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Caracterização estrutural da proteína spliceosomal de Trypanosoma brucei U5-15K / Structural characterization of Trypanosoma brucei\'s spliceossomal protein U5-15KAna Laura de Lima 23 February 2015 (has links)
A doença do sono é um dos maiores obstáculos para o desenvolvimento das áreas rurais da África Subsaariana. O diagnóstico positivo da doença do sono, bem como o estágio em que ela se encontra, é essencial perante a severidade da doença e toxicidade dos medicamentos disponíveis para o tratamento. A eficiência do tratamento e diagnóstico depende do conhecimento do ciclo de vida, biologia do parasito e seu metabolismo. Os tripanossomatídeos possuem mecanismos conservados entre si como a expressão gênica, neste contexto o Trypanosoma brucei pode ser considerado um organismo modelo e o estudo do processamento de RNA mensageiro por splicing neste parasito pode ser extrapolado para outros tripanosomatídeos. O processo de excisão dos íntrons e junção dos éxons é chamado splicing, e tanto o cis quanto o trans-splicing são reações de transesterificação realizados pelo spliceosomo, que consiste de 5 partículas nucleares, as snRNPs (small nuclear ribonucleoprotein) U1, U2, U4, U5 e U6 bem como proteínas não específicas de snRNPs. As snRNPs são complexos que consistem de pequenos RNAs ricos em uridina (U snRNAs) unidos fortemente a proteínas. São conhecidas oito proteínas específicas de U5 snRNP em humanos 220K, 200K, 116K, 102K, 100K, 52K, 40K e 15K. Foi mostrado que a U5-15K humana é essencial em diversos pontos da formação do spliceosomo. O objetivo desse trabalho foi a caracterização estrutural e da atividade de autoclivagem da proteína U5-15K de T. brucei. Essa proteína pertencente à família Dim1e é formada por 155 resíduos de aminoácido e massa molecular de 17,7 kDa. A proteína recombinante clonada no vetor de expressão pET SUMO (Invitrogen) foi expressa em E. coli por indução com IPTG e purificada por cromatografia de afinidade por íons metálico em resina de Cobalto. O produto foi usado para testes da atividade de auto-clivagem, contendo ou não inibidores de protease. A proteína pura também foi usada em estudos de suas propriedades em solução por experimentos de DLS, que mostrou uma maior homogeneidade da proteína na presença de MgCl2, DSF, que mostrou a estabilidade da U5-15K em solução com relação ao pH. As mudanças de conformação da estrutura secundária da U5-15K e U5-15K clivada foi estudada por experimentos de CD, que mostraram uma redução na porcentagem de folhas-β na estrutura terciária da U5-15K clivada, e foi construído um modelo tridimensional através da modelagem por homologia, que foi comparado com os resultados obtidos por experimentos de SAXS. Foram realizados ensaios de cristalização em diversas condições provenientes de kits comerciais com a proteína nas formas clivada e não clivada e em diferentes concentrações. Como perspectivas ficam a definição exata do ponto de clivagem por espectrometria de massas, proteólise da alça flexível para novos ensaios de cristalização e estudo das mutações nos possíveis sítios ativos e sítio de clivagem. / Sleep sickness is one the most important public health problem in the Africa and it causative agent, Trypanosma brucei, is an organism model for the study of different conserved process among the trypanosomatids. In trypanosomes, mRNAs are processed by trans-splicing, in which a common spliced leader sequence (SL) is acquired at the 5\' end of the mRNA to yield a mature transcript. RNA splicing is carried out by the spliceosome, which consists of the U1, U2, U4, U5 and U6 U snRNPs particles and non-snRNP proteins. The ribonucleoproteins are complexes that consist of small uridine-rich RNAs (U snRNAs) and interact with common Sm proteins and proteins that are specific for each snRNP. Seven U5 snRNP specific proteins are known in trypanosomes, 220K, 200K, 116K, 102K, Cwc21, 40K e 15K. The spliceosomal protein U5-15K is essential for the parasite viability and various evidences suggest participation of the member in spliceosome assembly. U5-15K presents a conserved domain dim1, its molecular weight is estimated of 17,7 kDa and 155 amino acids residues. In this work, U5-15K was cloned into pET SUMO (Invitrogen), transformed in BL21(DE3)pLysS and recombinant protein was purified by immobilized ion affinity chromatography. It was possible observe that U5-15K undergo self-cleavage, process inhibited by serine and cysteine protease inhibitors. Dynamic Light Scattering (DLS) experiments showed higher protein homogeneity in the presence of MgCl2 and Differential Scanning Fluorimetry (DSF) data demonstrated higher stability at neutral pH. Circular dichroism (CD) spectra obtained using U5-15K native and cleaved suggest a reduction in percentage of β-sheets at cleaved U5-15K secondary structure. Native and cleaved proteins at different concentrations were used in crystallization trials, however, no suitable protein crystals were observed. A tridimensional homology model for U5-15K from Trypanosoma brucei, using as template the human homologue, present a thioredoxin folding, although it has observed a possible central loop not present in the template. We intent to determine the exact cleavage point using mass spectrometry and new crystallization trials will be performed after removal of probable loops by limited proteolysis.
