The CsgD protein is the master regulator of a pathway leading to the formation of curli, in essence regulating the switch between a motile and a sessile lifestyle for bacteria. The 5’-UTR region of the csgD mRNA is a hotspot for multiple regulatory small RNAs (sRNA) involved in a complex regulatory network. Even though it is previously known how the interaction takes place it is unknown how sRNA binding affects the translational activity. In order to suggest a mode of regulation a mutational assay was performed by making changes in the csgD 5’-UTR and investigate what the translational effects were. Mutations in different regions are shown to affect the translation levels in various ways.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that affects several organs in the human body including the brain, heart, kidneys, eyes, skin, spleen, liver and lungs [Roach, et al., 1999]. TSC is characterized by hamartomas that rarely progress to malignancy in the affected organs. Clinical symptoms of TSC include cortical tubers and subependymal nodules in the brain, seizures, mental retardation, ungual and periungual fibromas, angiofibromas of the face, and angiomyolipomas in the kidneys [Roach, et al., 1999]. TSC displays genetic heterogeneity with two known loci: TSC1 on chromosome 9q34 [Fryer, et al., 1987a] and TSC2 on chromosome 16p13.3 [Kandt, et al., 1992]. The genes for both loci have been isolated and characterized [ The European Chromosome 16 Tuberous Sclerosis Consortium, 1993; van Slegtenhorst, et al., 1997]. The TSC1 gene contains 21 coding and two non-coding exons and encodes for an 8.6 kb mRNA. It spans 45 kb of genomic DNA and codes for hamartin, a 1,164 amino acid protein of 130 kDa. The TSC2 encodes for a 200 kDa protein, tuberin, and spans 43 kb of genomic DNA. The TSC2 gene consists of 41 coding exons and one non-coding exon and encodes for a 5.4 kb mRNA. Both genes are known to function as tumor suppressors [Carbonara, et al., 1994; Green, et al., 1994a; Green, et al., 1994b]. Several groups have performed mutation analysis of both the genes in patients mainly from the western and Japanese populations. A total of 133 mutations in the TSC1 gene and 350 mutations in the TSC2 gene have been reported so far (Human Gene Mutation Database; http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html). However, there is no report on the mutation analysis of the TSC genes from the Indian population. In this study, a total of 24 TSC cases were ascertained from the Indian population and a comprehensive mutation analysis of both the TSC genes was carried out in them to understand the function of both the genes, to locate important domains and also to find the mutational hotspots for molecular diagnosis of TSC. A total of 12 mutations, including seven novel mutations were identified. It was also shown that the most recurrent mutations (c.1831C>T and c.1832G>A) are, in part, due to methylation of the CpG dinucleotide. There are still 15-25% TSC cases in western populations with undetected mutations [Cheadle, et al., 2000a]. Further, there are familial TSC cases linked either to the TSC1 on 9q34 or TSC2 on 16p13.3 which fail to show any mutations in the coding sequences of both genes [Cheadle, et al., 2000a]. The failure to detect mutations in these cases could be due to several reasons. First, it could be that the mutations lie in the regulatory regions (promoters and enhancers) of both the genes, presently unidentified for the TSC1 gene [Cheadle, et al., 2000a]. Second, it is possible that the mutations lie outside of the coding sequences, within intronic sequences, or in the 5’ or 3’ UTRs [Cheadle, et al., 2000a]. Third, it may be due to the limitation of the techniques used to identify mutations [Cheadle, et al., 2000a]. In order to look for mutations in the promoter, the TSC1 gene promoter was characterized using luciferase reporter gene transfection assay. The promoter for the TSC2 gene is known [Kobayashi, et al., 1997]. The promoters of both TSC1 and TSC2 genes were sequenced in all the 24 cases to look for mutations. During the characterization of the TSC1 gene promoter, a novel isoform involving the non-coding exon 1 of the TSC1 gene was discovered serendipitously.

