While there has been an opulence of data and studies surrounding the study of the developing pancreas in mammals and other vertebrates, the focus has largely been in mice. The paucity of research in the development of the human pancreas has led to diminished knowledge in the area, compared to other species. Recent discoveries provide growing evidence for discrepancies between mouse and human pancreatic development and diseases and highlight the fact that developmental studies of the pancreas in humans are imperative. The need to develop therapies for diabetes, a growing and one of the leading health problems worldwide, further compels more exploration in this area to deepen our understanding in the different aspects of diabetes in humans and its underlying causes. Research involving modelling human diseases in vitro enables the investigation of the cellular and molecular mechanisms underlying these diseases as well as the development of therapies for treating them. The availability of hPSCs brings with it the advantage of overcoming the limitations of animal models for certain disorders such as pancreatic agenesis, the focus of my project. The use of site-specific nucleases such as TALENs for such a purpose represents a paradigm shift in disease modelling, where TALENs are capable of directly correcting disease-causing mutations, therefore permanently eliminating the symptoms with precise genome modifications. Alternatively, TALENs can also be used to inactivate specific genes by inducing site-specific mutations. Using these tools, I found that GATA6 is required for the formation of the definitive endoderm (DE) and pancreas in humans; hPSCs harbouring homozygous GATA6 mutations fail to form the definitive endoderm, and consequently the pancreas, whereas hPSCs harbouring heterozygous GATA6 mutations exhibited impairment in definitive endoderm development, although it remains unclear if this is a protocol dependent defect. At the pancreatic stage, heterozygous GATA6 mutations consistently compromised pancreas formation regardless of protocol used. I also found that GATA6 transcriptionally activates the development of the definitive endoderm and pancreatic endoderm, and possibly represses the development of mesoderm. Furthermore, I also established that GATA6 directly interacts with key definitive endoderm markers CXCR4 and SOX17, and pancreatic marker PDX1. Taken together, the work herein demonstrates the successful use of hPSCs coupled with the TALEN genome editing technology as a unique in vitro system for disease modelling. These findings also establish two developmental windows, the DE and pancreatic progenitor stages, where GATA6 haploinsufficiency can result in the impairment of pancreatic development leading to pancreatic hypoplasia observed in human GATA6 heterozygous patients. Lastly, my work also provides the molecular mechanism by which GATA6 regulates pancreatic development. Overall, this study provided new insights in the role of GATA6 during development of the human pancreas. These results will be important in developing new methods of differentiation for hPSCs and understanding the interconnection between early organogenesis and late onset of diabetes.

Chromosomal anomaly is an abnormality of the number or structure of the chromosome. Based on this, they are classified as either numerical or structural. These anomalies can have a mild or severe effect on the phenotype of the carrier which depends on the chromosomal region involved and the genes implicated. They can be sporadic or inherited. Thus, it is essential to investigate such anomalies both prenatally and postnatally alike. In a standard cytogenetic lab, these anomalies can be detected using low-resolution or high-resolution karyotype, usually by performing GTG (G-bands by trypsin using Giemsa) on chromosomes undergoing mitosis, derived from blood. If higher resolution is required, there are various other molecular cytogenetic techniques available like FISH and microsatellite analysis. More recently, microarrays have opened a new era in the field of molecular genetics by greatly increasing the resolution of screening for copy number gains and losses. Using these techniques, we characterized deletions in the long arm of chromosome 7 in three clinical cases, identified the breakpoint, studied the inheritance pattern and compared our cases with the other cases carrying similar deletion in the literature. It was observed that the deletion was located at 7q36.2, 7q35 and 7q36.1 for cases 1, 2 and 3 respectively. Case 3 also carried a duplication of Xq28.

