Durant la division cellulaire, la ségrégation des chromosomes et le partage du cytoplasme sont essentiels pour maintenir l'intégrité génomique. Cependant, les erreurs de ségrégation sont fréquentes chez l'embryon préimplantatoire de mammifère et entraînent un gain ou une perte de chromosomes, appelé aneuploïdie. L'aneuploïdie est préjudiciable au développement et est la principale cause de pertes de grossesse. La mitose est coordonnée par cycle cellulaire, notamment la Cycline-B. Comprendre comment la destruction de la Cycline-B contrôle la sortie de la mitose des embryons pourrait expliquer pourquoi l'aneuploïdie est courante en clinique de fertilité. Nous avons étudié la destruction de la Cycline-B en fonction du stade de développement et de l'aneuploïdie. La littérature suggère que l’aneuploïdie perturbe le cycle cellulaire conduisant les cliniques de fertilité à utiliser la durée du cycle cellulaire et la morphologie (morphocinétique) pour prédire la santé de l'embryon. Cependant, la prédiction de la ploïdie par morphocinétique reste à démontrer. Notre objectif était de savoir comment l'aneuploïdie affecte le cycle cellulaire et le développement de l'embryon. Après une micro-injection de CyclineB1:GFP (Cycline-B) et H2B:RFP (chromosomes), les embryons de souris furent imagés par microscopie confocale. Des cellules aneuploïdes furent générées chimiquement pour évaluer leurs morphocinétiques. Curieusement, l'apparition de la Cycline-B après nuclear envelope breakdown a été devancée avec la progression du développement indépendamment de la taille des cellules. De plus, les erreurs de ségrégation ont peu impacté le développement et la destruction de la Cycline-B. Nous concluons que la morphocinétique est un outil prédictif peu fiable pour identifier les embryons aneuploïdes. / During cell division, it is essential that chromosome segregation during mitosis, and the partitioning of the cytoplasm at cytokinesis occur in successive timing to maintain genomic integrity. However, segregation errors are frequently observed in the early mammalian embryo, causing daughter cells to inherit whole chromosome gains and losses, termed aneuploidy. Aneuploidy is detrimental to development, being the leading cause of pregnancy loss and developmental disorders. The timing of mitosis is coordinated by the cell cycle component, Cyclin B. Understanding how Cyclin B destruction temporally controls mitotic exit in embryos could help elucidate why aneuploidy is common in IVF clinics. We investigate how Cyclin B destruction changes in different developmental stages and the presence of aneuploidy. Literature suggests aneuploidy disrupts the cell cycle, leading IVF clinics to use cell cycle timings and morphology (morphokinetics) to predict embryo health. However, whether morphokinetics predicts embryo ploidy is uncertain. We seek to investigate how aneuploidy affects the cell cycle and embryo development. We used live-cell confocal imaging and microinjection of CyclinB1:GFP and H2B:RFP mRNA to visualise Cyclin B and chromosomes during mitosis in the 2-, 4- and 8-cell stage mouse embryo. Secondly, we pharmacologically-induced aneuploidy to assess aneuploid morphokinetics. Interestingly, we observe a developmental trend, independent of cell size, where Cyclin B onset begins progressively sooner after NEBD at the 2-, 4- and 8-cell stage. Additionally, chromosome segregation errors had little impact on Cyclin B destruction and development. Finally, we find morphokinetics to be a poor predictive tool in identifying aneuploid embryos.

