As maiores taxas de incidência de câncer de mama ocorrem em mulheres idosas, que apresentam tumores com expressão de receptores de estrógeno e/ou progesterona, de baixo estadiamento e menor taxa de proliferação, se comparado com as jovens. Um dos fatores de predisposição ao câncer de mama é mutação germinativa nos genes BRCA1 ou BRCA2, que podem compreender entre 5-10% das pacientes diagnosticadas. A grande maioria dos casos são ditos esporádicos, em que não há como estabelecer um único fator determinante. Dentre o escopo de possíveis causas estão as mutações somáticas, acumuladas no tecido mamário ao longo da vida. A identificação destas mutações permite melhor compreensão da carcinogênese e possibilita a criação de tratamentos cada vez mais personalizados. O gene PIK3CA, por exemplo, já está determinado como driver (responsáveis pela obtenção de vantagem seletiva de um determinado clone) para câncer de mama. As mutações patogênicas que ocorrem neste gene levam a ativação da via de Akt/mTOR, entre outras, que mantém o ciclo celular ativo. Um gene que vem sendo estudado recentemente é o PRKD1, cujas funções parecem estar ligadas à manutenção do fenótipo epitelial das células do tecido mamário. Assim, o objetivo desse trabalho identificar mutações germinativas nos genes BRCA1 e BRCA2, analisando também o histórico familiar para câncer de mama/ovário/próstata, e mutações somáticas no gene PRKD1 em pacientes pós menopausadas,. Foram incluídas quarenta e nove pacientes diagnosticadas com carcinoma ductal invasivo em idade superior a 54 anos, que preenchessem critérios da NCCN (National Comprehensive Cancer Network) para Síndrome de Câncer de Mama e/ou Ovário Hereditário e tinham disponível um fragmento tumoral emblocado em parafina coletado na ausência de tratamento neo adjuvante. A extração de DNA foi realizada a partir do sangue periférico para sequenciamento de BRCA1 e BRCA2, realizado através da plataforma Ion Torrent(TM) ou pelo método de Sanger. Os resultados obtidos por Ion Torrent(TM) foram analisados, primeiramente, através da ferramenta online Ion Reporter e os de Sanger através do programa Mutation Surveyor v.3.20. Para a caractetização das variantes encontradas foram utilizados: os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar além dos preditores in silico da conservação dos aminoácidos entre as espécies Polyphen-2, SIF, Provean e AlignGVGD e do preditor de efeito no splicing HSF e bancos de dados de frequência alélica ExAC, 1000 genomas e NHLBI GO Exome Sequencing Project, seguindo os critérios da American College of Medical Genetics and Genomics em conjunto com a Association for Molecular Pathology. Para caracterização de mutação somática do gene PRKD1 determinou-se duas regiões de maior importância para serem sequenciadas: Ser738/Ser742 e Ser910 que fosforilam o domínio quinase da proteína, ativando-o. Vinte e três amostras tumorais tiveram DNA extraído. Também foi realizada uma análise das informações sobre PRKD1 do banco de dados COSMIC (Catalogue of Somatic Mutations in Cancer) e a construção de curvas de sobrevida (Kaplan-Meier) da expressão de PRKD1 utilizando a ferramenta online KM Plotter. A idade mediana das pacientes foi de 62 anos ao diagnóstico e de 64 anos na época de inclusão no estudo. A maioria tinha tumores de grau histológico II (63,27%), estádio clinico II (20%) e do subtipo luminal B (53,06%). Trinta e duas relataram parentes de primeiro grau afetados com câncer de mama/ovário/ próstata. Trinta e oito pacientes tiveram sequenciamento completo de BRCA1 e BRCA2 por Ion Torrent(TM) e onze tiveram sequenciamento parcial de BRCA1 e BRCA2 por Sanger. Variantes patogênicas foram encontradas em quatro pacientes (BRCA1=2/BRCA2=2). Uma nova variante missense foi identificada em BRCA2: c.3371A > G (p.Q1124R). Para o sequenciamento de PRKD1 quinze foram sequenciadas para Ser910 e de oito foi possível analisar o resultado. Nenhuma variante patogênica foi encontrada. Os dados obtidos sobre PRKD1 no COSMIC foram: de 2773 amostras, em apenas 15 (0,54%) foram identificadas mutações em PRKD1, 46% (7/15) provém de mulheres com idade superior a 55 anos e subtipo molecular Luminal. PRKD1 apresenta maiores frequência de mutação em câncer de intestino grosso (4,22%) e pele (4,02%). As curvas de sobrevida construídas no KM Plotter demonstram a alta expressão do gene parece ter impacto positivo na sobrevida das pacientes. Apesar