Copy Number Variants (CNVs) are defined as DNA segments of 1kb or more in length and present in a variable number of copies in the human genome. It has been recently shown that many human genetic diseases including organ malformations are caused by CNVs in a patient's genome. However, the genetic and molecular basis for Renal Agenesis (RA), which is a medical condition whereby unilateral or bilateral fetal kidneys fail to develop, has not yet been extended to CNV studies. By using array-based Comparative Genomic Hybridization, we are analyzing DNA from patients who have RA in order to identify CNVs that are causative for RA; genes within the CNVs will then be assessed for their potential involvement in RA by altering their dose in Xenopus embryos.

No description available.

Made available in DSpace on 2015-07-22T20:50:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-27. Added 1 bitstream(s) on 2015-08-11T03:07:21Z : No. of bitstreams: 1 Publico-12807.pdf: 1925667 bytes, checksum: 40f94a2382fcb5e9e0c7155532f0079a (MD5) / Objetivo: Apresentar e avaliar os resultados histológicos, anatômicos e funcionais do procedimento de McIndoe modificado, utilizando a celulose oxidada (Surgicel®). Métodos: Onze mulheres com agenesia vaginal se submeteram à neovaginoplastia com ou sem canalização útero-vaginal utilizando um molde revestido com celulose oxidada. As cirurgias foram realizadas entre janeiro de 2009 e dezembro de 2010. Oito dessas pacientes tiveram o diagnóstico de Síndrome de Mayer-Rokitansky-Kuster-Hauser (MRKH), e as outras três tiveram o diagnóstico de Agenesia Cervico-vaginal (CVA). O tempo médio de seguimento foi 14 meses (variando de 6 a 24 meses), e incluiu exame físico e avaliação do questionário sexual (Female Sexual Function Index-FSFI). Amostras teciduais foram obtidas no momento da cirurgia e 1 a 12 meses após o procedimento. Biópsias vaginais para controle foram obtidas de um grupo de pacientes com doenças ginecológicas benignas. Avaliou-se a epitelização escamosa do tecido da neovagina, e a quantidade total de colágeno das neovaginas, e comparou-se com o grupo controle. Para análise estatística utilizou-se o teste ANOVA e o t-test. Resultados: Seis meses após a cirurgia, o sucesso anatômico foi encontrado em 100% das pacientes com síndrome MRKH (comprimento vaginal > 6 cm), e sucesso funcional em todas as pacientes que iniciaram atividade sexual (Score FSFI > 30). As biópsias mostraram epitelização da neovagina após 6 meses em todas as amostras, e o conteúdo total de colágeno foi comparável ao de uma vagina normal. Uma complicação maior ocorreu em uma paciente com CVA, que culminou em morte. O procedimento de canalização útero vaginal não foi bem sucedido em criar um pertuito para menstruações regulares mensais em nenhum dos casos. Nenhuma das pacientes com CVA obtiveram sucesso anatômico ou funcional. Conclusão: A neovaginoplastia com a técnica de McIndoe modificada, utilizando a celulose oxidada (Surgicel®), procedimento simples e de baixo custo, permite a formação de canal vaginal com epitelização e conteúdo de colágeno similar ao de uma vagina normal. É uma potencial alternativa terapêutica para síndrome de MRKH. / Objective: To present and evaluate the histological, anatomical and functional results of the McIndoe procedure, as modified by the application of oxidized cellulose (SurgicelTM) in women with vaginal agenesis. Study design: Eleven patients with vaginal agenesis underwent vaginoplasty with or without uterovaginal cannulation using a mold that had been wrapped with oxidized cellulose. The surgeries were performed between January 2009 and January 2010. Eight of the patients had been diagnosed with Mayer-Rokitansky- Kuster-Hauser (MRKH) syndrome, and the remaining three had been diagnosed with cervicovaginal agenesis (CVA). The mean follow-up time was 14 months (range, 6-24 months), and it included clinical examinations and evaluation of the Female Sexual Function Index (FSFI). Neovaginal biopsies were taken from the patients with MRKH syndrome at the time of surgery and 1-12 months after surgery. Control tissues were taken from a group of patients without vaginal disease. The histology of the samples was evaluated to determine squamous epithelialization of the neovaginal tissue over time, and the total collagen content of the neovaginas were compared with normal control subjects. For statistical analysis we employed the ANOVA test and the t-test. Results: At 6 months, anatomical success was achieved in 100% of the MRKH syndrome patients (neovaginal length . 