Includes vita. / Title from PDF of title page. / Document formatted into pages; contains 241 pages. / Thesis (Ph.D.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: Genetic changes likely occur in wild fish populations as a consequence of interactions with cultured fish, but to what extent do those changes threaten the maintenance of natural genetic diversity and population viability? Following a review and categorization of numerous processes suspected of being agents of post-release genetic change in recipient wild populations (Chapter 1), I focus on risks relating to the magnitude and duration of releases -- but with a twist. That is, I assume that the mean fitness of released, cultured individuals does not differ from that of the recipient natural population. Throughout, attention is devoted to potential post-release changes in inbreeding (NeI) and variance (NeV) effective population sizes -- indicators of expected rates of population-level change in inbreeding and drift variance, respectively. The reductive effect that large-scale releases exert on NeI in recipient populations can be significant. / ABSTRACT: The effect is shown to be a threshold process (Chapter 2) and thus suggestive of an approach for determining risk-adverse stocking (or release) rates. This approach is utilized in Chapter 3, which describes genetic recommendations for an incipient marine stocking program. Several discordant contemporary NeI models are examined mathematically and by computer simulation (Chapter 4). I show that certain published results pertaining to the effect of multiple paternity on NeI are erroneous; a general model is described which accounts for inbreeding and relatedness in and among parents. That model is utilized in an empirical study of gene correlation in a hatchery cohort (Chapter 5). Propagation-related causes of reductions in NeI are also investigated in this cohort. / ABSTRACT: Finally, extending mutational meltdown theory to accommodate fluctuating population sizes and recessive selective effects, I show that when large reductions in NeV occur (such as those that accompany admixtures of cultured and wild fish), the expected time to population inviability is significantly reduced (Chapter 6). Although a more comprehensive theoretical approach is needed, a precautionary inference may be drawn -- aquaculture-induced reductions in Ne, even though they may be transient, can lead to adverse genetic impacts. Avoidance of Ne-reductions cannot be accomplished, in a practical sense, without considering the stocking or release rates of cultured fish. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [115]-121).

Somatic mutations are the main triggers that initiate the formation of cancer. Large sequencing data sets in recent years revealed a substantial number of mutational processes, many of which are poorly understood or of completely unknown aetiology. These mutational processes leave characteristic sequence patterns, often called "signatures", in the DNA. Characterisation of the mutational patterns observed in cancer patients with respect to different genomic features and processes can help to unravel the aetiology and mechanisms of mutagenesis. Here, we explored the effects of DNA modifications and DNA replication on mutagenesis. The most common mutation type, C>T mutations in a CpG context, is thought to result from spontaneous deamination of 5-methylcytosine (5mC), the major DNA modification. Much less is known about the mutational properties of the second most frequent modification, 5-hydroxymethylcytosine (5hmC). Integrating multiple genomic data sets, we demonstrate a twofold lower mutagenicity of 5hmC compared to 5mC, present across multiple tissues. Subsequently, we show how DNA modifications may modulate various mutational processes. In addition to spontaneous deamination of 5mC, our analysis suggests a key role of replication in CpG > TpG mutagenesis in patients deficient in post-replicative proofreading or repair, and possibly also in other cancer patients. Together with an analysis of mutation patterns observed in cancers exposed to UV light, tobacco smoke, or editing by APOBEC enzymes, the results show that the role of DNA modifications goes beyond the well-known spontaneous deamination of 5mC. Finally, we explored which of the known mutational processes might be modulated by DNA replication. We developed a novel method to quantify the magnitude of strand asymmetry of different mutational signatures in individual patients followed by evaluation of these exposures in early and late replicating regions. More than 75 % of mutational signatures exhibited a significant replication strand asymmetry or correlation with replication timing. The analysis gives new insights into mechanisms of mutagenicity in multiple signatures, particularly the so far enigmatic signature 17, where we suggest an involvement of oxidative damage in its aetiology. In conclusion, our results suggest that DNA replication or replication-associated DNA repair interacts with most mutagenic processes.

Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T09:35:25Z (GMT). No. of bitstreams: 1 Ramalho_VanessaDomingues_M.pdf: 1157730 bytes, checksum: 0cd719050713e1b2340be47d4d5f5b38 (MD5) Previous issue date: 2010 / Resumo: A agamaglobulinemia ligada ao X (XLA; OMIM#300755) é uma imunodeficiência primária humoral caracterizada por um bloqueio na diferenciação dos linfócitos B na medula óssea, levando à profunda hipogamaglobulinemia e reduzido número ou ausência de células B periféricas. Os pacientes com XLA são susceptíveis a infecções recorrentes por bactérias encapsuladas e enterovírus devido à deficiência de anticorpos. Mutações no gene codificante da tirosina quinase de Bruton (Btk) são responsáveis pela doença. Btk é uma tirosina quinase citoplasmática da família Tec importante no desenvolvimento, na diferenciação e na sinalização dos linfócitos B. A detecção de mutações no gene btk possibilita o diagnóstico definitivo de XLA. O objetivo deste estudo foi identificar e caracterizar mutações em btk. Foram incluídos 6 pacientes conforme os critérios do PAGID e ESID: indivíduos do sexo masculino com menos de 2% de linfócitos B periféricos, hipogamaglobulinemia e história de infecções bacterianas de repetição. A triagem de mutações foi realizada com a técnica de SSCP e possíveis mutações foram confirmadas por seqüenciamento. A expressão de Btk nos pacientes e mães foi avaliada em monócitos por citometria de fluxo. Dentre os pacientes analisados as principais manifestações clínicas foram as infecções do trato respiratório. Todos tiveram início dos sintomas durante o primeiro ano de vida, linfócitos B periféricos abaixo de 2% e hipogamaglobulinemia anterior ao início da terapia de reposição de imunoglobulinas. Foram identificadas cinco mutações em btk, três novas (p.Ala347fsX55, p.I355T e p.Thr324fsX24) e duas já descritas na literatura (p.Q196X e p.E441X). A detecção das mutações nos pacientes permitiu a análise mutacional de mães, avós e tias maternas. Três mães e uma avó foram confirmadas portadoras de XLA. Em adição, os valores de expressão de Btk obtidos mostraram deficiência da proteína (4,5% a 65,2%) nos pacientes e um padrão bimodal de expressão de Btk foi observado nas mães, indicando o estado de portadora de XLA. Em um dos pacientes não foi identificada mutação, entretanto a expressão de Btk mostrou-se reduzida. O uso combinado da análise genética e da avaliação da expressão de Btk por citometria de fluxo possibilitou o diagnóstico definitivo de XLA e a identificação de portadoras da doença. / Abstract: X-linked agammaglobulinemia (XLA; OMIM# 300755) is a primary humoral immunodeficiency characterized by a block in early B cell differentiation, leading to profound hypogammaglobulinemia and few or no circulating B cells. Patients with XLA are susceptible to recurrent infections by encapsulated bacteria and enteroviruses due to antibody deficiency. Mutations in the Bruton tyrosine kinase (Btk) gene have been identified as responsible for XLA. Btk is a cytoplasmic tyrosine kinase of the Tec family important in B-lymphocyte development, differentiation, and signaling. Detection of a btk mutation allows definitive diagnosis of XLA. The aim of this study was to identify and characterize mutations in btk. Six patients were included according to the criteria of PAGID and ESID: males with less than 2% of circulating B cells, hypogammaglobulinemia and a history of recurrent bacterial infections. Mutation screening was performed with SSCP technique and possible mutations were confirmed by sequencing. Expression of Btk protein in patients and mothers was assessed in monocytes by flow cytometry. The major clinical manifestations among patients were respiratory tract infections. All had onset of symptoms during the first year of life, circulating B cells below 2% and hypogammaglobulinemia before the start of immunoglobulin replacement therapy. We identified five mutations in btk, three novel (p.Ala347fsX55, p.I355T and p.Thr324fsX24) and two recurrent mutations (p.Q196X and p.E441X). The btk mutations detection in patients enabled the screening of mothers, grandmothers and maternal aunts. Three mothers and one grandmother were confirmed XLA carriers. In addition, flow cytometric evaluation of Btk expression in monocytes revealed that Btk deficiency (4,5% a 65,2%) was present in patients and a bimodal pattern of Btk expression was observed in mothers, indicating that they were XLA carriers. In one patient no mutation was identified, but his Btk expression was reduced. The combined use of genetic analysis and flow cytometric assay of Btk protein expression allowed the definitive diagnosis of XLA and its carriers detection. / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente