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Padrão de Inativação do Cromossomo X e Expressão de microRNAs X-específicos na Pré-Eclâmpsia / X-chomosome Inactivation Pattern and of MicroRNAs X-specific Expression in PreeclampsiaOliveira, Adriane Araujo de 27 January 2010 (has links)
As síndromes hipertensivas gestacionais estão entre as maiores causas de morte materna e fetal. Entre elas destaca-se a pré-eclâmpsia (PE), que caracteriza-se pelo aumento da pressão arterial e proteinúria, a partir da 20ª semana de gestação. Embora sua etiologia seja ainda discutida, o papel dos fatores genéticos é amplamente aceito. Alterações do padrão da inativação do cromossomo X, processo epigenético encontrado em mamíferos com placenta, têm sido encontradas em algumas doenças que ocorrem exclusivamente em mulheres. O XIST é um gene chave nesse processo. Por outro lado, muitos microRNAs (pequenos RNAs não codificantes) são expressos abundantemente na placenta humana e alguns estão mapeados no cromossomo X. O objetivo do presente trabalho foi a verificação do padrão de inativação do cromossomo X e da expressão dos genes XIST e dos microRNAs X-específicos miR-221, miR-222 e mir-223 em mulheres com PE. O ensaio de HUMARA (receptor de andrógeno humano) utilizando PCR convencional e digestão com a enzima sensível à metilação HpaII foi analisado de forma qualitativa (visualização em gel de poliacrilamida) e semi-quantitativa (sequenciamento), sendo realizado a partir de sangue periférico (todas as amostras) e de tecido placentário [apenas das placentas de fetos femininos (17 amostras)]. Para o estudo do padrão de expressão foi obtido cDNA por transcrição reversa, a partir de RNA total extraído do tecido placentário (30 amostras).. A análise foi realizada por meio de PCR em tempo real. Foram utilizados os testes de Qui-quadrado e de t-Student, além do modelo linear generalizado para a análise estatística. Não houve diferença estatisticamente significativa para o parâmetro de inativação do cromossomo X entre os grupos controle e de PE, independente do tipo de tecido estudado (sangue ou placenta) quando foram aplicados os ensaios de HUMARA qualitativo e semi-quantitativo. Para o gene XIST e o microRNA miR-221 não foi evidenciada diferença estatisticamente significativa entre os grupos controle e de PE. O microRNA miR-223 não apresentou transcritos detectáveis em nenhum dos grupos de estudo. Para o microRNA miR-222, houve diferença estatisticamente significativa, sendo que no grupo de PE a expressão foi mais elevada. Não foi encontrada associação entre o padrão de inativação do cromossomo X e a expressão do gene XIST e dos microRNAs estudados. Embora a inativação preferencial do cromossomo X tenha sido encontrada nos dois grupos, padrões de inativação preferencial extrema foram verificados em um número maior de casos com PE. Os resultados mostram que o miR-222 apresenta potencial para ser utilizado como marcador molecular da PE, sugerindo também que exista uma diminuição na expressão de seus genes-alvo que devem ser estudados como candidatos na patogênese da doença. / The gestational hypertensive syndromes are among the major causes of maternal and fetal death. The Preeclampsia (PE) is the most prevalent of those syndromes and it is characterized by the increase of blood pressure and proteinury, which start from to 20th week of gestation. Although its ethiology is argued actually the genetics factors have been accepted. Alterations in pattern of chromosome X inactivation, epigenetic process of mammals with placenta have been found in some diseases that occur exclusively in women. XIST is a key gene in this process. On the other hand very microRNAs (small RNAs no coding) are overexpressed in human placenta and someones are located at X choromosome. The subject of this research was verify the patterns of chromosome X inactivation and the expression of the XIST gene and X-specific microRNAs in women affected by PE. In the HUMARA (human androgen receptor) assay was carried out with peripheral blood (all samples) and placental tissue [only female fetuses placentas (17 samples)]. The conventional PCR and digestion methodology with HpaII enzyme were employed and the result was analysed to qualitative (polyacrilamide gel) and semi quantitative (sequencing) form. The cDNA was obtained to gene expression study by reverse transcription reaction from placenta total RNA (30 samples). Gene expression assay was carried out with real time PCR. To statistical analyze was used the Qui-square and t-Student tests besides the widespread lineal model. There was not statistical significant differences to chormosome X inactivation parameter among the groups control and PE independently of the tissue studied (blood or placenta) when the qualitative and semi quantitative HUMARA assay were applied. To XIST and miR-221 were not evidenced significant statistical differences among the groups control and of PE. The miR-223 did not show detectable transcripts to any group studied. The miR-222 expression was more elevated in the PE group than control group and this difference was significant statistically. In the present study was not found association among the chromosome X inactivation pattern and the gene expression of XIST and the microRNAs studied. Although the chromosome X inactivation have been found in the two groups the preferential chromosome X inactivation patterns were verified in a great number of cases in PE group. The results showed that miR-222 has a potential to be employed as molecular marker of PE also suggesting the existence of a decrease in expression of its target genes which have to be investigated like candidates to disease pathogenesis.