6 cm), and functional success was achieved in 100% of the patients who started their sexual life (FSFI score . 30). Biopsy results showed complete epithelialization of the neovagina after 5 monthsin all samples, and the amount of collagen content in the neovaginas progressively increased. After 6 months, the collagen content was comparable to that of a normal vagina. One major postoperative complication occurred in a patient with CVA, which culminated in death. The uterovaginal canalization procedure was unsuccessful at creating an outflow tract for regular menses in all cases. None of patients with CVA achieved anatomical or functional success. Conclusions: The procedure described here offers patients a functional vagina by means of a simple and low-cost procedure that elicits squamous epithelialization of the neovaginal vault, with total collagen content similar to that of normal vaginal tissue. It is a potential alternative therapeutic approach for MRKH syndrome but not applicable to cases of CVA. / TEDE

A Síndrome de Van der Woude (VWS) se caracteriza pela presença de fossetas congênitas no lábio inferior, associadas ou não à presença de fissura de lábio e/ou palato. Esta entidade é a forma sindrômica mais comum nos pacientes portadores de fissura labiopalatal, mesmo assim, ela é subdiagnosticada. Após o seqüenciamento do DNA, no ano de 2001, houve um aumento no interesse e no desenvolvimento de pesquisas na área de Genética Molecular Humana. Em 2002, pesquisadores identificaram no cromossomo 1, o gene IRF6 como sendo o responsável pela VWS. Neste trabalho, foram utilizados e analisados os prontuários de pacientes cadastrados e portadores da VWS do Hospital de Reabilitação de Anomalias Craniofaciais - HRAC - USP, juntamente com seus familiares em primeiro-grau portadores de fissura de lábio e/ou palato, com ou sem fossetas, totalizando 22 pacientes. Foram realizados exames clínicos e radiográficos, medidas antropométricas, sialometria e heredograma de todos os pacientes. Os resultados obtidos foram confrontados com os dados resultantes do estudo genético e molecular realizado por LIMA (2005), em sua tese de Doutorado. Observamos que os achados bucais são bastante comuns no fenótipo da VWS, já que em nossa amostra tivemos 68,20% dos pacientes com alguma anomalia dentária do desenvolvimento e 45,45% dos portadores da síndrome apresentavam pelo menos ausência de um dente. Sendo assim, o cirurgião-dentista tem importância chave tanto no diagnóstico e na avaliação bucal, quanto no tratamento dos pacientes portadores da VWS. / The Van der Woude syndrome (VWS) is characterized by the presence of congenital lower lip pits associated or not to cleft lip and/or palate. This disorder is the most common syndrome affecting patients with cleft lip and palate; despite of that, it is underdiagnosed. After DNA sequencing in 2001, there was an increase in the interest and development of studies on Human Molecular Genetics. In 2002, investigators identified the gene IRF6 on chromosome 1 as being responsible for the VWS. The present study comprised analysis of records of patients with VWS registered at the Hospital for Rehabilitation of Craniofacial Anomalies - HRAC - USP, as well as their first-degree relatives with cleft lip and/or palate without lip pits, adding up to 22 patients. Analysis comprised clinical and radiographic examinations, anthropometric measurements, salivary flow rate and genealogy of all patients. The results were compared with data on genetic and molecular investigations conducted in the PhD thesis of LIMA (2005). The oral findings in the present sample are very common in the phenotype of VWS, since 68.20% of patients had some type of developmental dental anomaly, and agenesis of at least one tooth was observed in 45.45% of patients. Therefore, dental professionals play a fundamental role in the diagnosis, oral examination and treatment of patients with VWS.