Background: The accuracy of DNA image cytometry as an investigation method for potentially malignant disorders of the oral cavity is currently still a subject of controversy, due to inconsistently applied definitions of DNA aneuploidy, small cohorts and different application techniques of the method. The aim of this study was to examine the accuracy of the method as a supplementary diagnostic tool in addition to the cytological examination using internationally consented definitions for DNA aneuploidy. Methods: A total of 602 samples from 467 patients with various oral lesions were included in this prospective study. Brush biopsies from each patient were first cytologically examined and categorized by a pathologist, second evaluated using DNA image cytometry, and finally compared to either histological biopsy result or clinical outcome. Results: Using the standard definition of DNA aneuploidy, we achieved a sensitivity of 93.5%, a positive predictive value for the detection of malignant cells of 98.0%, and an area under the curve of 0.96 of DNA ploidy analysis for the detection of severe oral epithelial dysplasia, carcinoma in situ or oral squamous cell carcinoma. Importantly, using logistic regression and a two-step model, we were able to describe the increased association between DNA-ICM and the detection of malignant cells (OR = 201.6) as a secondary predictor in addition to cytology (OR = 11.90). Conclusion: In summary, this study has shown that DNA ploidy analysis based on conventional specimens of oral brush biopsies is a highly sensitive, non-invasive, patient-friendly method that should be considered as an additional diagnostic tool for detecting malignant changes in the oral cavity.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.

Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

Le centromère est le site chromosomal où le kinetochore se forme, afin d’assurer une ségrégation fidèles des chromosomes et ainsi maintenir la ploïdie appropriée lors de la mitose. L’identité du centromere est héritée par un mécanisme épigénétique impliquant une variante de l’histone H3 nommée centromere protein-A (CENP-A), qui remplace l’histone H3 au niveau de la chromatine du centromère. Des erreurs de propagation de la chromatine du centromère peuvent mener à des problèmes de ségrégation des chromosomes, pouvant entraîner l’aneuploïdie, un phénomène fréquemment observé dans le cancer. De plus, une expression non-régulée de CENP-A a aussi été rapportée dans différentes tumeurs humaines. Ainsi, plusieurs études ont cherchées à élucider la structure et le rôle de la chromatine contenant CENP-A dans des cellules en prolifération. Toutefois, la nature moléculaire de CENP-A en tant que marqueur épigénétique ainsi que ces dynamiques à l'extérieur du cycle cellulaire demeurent des sujets débat. Dans cette thèse, une nouvelle méthode de comptage de molécules uniques à l'aide de la microscopie à réflexion totale interne de la fluorescence (TIRF) sera décrite, puis exploitée afin d'élucider la composition moléculaire des nucléosomes contenant CENP-A, extraits de cellules en prolifération. Nous démontrons que les nucléosomes contenant CENP-A marquent les centromères humains de façon épigénétique à travers le cycle cellulaire. De plus, nos données démontrent que la forme prénucléosomale de CENP-A, en association avec la protéine chaperon HJURP existe sous forme de monomère et de dimère, ce qui reflète une étape intermédiaire de l'assemblage de nucléosomes contenant CENP-A. Ensuite, des analyses quantitatives de centromères lors de différenciation myogénique, et dans différents tissus adultes révèlent des changements globaux qui maintiennent la marque épigénétique dans une forme inactive suite à la différentiation terminale. Ces changements incluent une réduction du nombre de points focaux de CENP-A, un réarrangement des points dans le noyau, ainsi qu'une réduction importante de la quantité de CENP-A. De plus, nous démontrons que lorsqu'une dédifférenciation cellulaire est induite puis le cycle cellulaire ré-entamé, le phénotype "différencié" décrit ci-haut est