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Padrão de Inativação do Cromossomo X e Expressão de microRNAs X-específicos na Pré-Eclâmpsia / X-chomosome Inactivation Pattern and of MicroRNAs X-specific Expression in PreeclampsiaAdriane Araujo de Oliveira 27 January 2010 (has links)
As síndromes hipertensivas gestacionais estão entre as maiores causas de morte materna e fetal. Entre elas destaca-se a pré-eclâmpsia (PE), que caracteriza-se pelo aumento da pressão arterial e proteinúria, a partir da 20ª semana de gestação. Embora sua etiologia seja ainda discutida, o papel dos fatores genéticos é amplamente aceito. Alterações do padrão da inativação do cromossomo X, processo epigenético encontrado em mamíferos com placenta, têm sido encontradas em algumas doenças que ocorrem exclusivamente em mulheres. O XIST é um gene chave nesse processo. Por outro lado, muitos microRNAs (pequenos RNAs não codificantes) são expressos abundantemente na placenta humana e alguns estão mapeados no cromossomo X. O objetivo do presente trabalho foi a verificação do padrão de inativação do cromossomo X e da expressão dos genes XIST e dos microRNAs X-específicos miR-221, miR-222 e mir-223 em mulheres com PE. O ensaio de HUMARA (receptor de andrógeno humano) utilizando PCR convencional e digestão com a enzima sensível à metilação HpaII foi analisado de forma qualitativa (visualização em gel de poliacrilamida) e semi-quantitativa (sequenciamento), sendo realizado a partir de sangue periférico (todas as amostras) e de tecido placentário [apenas das placentas de fetos femininos (17 amostras)]. Para o estudo do padrão de expressão foi obtido cDNA por transcrição reversa, a partir de RNA total extraído do tecido placentário (30 amostras).. A análise foi realizada por meio de PCR em tempo real. Foram utilizados os testes de Qui-quadrado e de t-Student, além do modelo linear generalizado para a análise estatística. Não houve diferença estatisticamente significativa para o parâmetro de inativação do cromossomo X entre os grupos controle e de PE, independente do tipo de tecido estudado (sangue ou placenta) quando foram aplicados os ensaios de HUMARA qualitativo e semi-quantitativo. Para o gene XIST e o microRNA miR-221 não foi evidenciada diferença estatisticamente significativa entre os grupos controle e de PE. O microRNA miR-223 não apresentou transcritos detectáveis em nenhum dos grupos de estudo. Para o microRNA miR-222, houve diferença estatisticamente significativa, sendo que no grupo de PE a expressão foi mais elevada. Não foi encontrada associação entre o padrão de inativação do cromossomo X e a expressão do gene XIST e dos microRNAs estudados. Embora a inativação preferencial do cromossomo X tenha sido encontrada nos dois grupos, padrões de inativação preferencial extrema foram verificados em um número maior de casos com PE. Os resultados mostram que o miR-222 apresenta potencial para ser utilizado como marcador molecular da PE, sugerindo também que exista uma diminuição na expressão de seus genes-alvo que devem ser estudados como candidatos na patogênese da doença. / The gestational hypertensive syndromes are among the major causes of maternal and fetal death. The Preeclampsia (PE) is the most prevalent of those syndromes and it is characterized by the increase of blood pressure and proteinury, which start from to 20th week of gestation. Although its ethiology is argued actually the genetics factors have been accepted. Alterations in pattern of chromosome X inactivation, epigenetic process of mammals with placenta have been found in some diseases that occur exclusively in women. XIST is a key gene in this process. On the other hand very microRNAs (small RNAs no coding) are overexpressed in human placenta and someones are located at X choromosome. The subject of this research was verify the patterns of chromosome X inactivation and the expression of the XIST gene and X-specific microRNAs in women affected by PE. In the HUMARA (human androgen receptor) assay was carried out with peripheral blood (all samples) and placental tissue [only female fetuses placentas (17 samples)]. The conventional PCR and digestion methodology with HpaII enzyme were employed and the result was analysed to qualitative (polyacrilamide gel) and semi quantitative (sequencing) form. The cDNA was obtained to gene expression study by reverse transcription reaction from placenta total RNA (30 samples). Gene expression assay was carried out with real time PCR. To statistical analyze was used the Qui-square and t-Student tests besides the widespread lineal model. There was not statistical significant differences to chormosome X inactivation parameter among the groups control and PE independently of the tissue studied (blood or placenta) when the qualitative and semi quantitative HUMARA assay were applied. To XIST and miR-221 were not evidenced significant statistical differences among the groups control and of PE. The miR-223 did not show detectable transcripts to any group studied. The miR-222 expression was more elevated in the PE group than control group and this difference was significant statistically. In the present study was not found association among the chromosome X inactivation pattern and the gene expression of XIST and the microRNAs studied. Although the chromosome X inactivation have been found in the two groups the preferential chromosome X inactivation patterns were verified in a great number of cases in PE group. The results showed that miR-222 has a potential to be employed as molecular marker of PE also suggesting the existence of a decrease in expression of its target genes which have to be investigated like candidates to disease pathogenesis.