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As corajosas: etnografando experiências travestis na prostituição / The Brave: etnographing transvestites experiences in prostitutionLetizia Patriarca 13 November 2015 (has links)
Esta dissertação parte de experiências de travestis que são também profissionais do sexo para focar nas relações que estabelecem com donas de casas de prostituição. Há uma atenção para o fazer antropológico, percorrendo as construções das vivências das travestis que se prostituem, perpassando marcadores sociais da diferença a saber, gênero, sexualidade, classe, raça e geração. Em um primeiro momento, surgem as diversas construções identitárias das que circulam pelo bairro Jardim Itatinga (Campinas SP), para depois percorrer a especificidade deste. Estruturado por uma variedade de casas de prostituição, sua dinâmica engendra determinadas vivências do trabalho sexual ali realizado, que permite repensar relações com as donas de casas de prostituição, especificamente das experiências de travestis em casas de prostituição. A hipótese percorrida é a de que donas e suas casas de prostituição podem ser um suporte econômico-afetivo para construções identitárias do universo trans, assim como representam um apoio seguro diante de violências policiais e de clientes que acometem suas vivências na prostituição. / This dissertation is based on the experiences of transvestites who are also sex workers in order to focus on the relationships they establish with madams, brothels owners. Attention is paid to the anthropological doing, covering the experiences buildings of transvestite prostitutes, passing through social markers of difference - namely, gender, sexuality, class, race and generation. In a first moment, the various identity constructions of those who circulate around Jardim Itatinga (Campinas SP) are brought to discussion, and then the specificity of this neighborhood is handled. Structured by a variety of brothels, its dynamic engenders certain experiences of the sexual work performed there, allowing the rethinking of the relations with the brothels owners, specifically the experiences of transvestites in such establishments. The hypothesis sustained is that madams and their brothels can be an economic and emotional support for the identity constructions of the trans universe, at the same time that they represent a secure support in the face of police and customers violence that affect their experiences in prostitution.
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An unusual variant of perilunate fracture dislocationsMorin, Matthew L., Becker, Giles W. 04 March 2016 (has links)
Trans-scaphoid, trans-radial styloid, trans-triquetral perilunate fracture dislocations are rare. We describe a 19-year-old male who suffered this injury after crashing his bicycle. He underwent open reduction internal fixation and percutaneous pinning. Scaphoid union was achieved at 8 weeks. Near complete range of painless motion was achieved by 4 months.
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Psychological wellbeing among the treatment-seeking trans populationDavey, Amanda January 2016 (has links)
The overarching aim of this thesis was to explore psychological wellbeing among a treatment seeking population of trans individuals. Specifically, psychopathology and quality of life were studied as key dimensions of psychological wellbeing. The thesis begins with a proposed model of predictors of psychological wellbeing derived from a review of the literature, which includes social support, interpersonal problems, body dissatisfaction, self-esteem, experiences of transphobia, non-suicidal self-injury (NSSI), age and gender. Study 1 proceeds then to focus specifically on levels of perceived social support and its relationship to psychological wellbeing. Study 2 assesses interpersonal problems. Study 3 investigates prevalence rates of NSSI. Drawing on the findings from the previous three studies, Study 4 subsequently tests an amended model of predictors. In terms of the methodology employed across the studies, a cross-sectional questionnaire survey was conducted using standardised measures, a large sample of trans individuals recruited from a national gender identity clinic (GIC), and a matched control group of non-trans (hereafter referred to as cisgender) individuals. Throughout, consideration was given to differences between trans women and trans men as well as how trans individuals compare to cisgender individuals, with regards to each of the variables tested. The thesis concludes with a revised model of predictors, in addition to recommendations for preventing the development of poor psychological wellbeing and interventions for improving poor psychological wellbeing among the trans population.