récupéré, et les centromères reprennent leur phénotype "prolifératif". En somme, cet oeuvre décrit la composition structurale sous-jacente à l'identité épigénétique des centromères de cellules humaines lors du cycle cellulaire, et met en lumière le rôle de CENP-A à l'extérieur du cycle cellulaire. / The centromere is a unique chromosomal locus where the kinetochore is formed to mediate faithful chromosome partitioning, thus maintaining ploidy during cell division. Centromere identity is inherited via an epigenetic mechanism involving a histone H3 variant, called centromere protein-A (CENP-A) which replaces histone H3 in centromeric chromatin. Defects in the centromeric chromatin can lead to missegregation of chromosomes resulting in aneuploidy, a ¬¬frequently observed phenomenon in cancer. Moreover, deregulated CENP-A expression has also been documented in a number of human malignancies. Therefore, much effort has been devoted to uncover the structure and role of CENP-A-containing chromatin in proliferating cells. However, the molecular nature of this epigenetic mark and its potential dynamics during and outside the cell cycle remains controversial. In this thesis, the development of a novel single-molecule imaging approach based on total internal reflection fluorescence and the use of this assay to gain quantitative information about the molecular composition of CENP-A-containing nucleosomes extracted from proliferating cells throughout the cell cycle as well as the dynamics and cellular fate of CENP-A chromatin in terminal differentiation are described. Here, we show that octameric CENP-A nucleosomes containing core Histones H2B and H4 epigenetically mark human centromeres throughout the cell cycle. Moreover, our data demonstrate that the prenucleosomal form of CENP-A bound by the chaperone HJURP transits between monomeric and dimeric forms likely reflecting intermediate steps in CENP-A nucleosomal assembly. Moreover, quantitative analyses of centromeres in myogenic differentiation and adult mouse tissue sections revealed that centromeres undergo global changes in order to retain a minimal CENP-A epigenetic code in an inactive state, upon induction of terminal differentiation. These include a robust decrease in the number of centromeric foci, subnuclear rearrangement as well as extensive loss of CENP-A protein. Interestingly, we show that forced dedifferentiation under cell cycle reentry permissive conditions, rescued the above-mentioned phenotype concomitantly with the restoration of cell division. Altogether, this work delineates the structural basis for the epigenetic specification of human centromeres during the cell cycle and sheds light on the cellular fate of the CENP-A epigenetic code outside the cell cycle.

Objetivos: Descrever a frequência, e investigar fatores preditivos, de óbito fetal espontâneo (OF), em gestações com anomalias cromossômicas. Métodos: Trata-se de estudo retrospectivo, abrangendo o período de novembro de 2004 a maio de 2012, realizado na Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Foram incluídas gestações únicas com diagnóstico pré-natal de trissomia dos cromossomos 21 (T21), 18, 13 (T13/18) e monossomia do X (45X), realizado até a 26ª semana de gestação. Resultados: Foram incluídas 92 gestantes com idade materna média de 32,7 ± 8,7 anos. O diagnóstico das anomalias cromossômicas (T21 n=36, T13/T18 n=25, 45X n=31) foi realizado em idade gestacional média de 18,3 ± 3,7 semanas, por meio de biópsia de vilo corial (n=22, 24%), amniocentese (n=66, 72%) e cordocentese (n=4, 4%). Malformação major estava presente em 45 (49%); e hidropisia foi identificada em 32 (35%) fetos, sendo mais frequente no grupo 45X (n=24/31 (77%) versus T21: n=6/36 (17%) e T13/18: n=2/25 (8%), p < 0,001). Exame ecocardiográfico fetal especializado foi realizado em 60% (55/92) das gestações. Dessas, 60% (33/55) apresentaram alterações na morfologia e/ou função cardíaca, sendo o achado mais frequente a comunicação interventricular (39%). Fetos com T13/18 apresentaram incidência maior de anomalias cardíacas (60% versus 25% (T21) e 29% (45X), p= 