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Caractérisation de l'inactivation du chromosome X chez l'humain à la naissance : distribution et transmission des ratios d'inactivationBolduc, Véronique January 2006 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Využití metody paralelního sekvenování při stanovování zešikmení X inaktivace / Use of massive parallel sequencing in determination of skewed X inactivationVeselková, Tereza January 2016 (has links)
Skewed X chromosome inactivation has been often studied as a possible factor that influences manifestation of X-linked diseases in heterozygous women. Yet the association between phenotype and degree of skewing stays unclear for most disorders. Current works rely mostly on methods that are based on methyl-sensitive restriction while determining the X inactivation pattern and mainly the HUMARA assay which investigates the methylation profile in the AR gene. However those methods have some known disadvantages and therefore we are still seeking new methodical approaches. We used DNA isolated from whole blood and in some cases also buccal swabs to asses X inactivation patterns in 54 women using methylation-based methods for loci AR, CNKSR2 and RP2. Transcription-based assay was utilized to evaluate skewing of X inactivation in 32 of those women, whose samples were available for RNA extraction, using massive parallel sequencing and polymorphisms LAMP2 c.156A>T, IDS c.438C>T and ABCD1 c.1548G>A. Partly thanks to almost no stuttering during PCR the RP2 locus was the most informative in our study (71 % of women) and approximately the same number of women (69 %) were informative for the HUMARA assay. However when comparing the results of those two methods we determined difference greater than 10 % in...
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Genetic-epidemiologic analysis of X-inactivation skewing in human females : suggestive evidence for two distinct traitsMio, Robert January 2003 (has links)
Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.
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Étiologie du biais de l'inactivation du chromosome X (ICX) dans les cellules sanguines des femmes vieillissantes : sélection hémizygote et acquisition de mutations somatiquesAyachi, Sami 04 1900 (has links)
Les cellules souches hématopoïétiques (CSH) assurent une production constante des cellules
sanguines tout au long de la vie, mais sont vulnérables à l’acquisition de mutations pouvant
mener à une transformation maligne. Les mutations qui confèrent un avantage de croissance
entraîneront une prolifération clonale. L’étude de la clonalité est centrale à la compréhension
de ces phénomènes. Historiquement, l’analyse de la clonalité a été possible grâce au principe
de l’inactivation du chromosome X (ICX) chez les femmes qui entraîne la création de deux
populations cellulaires, celle avec le X-paternel actif et celle avec le X-maternel actif. Une
déviation (biais) de la proportion théorique de 1 :1 entre ces deux populations peut supposer
une dominance clonale.
Nous avons démontré un biais significatif de l’ICX chez les femmes avec l’âge. Ce
phénomène peut être expliqué par plusieurs causes dont la sélection hémizygote (un des deux
X possède des allèles plus forts que l’autre) et l’acquisition de mutations dans une CSH.
Nous posons l’hypothèse que ces deux phénomènes coexistent et peuvent être distingués par
une approche génomique.
