três departamentos colombianos e às populações europeias. A proporção de ancestralidade confirmou a miscigenação das populações estudadas, sendo identificado uma contribuição praticamente equilibrada para europeus, africanos e nativo-americanos. Conclui-se que o conhecimento acerca dos marcadores de inserção/deleção no cromossomo X pode ser ampliado, uma vez que na literatura ainda há pouco material disponível sobre o assunto; entretanto os dados deste trabalho já demonstram seu potencial como método complementar para a análise de amostras forenses, pois foram identificados elevados valores de poder de discriminação e exclusão / The analysis of DNA polymorphisms is the best tool found for solving cases of human identification, and the Short Tandem Repeat (STR) markers used in the autosomal regions are the main and most marker used for this purpose. Despite the undeniable reproducibility of these markers, when analyzed in degraded samples they may not present good results, which makes it difficult to solve the cases. Because of this, there is a need for an alternative tool and currently the most indicated is the use of the biallelic polymorphisms. In this way, the analysis of the sex chromosome X has gained significant importance in the forensic context, due to its pattern of transmission between the parents. In this work, we characterize the Brazilian populations of the state of Mato Grosso and the Federal District by the analysis of 32 insertion/deletion polymorphisms on the X chromosome (X-InDels), considering that the data of this polymorphism in these populations are still unpublished, and for that these markers may also in the future help the resolution of forensic cases in our country. To identify the genetic diversity, the genotypic profiles of 303 unrelated individuals born in the state of Mato Grosso and 179 unrelated individuals living in Brasília were analyzed. The results shows that the 32 X-Indels panel is very efficient to its purpose, being almost all markers highly informative for the studied populations. The Hardy-Weinberg equilibrium test was performed on the female samples from both populations and no significant deviations were observed. The panel demonstrated high forensic efficiency, confirmed by the high discriminatory power for Mato Grosso (PDF= 0.9999999999997 and PDM=0.999999997) and Brasília (PDF=0.9999999999998 and PDM=0.999999998), and by the high exclusion power observed in trios: supposed father/mother/daughter (0.999998/Mato Grosso and 0,999997/Brasília) and duos: supposed father/daughter (0.9996/Mato Grosso and 0,9995/Brasília). In the comparative study with other populations, Mato Grosso and Brasília are closer to the state of São Paulo, to three departments in Colombia and to European populations. The proportion of ancestry confirmed the admixture of the populations studied, with a practically balanced contribution being identified for Europeans, Africans and American natives. We could concluded that knowledge about insertion/deletion markers on the X chromosome can be amplified, since in the literature there is still fewer available material on the subject, however the data of this work already demonstrates its potential as a complementary method for the analysis of forensic samples, since were identified high values of power of discrimination and exclusion.

Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...

Submitted by JULIANA MARTINEZ null (jumrtz@hotmail.com) on 2018-01-30T20:15:12Z No. of bitstreams: 1 Tese de Doutorado - Juliana Martinez_versaofinal.pdf: 5281561 bytes, checksum: 3904c01cc47c8b76f9c22a3a88eeb574 (MD5) / Approved for entry into archive by Maria Irani Coito null (irani@fcfar.unesp.br) on 2018-02-02T16:30:55Z (GMT) No. of bitstreams: 1 martinez_j_dr_arafcf_int.pdf: 5281561 bytes, checksum: 3904c01cc47c8b76f9c22a3a88eeb574 (MD5) / Made available in DSpace on 2018-02-02T16:30:55Z (GMT). No. of bitstreams: 1 martinez_j_dr_arafcf_int.pdf: 5281561 bytes, checksum: 3904c01cc47c8b76f9c22a3a88eeb574 (MD5) Previous issue date: 2017-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na rotina da genética forense, o uso exclusivo dos marcadores STRs (Short Tandem Repeats) em situações que a amostra biológica apresenta-se degradada pode gerar um resultado final estatístico inconclusivo, tornando fundamental a análise de marcadores adicionais para a resolução do caso. Utilizado como método complementar, os polimorfismos InDels (inserção/deleção) têm mostrado grande potencial para superar as limitações dos marcadores tradicionais. A análise de regiões do cromossomo X também vem ganhando significativa importância nesses estudos, especialmente nos casos em que a análise dos autossômicos não é suficiente. Nessa perspectiva, este trabalho teve por objetivo geral caracterizar a população do estado de São Paulo para 32 polimorfismos de inserção/deleção no cromossomo X (32 