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Pd/azaborine-biaryl phosphine complexes: reaction development, mechanistic analysis, and investigations into metal-ligand coordination dynamicsZhang, Yuanzhe January 2021 (has links)
Thesis advisor: Shih-Yuan Liu / Described herein are three research projects that focused on 1) the catalytic activities of Pd/azaborine-derived biary phosphine (Senphos) complexes in 1,3-enyne difunctionalization reactions and 2) the coordination behaviors of these Pd/Senphos complexes. In the first chapter, expansion of the substrate scope and mechanistic studies of the reported Pd/Senphos catalyzed site-, regio- and trans-selective hydroboration of 1,3- enynes are described. In the second chapter, the first intermolecular site-, regio- and transselective chloroboration and cyanoboration of enynes that are enabled by the Pd/Senphos catalytic system are presented. The cyanoboration products, namely vicinal boronsubstituted alkenylnitriles, are demonstrated as versatile synthetic building blocks. In the last chapter, the κ2-P-η2-B,C coordination behavior in a series of 1,2-, 1,3- and 1,4-Senphos ligated Pd(0) or Pd(II) complexes are evaluated based on solid-state structures and variable-temperature NMR measurements. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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Complicating Normal: How Trans People Simultaneously Challenge and Reproduce HeteronormativityKassner, Katherine M. 22 September 2010 (has links)
No description available.
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Trans-community Approaches to Childhood Obesity Prevention and TreatmentHingle, Melanie Daniela January 2008 (has links)
The Trans-community Approaches to Childhood Obesity Prevention and Treatment Study (Activa Y Sana) was a two-year (August 2006 to May 2008) intervention in one hundred and forty-one 3rd and 4th grade children and their parents from four schools in the Sunnyside School District in Tucson, Arizona. Activa was designed to expand upon schools as an intervention venue, and was the first to test the feasibility and impact of a multi-level, or "trans-community" approach (involving children, their parents or caregivers, schools, and community agencies) on the prevention of childhood weight gain in an at-risk Mexican-American population.Families were assigned to one of three interventions, depending upon which school the child attended: Level 1, state-mandated nutrition- and physical activity-based health curricula; Level 2, Level 1 curricula plus an after-school program; or Level 3, Level 1 + Level 2 activities, plus a family intervention.Primary endpoints of this study were changes in child BMI z-score, % fat, abdominal circumference, activity levels and food intake, psychosocial characteristics and correlates of these measures.The three manuscripts contained herein represent the main findings of this pilot study. Identifying potential mediators and describing their influence on childhood overweight is essential to development of successful interventions. In Study #1, the findings for the examination of correlates of child BMI z-score and % fat are reported. Activa Y Sana was designed to test whether combining different levels of intervention would have a greater impact on child weight. In Study #2, the results of this trans-community intervention on child weight status is discussed. The majority of evidence-based obesity prevention programs in use today were not designed with minorities in mind, and the continued scarcity of research in Latino populations has hindered the development of culturally-competent interventions that might reduce overweight prevalence. The challenges encountered while implementing Activa Y Sana, a population-specific intervention, are described in Study #3.The results from this research may be used to help inform the design of future intervention programs with the goal of reducing the burden of obesity in Mexican-American children, currently the fastest-growing segment of the U.S. population.
