Abstract In Western countries, breast and ovarian cancer are among the most frequent malignancies affecting women. Approximately 5–10% of the cases in the general population have been suggested to be attributed to inherited disease susceptibility. BRCA1 and BRCA2 are the main genes associated with predisposition to breast and ovarian cancer. Mutations in these two genes explain a major part of the families displaying a large number of early-onset breast and/or ovarian cancers, but at least one third of the cases appear to be influenced by other, as yet unidentified genes. Therefore, it is likely that defects in other cancer predisposing genes, perhaps associated with lower disease penetrance and action in a polygenic context, will also be discovered. In the present study, the contribution of germline mutations in putative breast and/or ovarian cancer susceptibility genes, based on their biological function, has been investigated in Finnish breast cancer families. The role of large genomic deletions or other rearrangements in the BRCA1 and BRCA2 genes was evaluated by Southern blot analysis, and mutation analysis of TP53, RAD51, the BRC repeats of BRCA2, and 53BP1 was performed by conformation sensitive gel electrophoresis and DNA sequencing. Germline TP53 mutations were searched for in 108 Finnish breast cancer families without BRCA1 or BRCA2 alterations. In this study, the pathogenic TP53 germline mutation, Arg248Gln, was identified in only one family. This family showed a strong family history of breast cancer and other cancers also fulfilling the criteria for Li-Fraumeni-like syndrome. Germline TP53 mutations are expected to be found in cancer families with clinical features seen in Li-Fraumeni or Li-Fraumeni-like syndromes. In this study, large deletions in BRCA1 and BRCA2 were not observed in 82 breast and/or ovarian cancer families. Likewise, no disease-related aberrations were detected in RAD51, the BRC repeats of BRCA2 or 53BP1 in the 126 breast and/or ovarian cancer families studied. The obtained results were validated by comparing to the occurrence in 288–300 female cancer-free control individuals. These results do not support the hypothesis that alterations in these particular genomic regions play a significant role in breast cancer predisposition in Finland. Thus, there are still genes to be discovered to explain the molecular background of breast cancer.