0,01). Óbito fetal ocorreu em 55 (60%) gestações e foi mais frequente no grupo 45X (n=26/31 (84%) versus T21: n=13/36 (36%) e T13/18: n=16/25 (64%), p < 0,01). A análise multivariada stepwise demonstrou associação entre hidropisia e OF em fetos com trissomia 21 (LR= 4,29; IC95%= 1,9-8,0, p< 0,0001). Em fetos com monossomia X, a presença de alterações ecocardiográficas esteve associada com menor risco de OF (LR= 0,56; IC95% = 0,27-0,85, p= 0,005). Não foram identificados fatores preditores no grupo T13/18. Conclusão: A letalidade intrauterina de fetos com anomalias cromossômicas é elevada. A presença de hidropisia aumenta o risco de óbito fetal, em gestações com trissomia 21. Enquanto, em gestações com monossomia X, a ocorrência de alterações ecocardiográficas reduz esse risco / Objectives: To describe the frequency, and associated factors, of intrauterine fetal death (IUD), in pregnancies with chromosomal abnormality. Methods: This was a retrospective (November 2004 to May 2012) performed at de department of obstetrics, Hospital das Clínicas, São Paulo University Medical School. Inclusion criteria were: singleton pregnancies with prenatal diagnosis of trisomy 21 (T21), 18, 13 (T13/18) and X monosomy (45X), performed up to 26 weeks gestation. Results: 92 women were included in the study with a mean maternal age of 32.7 ± 8.7 years. Fetal chromosomal abnormalities (T21 n=36, T13/T18 n=25, 45X n=31) were diagnosed at a mean gestational age of 18.3 ± 3.7 weeks, by chorionic villus sampling (n=22, 24%), amniocentesis (n=66, 72%) and cordocentesis (n=4, 4%). Major fetal structural abnormality was present in 45 (49%) cases; hydrops was diagnosed in 32 (35%) fetuses, and was more common in 45X group (n=24/31 (77%) versus T21: n=6/36 (17%) and T13/18: n=2/25 (8%), p < 0.001). Specialist fetal echocardiography was performed in 55 (60%) pregnancies and showed structural and/or functional abnormalities in 33 (60%) fetuses; ventricular septal defect was the most common finding (39%). T13/18 fetuses showed a higher incidence of cardiac abnormalities (60% versus 25% (T21) and 29% (45X), p= 0.01). IUD occurred in 55 (60%) pregnancies and was more common in 45X group (n=26/31 (84%) versus T21: n=13/36 (36%) and T13/18: n=16/25 (64%), p < 0.01). Stepwise logistic regression analysis demonstrated an association between hydrops and IUD in T21 pregnancies (LR= 4.29; 95%CI= 1.9-8.0, p < 0.0001). In 45X pregnancies, cardiac abnormalities were associated with a lower risk of IUD (LR= 0.56; 95%CI = 0.27-0.85, p= 0.005). No predictors of IUD were identified in T13/18 group. Conclusion: Intrauterine death rate is high in pregnancies with a fetal chromosomal abnormality. Presence of hydrops increases the risk of this complication in trisomy 21 fetuses. Whereas the presence of a cardiac abnormality is protective in X monosomy pregnancies

INTRODUÇÃO: A Leucemia/linfoma de células T do Adulto (ATL) é uma doença maligna de fenótipo T CD3+/CD4+/CD25+/CD7- e, geneticamente, apresenta cariótipo complexo e aneuploidia. Clinicamente muito agressiva e ainda incurável, está associada ao vírus linfotrópico T humano do tipo-1 que, preferencialmente, infecta linfócitos T CD4+. Dos indivíduos portadores do HTLV-1, somente 3-5% irão evoluir para ATL e após longo período de latência. Entretanto, os fatores virais ou do hospedeiro que estão associados com a progressão para ATL permanecem desconhecidos. O proto-oncogene hSecurina é um regulador mitótico importante para o processo de segregação cromossômica durante a separação das cromátides irmãs e está envolvido na patogênese de vários tumores. Com o objetivo de avaliar o conteúdo de DNA, o ciclo celular e a expressão do gene hSecurina em células T CD4+ e CD8+ dos portadores assintomáticos do HTLV-1 em comparação com ATL e indivíduos saudáveis, nos propusemos a realizar o presente estudo. MÉTODOS: Foram avaliados 38 portadores assintomáticos do HTLV-1, 20 casos de ATL pareados por sexo e idade com 35 indivíduos saudáveis. Foram estudados, individualmente, os subtipos linfocitários T CD4+ e CD8+, sendo o ciclo celular avaliado por citometria