O objetivo deste estudo foi avaliar a associação entre agenesia dentária e padrões esqueléticos faciais. A documentação ortodôntica inicial de 347 indivíduos foi avaliada para verificar a presença de agenesia dentária de pelo menos 1 dente permanente (excluindo terceiros molares) por meio de radiografias panorâmicas. Três medidas angulares foram avaliadas de cada radiografia cefalométrica (SNA, SNB e ANB). Os participantes do estudo foram agrupados em Classe I, Classe II e Classe III esquelética. Para a análise estatística, os indivíduos foram divididos em 2 grupos, \"com agenesia dentária\" e \"sem agenesia dentária\". O teste do qui-quadrado ou teste exato de Fisher foram utilizados para comparar os dados categóricos. O teste ANOVA com Tukey foi utilizado para comparação de médias, com nível de significância de 5%. Vinte e oito indivíduos apresentaram agenesia dentária de pelo menos um dente permanente. Não houve diferença estatística entre os gêneros (p=0,27). O padrão esquelético mais prevalente foi o de Classe I (63,1%), seguido pelo de Classe II esquelética (25,9%) e Classe III esquelética (10,9%). O grupo com agenesia dentária apresentou o ângulo ANB menor (1,66±2,52) do que o grupo sem agenesia dentária (2,86 ±2,49), (p=0,01). Concluindo, os resultados encontrados neste estudo sugerem que a agenesia dentária está associada a alterações no ângulo ANB. / The aim of this study was to evaluate the association between tooth agenesis and skeletal facial pattern. Orthodontic records of 347 subjects were examined. The presence of tooth agenesis of at least 1 permanent (excluding third molars) tooth was evaluated in panoramic radiographs. Three angular measurements were taken from each cephalogram. The patients were also divided in skeletal Class I, skeletal Class II and skeletal Class III. For statistical analysis, the subjects were divided into 2 groups, with tooth agenesis and without tooth agenesis. Chi-square or Fisher exact test was used to compare categorical data. ANOVA with Tukeys post-test was used for means comparisons. An alpha of 5% was established. Twenty-eight subjects presented at least one tooth agenesis. There was no difference between genders (p=0.27). The most prevalent skeletal pattern was Class I (63.1%), followed by skeletal Class II (25.9%), and skeletal Class III (10.9%). The group with tooth agenesis presented smaller ANB angle (1.66 ± 2.52) than the group without tooth agenesis (2.86 ± 2.49), (p=0.01). In conclusion, our results suggested that tooth agenesis is associated with ANB angle alterations.

O objetivo deste estudo foi avaliar a associação entre agenesia dentária e padrões esqueléticos faciais. A documentação ortodôntica inicial de 347 indivíduos foi avaliada para verificar a presença de agenesia dentária de pelo menos 1 dente permanente (excluindo terceiros molares) por meio de radiografias panorâmicas. Três medidas angulares foram avaliadas de cada radiografia cefalométrica (SNA, SNB e ANB). Os participantes do estudo foram agrupados em Classe I, Classe II e Classe III esquelética. Para a análise estatística, os indivíduos foram divididos em 2 grupos, \"com agenesia dentária\" e \"sem agenesia dentária\". O teste do qui-quadrado ou teste exato de Fisher foram utilizados para comparar os dados categóricos. O teste ANOVA com Tukey foi utilizado para comparação de médias, com nível de significância de 5%. Vinte e oito indivíduos apresentaram agenesia dentária de pelo menos um dente permanente. Não houve diferença estatística entre os gêneros (p=0,27). O padrão esquelético mais prevalente foi o de Classe I (63,1%), seguido pelo de Classe II esquelética (25,9%) e Classe III esquelética (10,9%). O grupo com agenesia dentária apresentou o ângulo ANB menor (1,66±2,52) do que o grupo sem agenesia dentária (2,86 ±2,49), (p=0,01). Concluindo, os resultados encontrados neste estudo sugerem que a agenesia dentária está associada a alterações no ângulo ANB. / The aim of this study was to evaluate the association between tooth agenesis