da baixa frequência de mutações no PRKD1 este gene, outros dados demonstram que parece ter um papel de gene supressor de tumor no câncer de mama, que deve ser inibido de através de outros mecanismos como metilaçao de DNA / The highest rates of breast cancer incidence occur in elderly women, who present estrogen and / or progesterone receptor tumors, with a low clinical staging and lower proliferation rate compared to the young women. One of the factors predisposing to breast cancer is germline mutation in the BRCA1 or BRCA2 genes, which may comprise between 5-10% of the diagnosed patients. The vast majority of cases are said to be sporadic, in which there is no way to establish a single determining factor. Among the scope of possible causes are somatic mutations, accumulated in the breast tissue throughout life. The identification of these mutations allows a better understanding of carcinogenesis and enables the creation of increasingly personalized treatments. The PIK3CA gene, for example, is already determined as a driver (responsible for the selective advantage of a particular clone) for breast cancer. The pathogenic mutations that occur in this gene lead to the activation of Akt / mTOR pathway, among others, which keeps the cell cycle active. One gene that has recently been studied is PRKD1, whose functions seem to be linked to the maintenance of the epithelial phenotype of the mammary tissue cells. Thus, the objective of this work was to identify germline mutations in BRCA1 and BRCA2 genes, also analyzing the family history for breast / ovarian / prostate cancer, and somatic mutations in the PRKD1 gene in postmenopausal patients. Forty-nine patients diagnosed with ipsilateral ductal carcinomas over the age of 54 years who completed NCCN (National Comprehensive Cancer Network) criteria for Breast Cancer and / or Hereditary Ovarian Syndrome and had a tumor paraffin embedded in paraffin collected in the absence of neo adjuvant treatment available. DNA extraction was performed from the peripheral blood for sequencing of BRCA1 and BRCA2, performed through the Ion Torrent (TM) platform or by the Sanger method. The results obtained by Ion Torrent (TM) were first analyzed through the online tool Ion Reporter and those by Sanger through the program Mutation Surveyor v.3.20. The BIC, LOVD, LOVD-IARC, UMD and ClinVar databases were used in addition to the in silico predictors of amino acid conservation among Polyphen-2, SIF, Provean and AlignGVGD species and the effect predictor in the HSF splicing and allelic frequency databases ExAC, 1000 genomes and the NHLBI GO Exome Sequencing Project, following the criteria of the American College of Medical Genetics and Genomics in conjunction with the Association for Molecular Pathology. In order to characterize the somatic mutation of the PRKD1 gene, we determined two regions of greater importance to be sequenced: Ser738 / Ser742 and Ser910 that phosphorylate the protein kinase domain, activating it. Twenty-three tumor samples had DNA extracted. An analysis of PRKD1 information from the COSMIC (Catalog of Somatic Mutations in Cancer) database and the construction of survival curves (Kaplan-Meier) for PRKD1 expression using the online KM Plotter tool was also performed. The median age of the patients was 62 years at diagnosis and 64 years at the time of inclusion in the study. Most of them had tumors of histological grade II (63.27%), clinical stage II (20%) and molecular subtype luminal B (53.06%). Thirty-two reported first-degree relatives affected with breast / ovarian / prostate cancer. Thirty-eight patients had BRCA1 and BRCA2 complete sequencing by Ion Torrent (TM) and eleven had BRCA1 and BRCA2 partial sequencing by Sanger. Pathogenic variants were found in four patients (BRCA1 = 2 / BRCA2 = 2). For PRKD1 sequencing, fifteen patients tumors were sequenced for Ser910 and in eight samples it was possible to analyze the result. No pathogenic variant was found. The data obtained on PRKD1 in COSMIC were: from 2773 samples, in only 15 (0.54%) mutations were identified in PRKD1, 46% (7/15) came from women aged over 55 years and had tumor molecular subtype Luminal. PRKD1 shows higher mutation frequency in cancer of the large intestine (4.22%) and skin (4.02%). The survival curves constructed in KM Plotter demonstrate the high expression of the gene seems to have a positive impact on the patients survival . Despite the low frequency of mutations in PRKD1 gene, other data demonstrate that it appears to play a role of tumor suppressor gene in breast cancer, which must be inhibited by other mechanisms such as DNA methylation

Chez le maïs, la création de nouvelles variétés repose largement sur la technologie des plantes Haploïdes Doublées. Celle-ci s’appuie sur l’utilisation du pollen de lignées particulières, dites «inductrices d’haploïdes», qui déclenchent un processus de gynogenèse in vivo. Ce mécanisme permet la production d’embryons haploïdes contenant uniquement les informations génétiques maternelles. Après doublement des stocks chromosomiques des plantules haploïdes générées, il est possible d’obtenir des lignées fixées en seulement quelques générations, réduisant ainsi considérablement les schémas de sélection. Bien que les déterminismes génétiques à l’origine de la gynogenèse in vivo soient restés énigmatiques pendant près de soixante ans, nos travaux ont permis le clonage positionnel et l’identification du gène majeur causal de l’induction d’haploïdes. Ce gène, exprimé spécifiquement dans les grains de pollen, code pour une phospholipase A2, nommée NOT LIKE DAD (NLD). Il s’avère que chez l’ensemble des lignées inductrices le gène NLD présente une insertion de quatre paires de bases, conduisant à la formation d’une protéine tronquée responsable de la gynogenèse in vivo. Cette mutation n’entraine pas un défaut spatial ou temporel de l’expression du gène mais une délocalisation de la protéine. La protéine sauvage localise au niveau de l’unité mâle germinative, sur la membrane d’origine végétative entourant les deux cellules spermatiques, alors que la protéine tronquée perd cette localisation. Des approches pharmacologiques et de mutagenèses dirigées ont permis de révéler un adressage de NLD à la membrane grâce à des ancrages lipidiques et des interactions électrostatiques entre la membrane et la protéine. En parallèle, il a pu être observé que la troncation de NLD, en plus de conduire à la formation d’haploïdes maternels, entraînait de nombreux phénotypes de grains anormaux. Le génotypage des produits de fécondations (albumens et embryons) résultant de croisements avec des lignées inductrices ont permis de mettre en évidence l’existence d’anomalies chromosomiques paternelles, pouvant expliquer ces différents phénotypes de grains anormaux. L’ensemble de ces résultats indiquent que la phospholipase NLD intacte est nécessaire pour le bon déroulement de la reproduction sexuée. / In maize, plant breeding mainly relies on double haploid technology. The main principle of this technology is the use of special lines called “inducer lines”, which induce in vivo gynogenesis. This mechanism allows haploid embryos production which only contain maternal genetics information. After chromosome doubling of the haploid seedlings, it is possible to obtain fixed lines in only few generations, thus considerably reducing breeding cycles. Although genetic determinants behind in vivo gynogenesis remained enigmatic for nearly sixty years, our work allowed the positional cloning and the identification of the major gene controlling haploid induction. This gene, specifically expressed in pollen grain, encodes for a phospholipase A2 called NOT LIKE DAD (NLD). It turns out that in all the inducer lines the NLD gene has a 4-bp insertion, leading to the formation of a truncated protein responsible for gynogenesis in vivo. This mutation did not result in a spatial or temporal defect in the expression of the gene but in the delocalization of the protein. The wild type protein localizes at the male germ unit on the vegetative membrane surrounding the two sperm cells, while the truncated protein loses this location. Pharmacological and mutagenic approaches revealed that NLD is targeted to the membrane through lipid anchoring and electrostatic interactions between membrane and protein. In parallel, it has been observed that the truncation of NLD; in addition to leading to the formation of maternal haploids; led to many pleiotropic phenotypes of abnormal kernels. The genotyping of fertilization products (endosperms and embryos), shows the existence of paternal chromosomal anomalies, which can explain these different phenotypes of abnormal kernels. All these results indicate that the intact NLD phospholipase is therefore necessary for the proper course of sexual reproduction.

O Pseudohermafroditismo masculino disgenético (Anomalia da diferenciação sexual 46,XY ADS 46,XY) é definido como ambigüidade genital num paciente com testículos e/ou cariótipo 46,XY com uma das seguintes características: alteração histológica testicular, ausência ou hipoplasia das células de Leydig em tecido previamente estimulado com gonadotrofina coriônica humana(hCG), falta de resposta de testosterona ao estímulo com hCG sem acúmulo de precursores, ausência de células germinativas, presença de derivados müllerianos indicando inadequada produção do hormônio antiMülleriano (HAM) ou resistência de seus receptores. Esse estudo apresenta uma avaliação clínica, laboratorial, anátomopatológica, morfométrica e molecular de 10 pacientes com ADS 46,XY; dois pacientes apresentaram mutação no SF1 (fator esteroidogênico 1), duas mutações no domínio hingee uma terceira produziu um stop códon na posição 404; três pacientes com deleção da cópia do DAZ2. A morfometria testicular mostrou todos os diâmetros tubulares médios (DTM) moderado a gravemente diminuídos e os índices de fertilidade tubular leve a moderadamente diminuídos. Devido à dificuldade do diagnóstico diferencial e etiológico, o estudo morfométrico e molecular deve sempre acompanhar esses casos de ADS 46,XY. / The dysgenetic male pseudohermaphroditism 46,XY ; disorders of sex development (DSD 46,XY) is defined as sexual ambiguity in patients with testis and/or 46,XY karyotype and one of the characteristics: hystologic alteration of the testis; absence or hypoplasia of Leydig cells; a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors; absent germ cells; presence of müllerian duct derivatives showing inappropriate production of antimüllerian hormone (AMH) or resistance to its receptors. This study shows the clinic, laboratory, histologic, morphometric and molecular evaluation of 10 patients with DSD 46,XY; two patients showed mutations in the SF1 gene (steroidogenic factor-1); two in the hinge domain and one stop codon at the position 404 of the protein; three patients exhibited deletion of DAZ2. The testis morphometry showed reduction: marked to severe of all mean tubular diameter (MTD) while the reduction of the tubular fertility index (TFI) were slight to marked. Due to difficulties establishing the differential diagnosis and the etiology, the morphometric and molecular evaluation must be always done in the patients with DSD 46,XY.

<p>Zadatak ove teze je da se ispita uticaj zamene dela p&scaron;eničnog bra&scaron;na ekstrudiranim snek proizvodima obogaćenim dodatkom p&scaron;enične klice, pivskog tropa i tropa jabuke na svojstva testa (boja, fizička, reolo&scaron;ka) kao i na karakteristike čajnog peciva (fizičke, senzorske, nutritivne, trajnost, mikrobiolo&scaron;ke). U toku preliminarnih ispitivanja (prva faza eksperimentalnog rada) ispitan je uticaj obezma&scaron;ćene p&scaron;enične klice na kvalitet testa i čajnog peciva u cilju definisanja optimalnog nivoa zamene p&scaron;eničnog bra&scaron;na ekstrudatima kukuruzne krupice sa dodatkom sporednih proizvoda prehrambene industrije i optimalnog sadržaja vlage testa. P&scaron;enično bra&scaron;no je u količini od 5, 10 i 15 % zamenjeno obezma&scaron;ćenom p&scaron;eničnom klicom. Pored uticaja nivoa zamene p&scaron;eničnog bra&scaron;na p&scaron;eničnom klicom, ispitan je i uticaj veličine čestica p&scaron;enične klice (&lt;150 &mu;m, 150-1000 &mu;m i 800-2000 &mu;m), kao i vlage testa pri zamesu (20, 22 i 24 %). U toku druge faze eksperimentalnog rada ove doktorske disertacije, pripremljeni su ekstrudati kukuruzne krupice sa dodatkom sporednih proizvoda prehrambene industrije i to: p&scaron;enične klice, pivskog tropa i tropa jabuke u udelima 15, 30 i 45 % (odnos kukuruzna krupica:sporedni proizvod bio je 85:15, 70:30 i 55:45). Dobijeni ekstrudati su samleveni i prosejani na sitima u cilju dobijanja tri frakcije sa različitim veličinama čestica (&lt; 250 &mu;m, 250 - 1000 &mu;m i 1000 - 2000 &mu;m). Izvr&scaron;ena je karakterizacija ekstrudata u smislu određivanja hemijskog sastava, raspodele veličine čestica, boje i mikrobiolo&scaron;ke analize. Ovako dobijeni ekstrudati su zatim kori&scaron;ćeni u proizvodnji čajnog peciva kao zamena p&scaron;eničnog bra&scaron;na u količinama od 5, 10 i 15 % računato na masu bra&scaron;na. Za procenu uticaja veličine čestica ekstrudata, udela sporednog proizvoda u ekstrudatu i udela ekstrudata u čajnom pecivu na osobine testa i čajnog peciva takođe je kori&scaron;ćen Box-Behnken eksperimentalni dizajn. Rezultati su pokazali da je moguća proizvodnja funkcionalnog čajnog peciva sa dodatkom ekstrudata kukuruzne krupice obogaćene sporednim proizvodima prehrambene industrije, pri čemu se pobolj&scaron;ava nutritivni profil čajnog peciva, bez negativnih efekata na fizičke i senzorske karakteristike gotovog proizvoda.