The D1-class receptors (D1R, D5R) each possess distinct signaling characteristics; however, pharmacological selectivity between them remains elusive. The third intracellular loops (IL3) of D1R and D5R harbour divergent residues that may contribute to their individual signalling phenotypes. Here we probe the function of central region of IL3 of D1R and D5R using deletion mutagenesis. Radioligand binding and whole cell cAMP assays suggest that the N-terminal and C-terminal moieties of the central IL3 oppositely contribute to the constitutive and agonist-dependant activity of D1-Class receptors. Whereas the N-terminal deletions ablated constitutive activity and decreased DA-induced activation, C-terminal deletions induced robust increases. These data, interpreted in concert with structural predictions generated from homology modeling implicate the central IL3 as playing an important role in the activation and subtype-specific characteristics of the D1-class receptors. This study may serve as a basis for the development of novel drugs targeting the central IL3 region.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T16:38:13Z (GMT). No. of bitstreams: 1 Neves_MeirieledaSilvadas_M.pdf: 2620199 bytes, checksum: 70b2df72cfdb93196c91a87c813e12e9 (MD5) Previous issue date: 2015 / Resumo: O gênero Salmonella pertence à família Enterobacteriaceae que agrupa bacilos Gram-negativos, anaeróbios facultativos, fermentadores e geralmente flagelados. S. enterica é um dos patógenos de origem alimentar mais prevalente, sendo que infecções causadas por essa bactéria podem estar relacionadas a praticamente todos os tipos de alimentos. O trabalho foi proposto com o intuito de realizar a caracterização fenotípica e molecular de linhagens atenuadas de Salmonella enterica Typhimurium para genes codificadores de proteínas associadas ao nucleóide (NAPs Nucleoid associated Proteins). As características fenótipicas dos mutantes nulos de Salmonella enterica para os genes ihfA ou ihfB, codificadores das subunidades A e B de IHF, foram avaliadas através de crescimento in vitro, motilidade, sobrevivência frente ao estresse nutricional (sobrevivência em fase estacionária), sob condições ácidas, na presença de sais biliares e quanto à capacidade de invasão e sobrevivência em macrófagos (linhagem J774A.1). Testes de confirmação da atenuação e avaliação da capacidade de induzir proteção em caso de infecção por S. enterica foram realizados utilizando o modelo murino. Os mutantes não apresentaram diferença no crescimento in vitro e na capacidade de sobreviver na presença de sais biliares em comparação com a linhagem selvagem. As linhagens mutantes para os genes ihfA ou ihf ihf ihfB) apresentaram uma menor capacidade de sobrevivência sob condições ácidas quando comparadas com a linhagem selvagem. A motilidade dos mutantes simples também foi reduzida. Os mutantes simples e duplo apresentaram maior capacidade de sobreviver sob estresse nutricional quando comparados com a linhagem selvagem. O mutante para o gene ihfA e o duplo mutante apresentaram um aumento na capacidade de invadir macrófagos. ihf ihfB mostraram uma capacidade aumentada em sobreviver no interior de macrófagos quando comparadas com a linhagem selvagem. Os mutantes nulos viii de Salmonella enterica para os genes ihfA ou ihfB apresentam atenuação, em diferentes graus, quanto à virulência e apresentaram capacidade de induzir proteção no modelo murino de infecção por S. enterica. Esses resultados demonstram que essa proteína apresenta função relacionada com a virulência bacteriana, sendo um importante alvo de estudo na busca de linhagens atenuadas / Abstract: The genus Salmonella belongs to the Enterobacteriaceae family that comprises Gram-negative bacillus, facultative anaerobe, fermenting and generally flagellate. S. enterica is one of the most prevalent food-borne pathogen, and infections caused by this bacterium can be associated to almost all types of food. The work was proposed with the purpose of performing phenotypic and molecular characterization of attenuated strains of Salmonella enterica Typhimurium for genes encoding proteins associated with the nucleoid (NAPs - Nucleoid associated Proteins). The phenotypic characteristics of the null mutants of Salmonella enterica for genes ihfA or ihfB, encoding the A and B subunits of IHF, were evaluated by in vitro growth, motility, survival under nutritional stress (survival in the stationary phase), under acidic conditions, in the presence of bile salts and for the ability of invasion and survival in macrophages (J774A.1 strain). Attenuation tests and evaluation of the capacity to induce protection in case of infection by S. enterica were performed using the murine model. The mutants showed no difference in the in vitro growth and the ability to survive in the presence of bile salts in comparison with the wild type strain. The single mutant for ihfA or ihf ihf ihfB) showed decreased survival under acidic conditions when compared to the wild type strain. Motility of single mutants was also reduced. Single and double mutants showed higher ability to survive under nutritional stress when compared with the wild type strain. The mutant gene for ihfA and the double mutant showed an increased ability to invade ihf ihfB mutants showed an increased ability to survive within macrophages when compared with the wild type strain. Null mutants of Salmonella enterica for ihfA or ihfB genes exhibited attenuation, to varying degrees, for virulence and showed ability to induce protection in a murine model of infection by S. enterica. x These results demonstrate that this protein has function associated to bacterial virulence and is an important subject of study in search for attenuated strains / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular

Gliomas are brain tumors arising from neuroglia. In most cases astrocytic or oligodendroglial component is the main element of the tumor. Non-random chromosomal abberations are found in tumor cells as was revealed previously. The aim of this study was a fluorescence in-situ hybridisation analysis (FISH) of tissue samples obtained during neurosurgical procedures, determine the frequence of selected chromosomal abberations, further correlation with morphological and clinical data and statistical analysis of the results. During six years 264 tissue samples were gained in which FISH with defined probes was performed. The acquired results were compared with histological analysis and selected clinical data (age, Karnofsky score, extent of resection, overall survival). The whole series was divided into 7 groups by tumor type for further statistical analysis. In every group median and mean survival time was calculated, Kaplan-Meier analysis was focused on influence of selected parameters to overall survival. In some categories Cox regression model was created to achieve a hazard ratio of selected parameters. In WHO Grade II and III tumors the risk of malignant progression and tumor upgrading is significantly higher in comparison with samples where specific abberations were not found (EGFR amplification, CDKN2A and...

Chronic Granulomatous Disease (CGD), a disorder of the NADPH oxidase system, results in phagocyte functional defects and subsequent infections with bacterial and fungal pathogens (such as Aspergillus species and Candida albicans). Deletions and missense, frameshift, or nonsense mutations in the gp91 phox gene (also termed CYBB), located in the Xp21.1 region of the X chromosome, are associated with the most common form of CGD. When larger X-chromosomal deletions occur, including the XK gene deletion, a so-called "Contiguous Gene Deletion Syndrome" may result. The contiguous gene deletion syndrome is known to associate the Kell phenotype/McLeod syndrome with diseases such as X-linked chronic granulomatous disease, Duchenne muscular dystrophy, and X-linked retinitis pigmentosa. These patients are often complicated and management requires special attention to the various facets of the syndrome. © 2011 Watkins et al; licensee BioMed Central Ltd.