de fluxo e a expressão do gene hSecurina pela reação em cadeia da polimerase quantitativa em Tempo Real. RESULTADOS: Neste estudo, observamos parada de maturação de linfócitos T CD4+ na fase G0/G1 em portadores assintomáticos do HTLV-1 com diferença estatisticamente significante em comparação aos grupos-controle (p=0,041) e ATL (p=0,023). No grupo de portadores assintomáticos, observamos correlação inversa entre a porcentagem de células em G0/G1 e expressão de hSecurina (p=0,018) em linfócitos T CD4+. Porém, neste mesmo grupo, houve correlação direta entre porcentagem de células em fase S e expressão de hSecurina em linfócitos T CD4+ (p=0,001). Como esperado, observou-se maior fase S em ATL em comparação aos grupos-controle (p=0,020) e portador do HTLV-1 (p < 0,001). CONCLUSÃO: Neste estudo, demonstramos que linfócitos T CD4+ de portadores assintomáticos do vírus HTLV-1 apresentam atraso no ciclo celular com aumento de células na fase G0/G1. Este retardo da progressão do ciclo celular correlacionou-se de forma inversamente proporcional à expressão do gene hSecurina / INTRODUCTION: Adult T-Cell Leukemia (ATL) is a malignant disease of the CD3+/CD4+/CD25+/CD7- T-lymphocytes and genetically features complex karyotypes and aneuploidy. It is a clinically aggressive disease, which is yet incurable. It is associated with the human T-cell leukemia virus type 1 (HTLV-1) that preferentially infects CD4+ T-lymphocytes. Among all individuals that carry HTLV-1, only 3-5% will develop ATL and that too after a long latency period. However, the viral or host factors that are associated with the progression of ATL remain unknown. The proto-oncogene hSecurin is an important mitotic regulator for the process of chromosome segregation during sister chromatid separation and is involved in the pathogenesis of various tumors. We decided to conduct this study in order to analyze the DNA content, cell cycle, and expression of the hSecurin gene in CD4+ and CD8+ T cells of asymptomatic HTLV-1 carriers compared with that in ATL and healthy individuals. METHODS: We evaluated 38 asymptomatic HTLV-1 carriers, 20 patients with ATL, and 35 healthy subjects paired by sex and age. We individually studied the lymphocyte subtypes T CD4+ and CD8+; their cell cycles were evaluated by flow cytometry, and the expression of the hSecurin gene was analyzed using quantitative real time polymerase chain reaction. RESULTS: We observed lymphocyte maturation arrest in CD4+ T cells in the G0/G1 phase of asymptomatic HTLV-1 carriers with a statistically significant difference compared to that in the control (p = 0.041) and ATL (p = 0.023) groups. In the asymptomatic HTLV-1 carrier group, we also found an inverse correlation between the percentage of cells in G0/G1 phase and the hSecurin expression (p = 0.018) in TCD4+ lymphocytes. However, in this same group, there was also a direct correlation between the percentage of S phase cells and hSecurin expression in TCD4+ lymphocytes (p = 0.001). As expected, there was a higher number of S phase cells in the ATL group compared to that in the control (p = 0.020) and asymptomatic HTLV-1 carrier (p < 0.001) groups. CONCLUSION: In this study, we demonstrated that CD4 + T lymphocytes from asymptomatic HTLV-1 virus carriers present cell cycle arrest with increased G0/G1 phase cells. This delay in cell cycle progression correlated inversely with the expression of the hSecurin gene

INTRODUÇÃO: A Leucemia/linfoma de células T do Adulto (ATL) é uma doença maligna de fenótipo T CD3+/CD4+/CD25+/CD7- e, geneticamente, apresenta cariótipo complexo e aneuploidia. Clinicamente muito agressiva e ainda incurável, está associada ao vírus linfotrópico T humano do tipo-1 que, preferencialmente, infecta linfócitos T CD4+. Dos indivíduos portadores do HTLV-1, somente 3-5% irão evoluir para ATL e após longo período de latência. Entretanto, os fatores virais ou do hospedeiro que estão associados com a progressão para ATL permanecem desconhecidos. O proto-oncogene hSecurina é um regulador mitótico importante para o processo de segregação cromossômica durante a separação das