X-InDels) e avaliar a utilidade desse multiplex na resolução de casos forenses. Para tanto, buscou-se identificar a diversidade genética desses polimorfismos nessa população, a segregação dos alelos entre os genitores (pai e mãe) para as suas respectivas filhas e a eficiência desse painel na amplificação de DNA extraído de amostras ósseas. Para identificar a diversidade genética, foram analisados os perfis genotípicos de 500 indivíduos não aparentados nascidos no estado de São Paulo. Todos os marcadores mostraram-se polimórficos para a população, sendo MID3701 o que apresentou maior diversidade e somente MID2637 se mostrou pouco informativo. O marcador MID1361 apresentou-se em desequilíbrio de Hardy-Weinberg e uma variante alélica foi identificada em seu alelo curto. O painel demonstrou alta eficiência forense, confirmado pelo poder acumulado de discriminação (0,999999999993 em mulheres e 0,99999993 em homens) e pelo valor acumulado da chance média de exclusão (0,999996 em trios e 0,9995 em duos). No comparativo com outras populações, valores significativos da distancia genética foram obtidos, verificando-se que São Paulo está mais próximo à três departamentos colombianos e às populações européias. A proporção de ancestralidade identificada foi 41,8% para europeus, 31,6% para africanos e 26,6% para nativo-americanos. Na análise de segregação realizada em 101 trios, o padrão de transmissão esperado entre pai-mãe/filha foi o observado, o que confirma a baixa taxa de mutação desses marcadores. Por fim, os 32 X-InDels apresentaram as características necessárias para a análise de amostras biológicas em baixa concentração e/ou degradadas, mas algumas dificuldades na amplificação podem ser encontradas a depender das condições ambientais a que as amostras foram expostas. Conclui-se que o conhecimento acerca dos marcadores de inserção/deleção no cromossomo X pode ser ampliado, uma vez que na literatura ainda há pouco material disponível sobre o assunto; entretanto os dados deste trabalho já demonstram seu potencial como método alternativo para a análise de amostras forenses, pois foram identificados elevados valores de poder de discriminação e exclusão, baixa taxa de mutação e um elevado potencial de amplificação de amostras biológicas degradadas. / In forensic genetics routine, the exclusive use of STRs (Short Tandem Repeats) markers when the biological sample is degraded can generate an inconclusive final statistical result, making essential the analysis of additional markers for case resolution. Used as a complementary method, InDels (insertion/deletion) polymorphisms have shown great potential to overcome the limitations of traditional markers. Polymorphisms in the X chromosome is also gaining significant importance in these studies, especially in those cases in which the analysis of the autosomal markers is not enough. In this perspective, this study aimed to characterize the São Paulo state population for 32 X chromosome insertion/deletion polymorphisms (32 X-InDels) and to evaluate the utility of this multiplex in the resolution of forensic cases. Therefore, it was analyzed the genetic diversity of this population, the alleles segregation between the parents and their respective daughters, and the amplification efficiency of this panel in DNA extracted from human bones. To identify genetic diversity, the genotypic profiles of 500 unrelated individuals born in São Paulo state was analysed. All markers were polymorphic for the population, with MID3701 being the most diverse, and MID2637 the less informative. The MID1361 marker was in Hardy-Weinberg disequilibrium and an allelic variant was identified in its short allele. The panel showed high forensic efficiency, confirmed by the accumulated power of discrimination (0.9999999999993 in females and 0.99999993 in males) and by the accumulated mean exclusion chance (0.999996 in trios and 0.99995 in duos). Comparing with other populations, significant values of genetic distance were obtained and São Paulo is closer to three Colombian departments and to European populations. The ethnic contributions identified 41.8% of Europeans, 31.6% of Africans and 26.6% of Native Americans admixture. In segregation analysis performed in 101 trios, the expected transmission pattern between parent/daughter was observed, which confirms the low mutation rate of these markers. Finally, the 32 X-InDels presented the necessary characteristics for the analysis of degraded biological samples, but some amplification difficulties can be found depending on the environmental conditions in which the samples were exposed. It can be concluded that knowledge about the X chromosome insertion/deletion markers can be expanded, because there is still little information available in the literature; meanwhile data from this work demonstrate its potential as an alternative method for the analysis of forensic samples.