Chronic Granulomatous Disease (CGD), a disorder of the NADPH oxidase system, results in phagocyte functional defects and subsequent infections with bacterial and fungal pathogens (such as Aspergillus species and Candida albicans). Deletions and missense, frameshift, or nonsense mutations in the gp91 phox gene (also termed CYBB), located in the Xp21.1 region of the X chromosome, are associated with the most common form of CGD. When larger X-chromosomal deletions occur, including the XK gene deletion, a so-called "Contiguous Gene Deletion Syndrome" may result. The contiguous gene deletion syndrome is known to associate the Kell phenotype/McLeod syndrome with diseases such as X-linked chronic granulomatous disease, Duchenne muscular dystrophy, and X-linked retinitis pigmentosa. These patients are often complicated and management requires special attention to the various facets of the syndrome.

There has been tremendous amount of effort focused on the development and improvement of genome editing applications over the decades. Particularly, the development of programmable nucleases has revolutionized genome editing with regards to their improvements in mutagenesis efficacy and targeting feasibility. Programmable nucleases are competent for a variety of genome editing applications. There is growing interest in employing the programmable nucleases in therapeutic genome editing applications, such as correcting mutations in genetic disorders. Type II CRISPR-Cas9 bacterial adaptive immunity systems have recently been engineered as RNA-guided programmable nucleases. Native CRISPR-Cas9 nucleases have two stages of sequence-specific target DNA recognition prior to cleavage: the intrinsic binding of the Cas9 nuclease to a short DNA element (the PAM) followed by testing target site complementarity with the programmable guide RNA. The ease of reprogramming CRISPR-Cas9 nucleases for new target sequences makes them favorable genome editing platform for many applications including gene therapy. However, wild-type Cas9 nucleases have limitations: (i) The PAM element requirement restricts the targeting range of Cas9; (ii) despite the presence of two stages of target recognition, wild-type Cas9 can cleave DNA at unintended sites, which is not desired for therapeutic purposes; and (iii) there is a lack of control over the mutagenic editing product that is procuded. In this study, we developed and characterized chimeric Cas9 platforms to provide solutions to these limitations. In these platforms, the DNA-binding affinity of Cas9 protein from S. pyogenes is attenuated such that the target site binding is dependent on a fused programmable DNA-targeting-unit that recognizes a neighboring DNA-sequence. This modification extends the range of usable PAM elements and substantially improves the targeting specify of wild type Cas9. Furthermore, one of the featured chimeric Cas9 variants developed in this study has both robust nuclease activity and ability to generate predictable uniform editing products. These superior properties of the chimeric Cas9 platforms make them favorable for various genome editing applications and bring programmable nucleases one step closer to therapeutic applications.

The Boston accent is one of the most famous accents in the United States and is known for its non-rhoticity, which essentially means that Bostonians do not normally pronounce their r’s after vowels. While most Boston locals would tell you to ‘pahk the cah ova hea’ when you arrive in the city, not every Bostonian has the same level of non-rhoticity; this variation is due to a number of different factors, but arguably one of the most interesting factors, which this paper focuses on, is gender. This study looks into how Boston non-rhoticity differs between males and females, as well the theories that explain these potential differences. This is done by collecting and analyzing the speech of Boston locals, following two previous studies on the same topic. In addition to gender, types of speech and other social factors are also analyzed. The biggest finding of this study is that there is a statistically significant difference in non-rhoticity between males and females, with females pronouncing more r’s, which supports one previous study and opposes another, and also supports the linguistic theory that women tend to exhibit more standardized speech than men.