and skeletal facial pattern. Orthodontic records of 347 subjects were examined. The presence of tooth agenesis of at least 1 permanent (excluding third molars) tooth was evaluated in panoramic radiographs. Three angular measurements were taken from each cephalogram. The patients were also divided in skeletal Class I, skeletal Class II and skeletal Class III. For statistical analysis, the subjects were divided into 2 groups, with tooth agenesis and without tooth agenesis. Chi-square or Fisher exact test was used to compare categorical data. ANOVA with Tukeys post-test was used for means comparisons. An alpha of 5% was established. Twenty-eight subjects presented at least one tooth agenesis. There was no difference between genders (p=0.27). The most prevalent skeletal pattern was Class I (63.1%), followed by skeletal Class II (25.9%), and skeletal Class III (10.9%). The group with tooth agenesis presented smaller ANB angle (1.66 ± 2.52) than the group without tooth agenesis (2.86 ± 2.49), (p=0.01). In conclusion, our results suggested that tooth agenesis is associated with ANB angle alterations.

A Síndrome de Van der Woude (VWS) se caracteriza pela presença de fossetas congênitas no lábio inferior, associadas ou não à presença de fissura de lábio e/ou palato. Esta entidade é a forma sindrômica mais comum nos pacientes portadores de fissura labiopalatal, mesmo assim, ela é subdiagnosticada. Após o seqüenciamento do DNA, no ano de 2001, houve um aumento no interesse e no desenvolvimento de pesquisas na área de Genética Molecular Humana. Em 2002, pesquisadores identificaram no cromossomo 1, o gene IRF6 como sendo o responsável pela VWS. Neste trabalho, foram utilizados e analisados os prontuários de pacientes cadastrados e portadores da VWS do Hospital de Reabilitação de Anomalias Craniofaciais - HRAC - USP, juntamente com seus familiares em primeiro-grau portadores de fissura de lábio e/ou palato, com ou sem fossetas, totalizando 22 pacientes. Foram realizados exames clínicos e radiográficos, medidas antropométricas, sialometria e heredograma de todos os pacientes. Os resultados obtidos foram confrontados com os dados resultantes do estudo genético e molecular realizado por LIMA (2005), em sua tese de Doutorado. Observamos que os achados bucais são bastante comuns no fenótipo da VWS, já que em nossa amostra tivemos 68,20% dos pacientes com alguma anomalia dentária do desenvolvimento e 45,45% dos portadores da síndrome apresentavam pelo menos ausência de um dente. Sendo assim, o cirurgião-dentista tem importância chave tanto no diagnóstico e na avaliação bucal, quanto no tratamento dos pacientes portadores da VWS. / The Van der Woude syndrome (VWS) is characterized by the presence of congenital lower lip pits associated or not to cleft lip and/or palate. This disorder is the most common syndrome affecting patients with cleft lip and palate; despite of that, it is underdiagnosed. After DNA sequencing in 2001, there was an increase in the interest and development of studies on Human Molecular Genetics. In 2002, investigators identified the gene IRF6 on chromosome 1 as being responsible for the VWS. The present study comprised analysis of records of patients with VWS registered at the Hospital for Rehabilitation of Craniofacial Anomalies - HRAC - USP, as well as their first-degree relatives with cleft lip and/or palate without lip pits, adding up to 22 patients. Analysis comprised clinical and radiographic examinations, anthropometric measurements, salivary flow rate and genealogy of all patients. The results were compared with data on genetic and molecular investigations conducted in the PhD thesis of LIMA (2005). The oral findings in the present sample are very common in the phenotype of VWS, since 68.20% of patients had some type of developmental dental anomaly, and agenesis of at least one tooth was observed in 45.45% of patients. Therefore, dental professionals play a fundamental role in the diagnosis, oral examination and treatment of patients with VWS.