Nous avons recruté une cohorte de 2996 femmes canadiennes-françaises âgées entre 37 et
101 ans composée de 2172 individus issus de 321 familles et de 824 individus non
apparentés. Deux tissus biologiques ont été recueillis : le sang périphérique (PMN,
monocytes, lymphocytes T, lymphocytes B) et des cellules buccales. Le ratio de l’ICX a été
déterminé par la méthode HUMARA, l’analyse de gènes associés à l’hématopoïèse clonale
(19 gènes) a été faite par la méthode de séquençage NGS, et la cohorte a été génotypée à
700 625 loci polymorphiques de l’ADN (SNP). Des analyses bioinformatiques ont été
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appliquées pour étudier la contribution génétique au biais de l’ICX. Nous démontrons que :
(i) le biais de l’ICX est plus prévalent dans les cellules sanguines par rapport aux cellules
épithéliales et maximal dans les cellules myéloïdes; (ii) le biais augmente avec l’âge
seulement dans les cellules sanguines et que cette influence est plus marquée pour les
neutrophiles; (iii) la concordance du biais est très importante pour les différents types
cellulaires sanguins, suggérant un mécanisme opérant au niveau de la CSH ; (iv) il y a une
composante héréditaire liée au biais de l’ICX; (v) la présence de mutations acquises (TET2,
DNMT3A, etc.) explique seulement une partie du biais ; (vi) à l’aide d’analyses par liaison
génétique la présence d’une région sur le chromosome X à Xq21 (LOD score 4.9) qui est
associée au biais des lymphocytes T et une autre sur le chromosome 1 à 1q21 (LOD score
6) qui est associée au biais des neutrophiles.
Nous avons départagé la contribution liée à l’acquisition de mutations somatiques et identifié
pour la première fois des régions liées à une prédisposition génétique. Nos travaux se
poursuivront d’une part par l’analyse de gènes candidats dans les régions identifiées, et
d’autre part nous tenterons d’identifier les cibles génétiques qui confèrent un potentiel de
transformation maligne en utilisant une approche basée sur l’analyse du méthylome, de
l’hydroxyméthylome et du transcriptome que nous venons de valider.
Notre étude démontre la complexité de l’adaptation de l’hématopoïèse au vieillissement et
ouvre des portes sur l’identification de facteurs prédisposant aux cancers hématologiques. / Hematopoietic stem cells (HSC) ensure a constant lifelong production of blood cells, but are
vulnerable to acquisition of mutations, which may lead to malignant transformation.
Mutations that confer a growth advantage will lead to clonal derivation of cells. The study
of clonality is central to the understanding of hematopoiesis adaptation to aging. Historically,
the first clonality assays were based on the principle of X-chromosome inactivation (XCI)
in women. Women are mosaics with half the cells with the paternal X active and the other
half with the maternal one. A skewing from the theoretical 1:1 ratio between these two
populations of cells could infer clonal derivation of cells.
More than 20 years ago, our team demonstrated, through analysis of (XCI) in women, that
skewing increases with age. This intriguing phenomenon can be explained by several
etiology including hemizygous selection (one of the 2 Xs has stronger alleles) or the
acquisition of mutations giving a growth advantage. The first etiology is genetically
predetermined and the second, acquired in somatic cells of bone marrow. We hypothesize
that these two phenomena coexist and can be distinguished with a genomic approach.
To test our hypothesis, we investigated skewing in a cohort of 2996 French-Canadian women
aged 37 to 101 comprised of 2172 related individuals from 321 families and 824 unrelated
individuals. We analyzed XCI ratios at the HUMARA locus in epithelial cells, neutrophils,
T-cells, monocytes, B-lymphocytes. We genotyped the cohort for clonal hematopoiesis and
looked for germline heritable components by genome wide association studies and linkage
analyses. We document that skewing was more prevalent in blood cells than in epithelial
cells, and maximal in myeloid cells. Skewing increases with age only in blood cells. Intra-
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individual correlation of skewing blood cell types was strongly correlated, suggesting
selection influences operating at the HSC. Sibship analyses demonstrated heritability which
was strongest when parental origin of skewing was taken into account. Clonal hematopoiesis
accounted only for a small proportion of the skewing trait but its importance increased in the
very old. Linkage analysis identified a region at Xq21 for skewing occurring in T-cells (LOD
score 4.9) suggesting a hemizygous cell selection influence. We also identified a region at
1q21 for skewing in neutrophils (LOD score 6) suggesting a gene-gene interaction with Xlinked
genes.
We have demonstrated that age-associated skewing is a complex trait caused in part by
acquired mutations and genetic predisposition variants. We will pursue our investigation
using a candidate gene approach in the two identified regions and will try to identify genetic
targets of oncogenic potential by a method based on analysis of the methylome,
hydroxymethylome and transcriptome that was have validated in this cohort.
This thesis demonstrates the complexity of the adaptation mechanisms of hematopoiesis to
aging and set the stage to identification of factors predisposing to hematological cancers.
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