cromátides irmãs e está envolvido na patogênese de vários tumores. Com o objetivo de avaliar o conteúdo de DNA, o ciclo celular e a expressão do gene hSecurina em células T CD4+ e CD8+ dos portadores assintomáticos do HTLV-1 em comparação com ATL e indivíduos saudáveis, nos propusemos a realizar o presente estudo. MÉTODOS: Foram avaliados 38 portadores assintomáticos do HTLV-1, 20 casos de ATL pareados por sexo e idade com 35 indivíduos saudáveis. Foram estudados, individualmente, os subtipos linfocitários T CD4+ e CD8+, sendo o ciclo celular avaliado por citometria de fluxo e a expressão do gene hSecurina pela reação em cadeia da polimerase quantitativa em Tempo Real. RESULTADOS: Neste estudo, observamos parada de maturação de linfócitos T CD4+ na fase G0/G1 em portadores assintomáticos do HTLV-1 com diferença estatisticamente significante em comparação aos grupos-controle (p=0,041) e ATL (p=0,023). No grupo de portadores assintomáticos, observamos correlação inversa entre a porcentagem de células em G0/G1 e expressão de hSecurina (p=0,018) em linfócitos T CD4+. Porém, neste mesmo grupo, houve correlação direta entre porcentagem de células em fase S e expressão de hSecurina em linfócitos T CD4+ (p=0,001). Como esperado, observou-se maior fase S em ATL em comparação aos grupos-controle (p=0,020) e portador do HTLV-1 (p < 0,001). CONCLUSÃO: Neste estudo, demonstramos que linfócitos T CD4+ de portadores assintomáticos do vírus HTLV-1 apresentam atraso no ciclo celular com aumento de células na fase G0/G1. Este retardo da progressão do ciclo celular correlacionou-se de forma inversamente proporcional à expressão do gene hSecurina / INTRODUCTION: Adult T-Cell Leukemia (ATL) is a malignant disease of the CD3+/CD4+/CD25+/CD7- T-lymphocytes and genetically features complex karyotypes and aneuploidy. It is a clinically aggressive disease, which is yet incurable. It is associated with the human T-cell leukemia virus type 1 (HTLV-1) that preferentially infects CD4+ T-lymphocytes. Among all individuals that carry HTLV-1, only 3-5% will develop ATL and that too after a long latency period. However, the viral or host factors that are associated with the progression of ATL remain unknown. The proto-oncogene hSecurin is an important mitotic regulator for the process of chromosome segregation during sister chromatid separation and is involved in the pathogenesis of various tumors. We decided to conduct this study in order to analyze the DNA content, cell cycle, and expression of the hSecurin gene in CD4+ and CD8+ T cells of asymptomatic HTLV-1 carriers compared with that in ATL and healthy individuals. METHODS: We evaluated 38 asymptomatic HTLV-1 carriers, 20 patients with ATL, and 35 healthy subjects paired by sex and age. We individually studied the lymphocyte subtypes T CD4+ and CD8+; their cell cycles were evaluated by flow cytometry, and the expression of the hSecurin gene was analyzed using quantitative real time polymerase chain reaction. RESULTS: We observed lymphocyte maturation arrest in CD4+ T cells in the G0/G1 phase of asymptomatic HTLV-1 carriers with a statistically significant difference compared to that in the control (p = 0.041) and ATL (p = 0.023) groups. In the asymptomatic HTLV-1 carrier group, we also found an inverse correlation between the percentage of cells in G0/G1 phase and the hSecurin expression (p = 0.018) in TCD4+ lymphocytes. However, in this same group, there was also a direct correlation between the percentage of S phase cells and hSecurin expression in TCD4+ lymphocytes (p = 0.001). As expected, there was a higher number of S phase cells in the ATL group compared to that in the control (p = 0.020) and asymptomatic HTLV-1 carrier (p < 0.001) groups. CONCLUSION: In this study, we demonstrated that CD4 + T lymphocytes from asymptomatic HTLV-1 virus carriers present cell cycle arrest with increased G0/G1 phase cells. This delay in cell cycle progression correlated inversely with the expression of the hSecurin gene

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 155 pages. Includes vita. Includes bibliographical references.