During the Human Genome Project the first hundred billion bases were sequenced in four years, however, the second hundred billion bases were sequenced in four months (NHGRI, 2013). As efforts were made to improve every aspect of sequencing in this project, cost became inversely proportional to the speed (NHGRI, 2013). Human Genome Project ended in April 2003 but research in faster and cheaper ways to sequence the DNA is active to date (NHGRI, 2013). On the one hand, these advancements have allowed the convenient and unbiased generation and interrogation of a variety of omics datasets; on the other hand, they have substantially contributed towards the ever-increasing size of biological data. Therefore, informatics techniques are indispensable tools in the field of biology and medicine due to their ability to efficiently store and probe large datasets. Bioinformatics is a specialized domain under informatics that focusses on biological data storage, organization and analysis (NHGRI, 2013). Here, I have applied informatics approaches such as database designing and web development in the context of biological datasets or bioinformatics, to create a novel web-based resource that allows users to explore the comprehensive transcriptome of common aquatic tunicate named Oikopleura dioica (O .dioica), and access their associated annotations across key developmental time points, conveniently. This unique resource will substantially contribute towards studies on development, evolution and genetics of chordates using O. dioica as a model. Mendelian or single-gene disorders such as cystic fibrosis, sickle-cell anemia, Huntington’s disease, and Rett’s syndrome run across generations in families (Chial, 2008). Allelic variations associated with Mendelian disorders primarily reside in the protein-coding regions of the genome, collectively called an exome (Stenson et al., 2009). Therefore, sequencing of exome rather than whole genome is an efficient and practical approach to discover etiologic variants in our genome (Bamshad et al., 2011). Renal agenesis (RA) is a severe form of congenital anomalies of the kidney and urinary tract (CAKUT) where children are born with one (unilateral renal agenesis) or no kidneys (bilateral renal agenesis) (Brophy et al., 2017; Yalavarthy & Parikh, 2003). In this study, we have applied exome-sequencing technique to selective human patients in a renal agenesis (RA) pedigree that followed a Mendelian mode of disease transmission. Exome sequencing and molecular techniques combined with my bioinformatics analysis has led to the discovery of a novel RA gene called GREB1L (Brophy et al., 2017). In this study, we have successfully demonstrated the validation of exome sequencing and bioinformatics techniques to narrow down disease-associated mutations in human genome. Additionally, the results from this study has substantially contributed towards understanding the molecular basis of CAKUT. Discovery of novel etiologic variants will enhance our understanding of human diseases and development. High-throughput sequencing technique called RNA-Seq has revolutionized the field of transcriptome analysis (Z. Wang, Gerstein, & Snyder, 2009). Concisely, a library of cDNA is prepared from a RNA sample using an enzyme called reverse transcriptase (Nottingham et al., 2016). Next, the cDNA is fragmented, sequenced using a sequencing platform of choice and mapped to a reference genome, assembled transcriptome, or assembled de novo to generate a transcriptome (Grabherr et al., 2011; Nottingham et al., 2016). Mapping allows detection of high-resolution transcript boundaries, quantification of transcript expression and identification of novel transcripts in the genome. We have applied RNA-Seq to analyze the gene expression patterns in water flea otherwise known as D. pulex to work out the genetic details underlying heavy metal induced stress (unpublished) and predator induced phenotypic plasticity (PIPP) (Rozenberg et al., 2015), independently. My bioinformatics analysis of the RNA-Seq data has facilitated the discovery of key biological processes participating in metal induced stress response and predator induced defense mechanisms in D. pulex. These studies are great additions to the field of ecotoxicogenomics, phenotypic plasticity and have aided us in gaining mechanistic insight into the impact of toxicant and predator exposure on D. pulex at a bimolecular level.