User Experience (UX) Writing is part of the product’s overall user experience design. It can be a part of a product content strategy as much as the visual design. The base for any UX centered profession is the understanding of usability — the practice of making products or services easy to use for the intended target group. The difference between the UX designer and UX writer is that the writer contributes with an in-depth understanding that textual content contributes to an overall better user experience along with the product brand. This study includes two UX writing usability evaluation methods for a defined functional service application. By performing the UX writing methods through an online form as well as in an in-person user interview, the validity of transforming an established method into an online tool was investigated. The liabilities of both contexts were evaluated and future developments are suggested. To accomplish this, a literature review was conducted and an online tool was developed. This was used to gather data from 20 users, 10 in the online context and 10 in the in-person context. The datasets were then compared for validation, which in turn served as a foundation for further discussing the possibilities of using an online tool in this situation. The conclusion is multifaceted. The in-person user tests require resources in terms of time for the UX writer, finances in terms of compensation for the users and result in a smaller data set. Digital user tests also require time and resources, but can generate a much larger data set, seeing as it provides a data set of the same quality level as the in-person user tests. Considering the advantages of an online tool, it can provide a valid replacement for in-person user tests, if one considers that the assets outweigh the minor liabilities. However, the potential of a future collaborative dataset and all the further developments suggested in this paper are what is truly worthwhile investigating further. / User Experience (UX) Writing, att anpassa text efter användaren, är en vital del av produktens övergripande användarupplevelse. Det kan vara en del av en produkts innehållsstrategi lika mycket som den visuella designen. Basen för alla användarcentrerade yrken är förståelsen för användbarhet den praxis att göra produkter eller tjänster lättanvända för den avsedda målgruppen. Skillnaden mellan en UX-designer och en UX-skribent är att skribenten bidrar med en djupgående förståelse för att även textinnehåll bidrar till en övergripande förbättrad användarupplevelse, tillsammans med produktens varumärke. Denna studie innehåller två utvärderingsmetoder inom UX writing, för en definierad service-applikation. Genom att utföra metoderna genom en online-formulär samt i en personintervju undersöktes validiteten kring att omvandla en etablerad intervjumetod till ett onlineverktyg. Svagheterna i båda sammanhangen utvärderas och framtida utveckling föreslås. För att uppnå detta genomfördes en litteraturöversikt och ett onlineverktyg utvecklades. Detta användes för att samla in data från 20 användare, 10 i ett onlinesammanhang och 10 i ett intervjusammanhang. Datan jämfördes sedan för validering, vilket i sin tur fungerade som grund för att ytterligare diskutera möjligheterna att använda ett onlineverktyg i denna situation. Slutsatsen är mångfacetterad. De personliga användartesterna kräver resurser i form av tid för UX-skribenten, kostnader vad gäller ersättning för deltagare, och resulterar i ett mindre dataset. Digitala användartester kräver också tid och resurser, men kan generera ett mycket större dataset, eftersom den ger ett dataset av samma kvalitetsnivå. Med tanke på fördelarna med ett onlineverktyg kan det fungera som en giltig ersättning för personliga användartester, om man anser att styrkorna överstiger de mindre svagheterna. Dock är potentialen i en framtida kollaborativt dataset och all ytterligare utveckling som föreslås i denna studie det som är verkligen värt att undersöka ytterligare.

Stem cells, with the ability to self-renew and differentiate into intended cell types, are a valuable tool for disease modeling and mechanistic study. CRISPR-Cas9 has been widely used for genome editing due to its high efficiency and convenience. However, CRISPR-Cas9 has large-deletion safety issues that dramatically restrict its applications. Wiskott-Aldrich syndrome (WAS) is an inborn immunological disorder caused by WASP deficiency. WASP functions in the nucleus, which may help to understand WAS pathology, are poorly defined. Pannexin 1 (PANX1) forms large plasma membrane pores to exchange intracellular small molecules with the extracellular environment and functions in inflammatory processes. The regulatory mechanisms of the PANX1 channel remain obscure. In this dissertation, I focused on mechanistic studies of CRISPR-Cas9 genome editing, and two immune disease relevant genes, WASP and PANX1 using stem cell-derived immune cells. We first found that CRISPR-induced large deletions (LDs) are predominantly mediated by the MMEJ repair pathway through statistical studies. Further, we found POLQ and RPA play vital roles in CRISPR-induced LDs. Modulation of POLQ and RPA can decrease CRISPR-induced LDs and increase HDR efficiency. Using three isogenic WAS iPSC models generated via gene editing, we successfully recapitulated WAS phenotypes, and for the first time, revealed that WASP regulates RNA splicing via epigenetically controlling the transcription of splicing factors and directly participating in the splicing machinery through a liquid-liquid phase separation process. We established a full-length human PANX1 (hPANX1) channel model via cryo-electron microscopy experiments and molecular dynamics simulation study, and found that hPANX1 channel is a homo-heptamer with both the N- and C-termini stretching deeply into the pore funnel. Functional studies of three selected residues support the new hPANX1 channel model and suggest the potential regulatory role of hPANX1 in pyroptosis upon immune responses. Overall, the mechanistic studies of WASP, PANX1 and CRISPR genome editing revealed new roles of WASP in regulating RNA splicing, new functional insights of PANX1 in pyroptosis, and uncovered two critical players POLQ and RPA in CRISPR-induced LDs.