Objetivos: Descrever a frequência, e investigar fatores preditivos, de óbito fetal espontâneo (OF), em gestações com anomalias cromossômicas. Métodos: Trata-se de estudo retrospectivo, abrangendo o período de novembro de 2004 a maio de 2012, realizado na Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Foram incluídas gestações únicas com diagnóstico pré-natal de trissomia dos cromossomos 21 (T21), 18, 13 (T13/18) e monossomia do X (45X), realizado até a 26ª semana de gestação. Resultados: Foram incluídas 92 gestantes com idade materna média de 32,7 ± 8,7 anos. O diagnóstico das anomalias cromossômicas (T21 n=36, T13/T18 n=25, 45X n=31) foi realizado em idade gestacional média de 18,3 ± 3,7 semanas, por meio de biópsia de vilo corial (n=22, 24%), amniocentese (n=66, 72%) e cordocentese (n=4, 4%). Malformação major estava presente em 45 (49%); e hidropisia foi identificada em 32 (35%) fetos, sendo mais frequente no grupo 45X (n=24/31 (77%) versus T21: n=6/36 (17%) e T13/18: n=2/25 (8%), p < 0,001). Exame ecocardiográfico fetal especializado foi realizado em 60% (55/92) das gestações. Dessas, 60% (33/55) apresentaram alterações na morfologia e/ou função cardíaca, sendo o achado mais frequente a comunicação interventricular (39%). Fetos com T13/18 apresentaram incidência maior de anomalias cardíacas (60% versus 25% (T21) e 29% (45X), p= 0,01). Óbito fetal ocorreu em 55 (60%) gestações e foi mais frequente no grupo 45X (n=26/31 (84%) versus T21: n=13/36 (36%) e T13/18: n=16/25 (64%), p < 0,01). A análise multivariada stepwise demonstrou associação entre hidropisia e OF em fetos com trissomia 21 (LR= 4,29; IC95%= 1,9-8,0, p< 0,0001). Em fetos com monossomia X, a presença de alterações ecocardiográficas esteve associada com menor risco de OF (LR= 0,56; IC95% = 0,27-0,85, p= 0,005). Não foram identificados fatores preditores no grupo T13/18. Conclusão: A letalidade intrauterina de fetos com anomalias cromossômicas é elevada. A presença de hidropisia aumenta o risco de óbito fetal, em gestações com trissomia 21. Enquanto, em gestações com monossomia X, a ocorrência de alterações ecocardiográficas reduz esse risco / Objectives: To describe the frequency, and associated factors, of intrauterine fetal death (IUD), in pregnancies with chromosomal abnormality. Methods: This was a retrospective (November 2004 to May 2012) performed at de department of obstetrics, Hospital das Clínicas, São Paulo University Medical School. Inclusion criteria were: singleton pregnancies with prenatal diagnosis of trisomy 21 (T21), 18, 13 (T13/18) and X monosomy (45X), performed up to 26 weeks gestation. Results: 92 women were included in the study with a mean maternal age of 32.7 ± 8.7 years. Fetal chromosomal abnormalities (T21 n=36, T13/T18 n=25, 45X n=31) were diagnosed at a mean gestational age of 18.3 ± 3.7 weeks, by chorionic villus sampling (n=22, 24%), amniocentesis (n=66, 72%) and cordocentesis (n=4, 4%). Major fetal structural abnormality was present in 45 (49%) cases; hydrops was diagnosed in 32 (35%) fetuses, and was more common in 45X group (n=24/31 (77%) versus T21: n=6/36 (17%) and T13/18: n=2/25 (8%), p < 0.001). Specialist fetal echocardiography was performed in 55 (60%) pregnancies and showed structural and/or functional abnormalities in 33 (60%) fetuses; ventricular septal defect was the most common finding (39%). T13/18 fetuses showed a higher incidence of cardiac abnormalities (60% versus 25% (T21) and 29% (45X), p= 0.01). IUD occurred in 55 (60%) pregnancies and was more common in 45X group (n=26/31 (84%) versus T21: n=13/36 (36%) and T13/18: n=16/25 (64%), p < 0.01). Stepwise logistic regression analysis demonstrated an association between hydrops and IUD in T21 pregnancies (LR= 4.29; 95%CI= 1.9-8.0, p < 0.0001). In 45X pregnancies, cardiac abnormalities were associated with a lower risk of IUD (LR= 0.56; 95%CI = 0.27-0.85, p= 0.005). No predictors of IUD were identified in T13/18 group. Conclusion: Intrauterine death rate is high in pregnancies with a fetal chromosomal abnormality. Presence of hydrops increases the risk of this complication in trisomy 21 fetuses. Whereas the presence of a cardiac abnormality is protective in X monosomy pregnancies