</p> / <p>The aim of this study is to examine the effect of replacing a portion of wheat flour with extruded snack products enriched with the addition of wheat germ, brewer&#39;s spent grain and apple pomace on the rheological and textural properties of dough and on characteristics of cookies (physical, sensory, nutritive, microbiological). During the preliminary analysis (the first phase of the experimental work), the influence of defatted wheat germ particle size, wheat germ content and dough moisture content on the quality of the dough and the cookies was investigated using the Box-Behnken experimental design. During the second phase of the experimental work, the extruded corn meal with the addition of by-products of the food industry (wheat germ, brewer&#39;s spent grain and apple pomace) were prepared (the ratio of corn meal: by-product was 85:15, 70:30 and 55:45). The obtained snack products are milled and sieved to obtain 3 fractions with different particle sizes (&lt;250 &mu;m, 250-1000 &mu;m and 1000-2000 &mu;m). Characterization of the extrudates in terms of determining the chemical composition, particle size distribution, color and microbiological analysis was performed. The obtained extrudates were then used in the production of cookies for the replacement of wheat flour in quantities of 5, 10 and 15%. The Box-Behnken experimental design was used to evaluate the influence of the extrudate particle size, the share of the by-product in the extrudate, and the share of extrudates in the cookies, on the dough properties and cookies characteristics.<br />The results showed that the corn snack products enriched with food industry by-products (brewer&#39;s spent grain, wheat germ and apple pomace) can be used for production of functional cookies. These additives in an amount up to 15% improving the nutritional profile of the cookies, without adverse effects on the physical and sensory characteristics of the final product.</p>

Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DRR) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DRL), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and the integrated virus can be induced to lytic replication. We sought, first, to definitively illustrate the in vitro and in vivo integration of HHV-6A and HHV-6B. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6A and Molt3 cell line by HHV-6B, the virus integrated into telomere of chromosomes. Next, peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per ml of blood. FISH confirmed that HHV-6 DNA co-localized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, while the integration site was identical among members of the same family. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome through the germ-line [inherited HHV-6 (iHHV-6)]. Amplification and sequencing of the HHV-6A and more recently HHV-6B viral-chromosome junction identified DRR integrated into the telomere directly adjacent to the subtelomere of the chromosome. After mapping the DRR of iHHV-6, we subsequently focused on determining if the DRL was present in the integrated genome and whether the remaining telomere sequence of the chromosome was extended beyond the DRL. Southern hybridization of PCR amplified HHV-6 integrated cell lines and iHHV-6 patients PBMCs indicate the presence of DRL within the integrated viral genome. Therefore, the genomic structure of the iHHV-6 is as follows: chromosome-subtelomere-(TTAGGG)5-41-DRR-U-DRL-(TTAGGG)n. During latent integration, no circular episomes were detected even by PCR. However, trichostatin-A treatment of PBMCs and in vitro integrated HEK-293 cells induced the reactivation of iHHV-6 from its latent integrated state. We demonstrated the induction of integrated iHHV-6 with trichostatin-A lead to the excision of the integrated genome and generation of the U-DR-U junction which signifies circularization and/or concatemer formation of the viral genome through rolling-circle replication. Taken together, the data suggests that HHV-6A and HHV-6B are unique among human herpesviruses: they specifically and efficiently integrate into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

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