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Determinação do ácido trans, trans-mucônico urinário por chromatografia líquida de alta eficiência visando a biomonitorização de trabalhadores expostos ao benzeno / Determination of trans, trans-muconic acid by liquid chromatografia high efficiency aiming biomonitoring of workers exposed to benzeneMartins, Isarita 06 December 1999 (has links)
O benzeno é um solvente comprovadamente cancerígeno e, para substâncias com tal característica, não há limites de exposição considerados seguros. Em vista disso, as discussões internacionais e nacionais visam a diminuir, cada vez mais, os níveis de exposição ocupacional permitidos. O ácido trans, trans-mucônico (ttAM) , um produto de biotransformação do benzeno, tem sido preconizado como um bioindicador sensível da exposição ao solvente. Este trabalho foi desenvolvido com o propósito de validar método capaz de detectar o ttAM em urina de indivíduos expostos ao benzeno, bem com estabelecer o melhor período de coleta das amostras. A técnica escolhida foi a cromatografia líquida de alta eficiência (CLAE) com coluna de fase-reversa, Lichrosorb RP 18, e detector de ultra-violeta. O método mostrou-se linear entre 0,2 a 5,0 mg/L (r2 = 0,9943). Os limites de detecção e de quantificação obtidos foram, respectivamente, 0,1 e 0,2 mg/L. A porcentagem de recuperação absoluta média foi de 77,1% e de inexatidão de 27,9%. Os coeficientes de variação médios foram, para a precisão intra-ensaio 7,7 % e, para a interensaio 10,6%. O analito permaneceu estável na matriz por um período de 6 semanas para a concentração de 0,2 mg/L e de 15 semanas, para a 2,0 mg/L, se armazenada em freezer (-20°C) e por até dez dias sob refrigeração (4°C) para os adicionados de 0,2, 2,0 e 5,0 mg/L. Com estes resultados, a validação foi considerada satisfatória. O valor médio obtido nas amostras de trabalhadores ocupacionalmente expostos no final da jornada de trabalho foi de 0,81 mg/g creatinina (mediana=0,62 mg/g creatinina). Estes resultados foram superiores e estatisticamente diferentes (p=0,025) daqueles encontrados em amostras do início da jornada e do dia seguinte, mostrando ser o final da jornada o período que melhor reflete a exposição do dia de trabalho. / Benzene is a carcinogenic chemical used in many plants. The national and international discussions aim to low more and more the permitted occupational exposure limit to benzene, although in fact, there is no safe limit to carcinogens. The trans, trans-muconic acid (ttMA), a benzene metabolite, has been recommended as a sensitive bioindicator in the biological monitoring of workers exposed to this solvent. This work was developed in order to validate a method for ttMA analysis in samples of benzene-exposed workers as well as to establish the best sample collecting time. The chosen technique was the high pressure liquid chromatography with reverse-phase column, Lichrosorb RP 18, and UV detection. A linear relationship (r2 =0.9943) was observed in the range from 0.2 to 5.0 mg/L. The detection and quantification limits were respectively 0.1 and 0.2 mg/L. The average recovery was 77.1 % and the inaccuracy (bias) was 27.9 %. Precision, evaluated by variation coefficient, were 7.7 % (intraassay) and 10.6 % (inter-assay). The ttMA remained stable in the matrix during a period of six weeks for the 0.2 mg/L samples and fifteen weeks for the 2.0 mg/L samples, in both cases when stored at - 20°C. In 0.2, 2.0 and 5.0 spiked samples no significant differences were found when conserved at 4°C for ten days. In the occupationally exposed workers, ttMA values were significantly higher in post-shift samples (p = 0.025) than in pre-shift or in those collected in the following day. These results reveal that the end of the shift is the best period to take urine samples for benzene biomonitoring.
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Comparative analysis of trans-fats and alpha-linolenic acid administration on cardiomyocyte viability during ischemia/reperfusion injuryGanguly, Riya 03 July 2015 (has links)
Ischemic heart disease is the largest cause of death due to cardiovascular origins. A better understanding of the mechanisms responsible for ischemic heart disease increases the potential for therapies. This will lead to decreased mortalities in Canada and around the world. Nutritional interventions have gained increasing attention as causes or treatments for cardiovascular disease. For example, trans fats (TFAs) have both beneficial and deleterious effects on cardiovascular disease [1]. In this study, we would like to examine this phenomenon. We contrast the effects of two different TFAs on cardiomyocyte viability. We compare the industrially produced trans-fat elaidic acid (EA) and the ruminant trans-fat vaccenic acid (VA) on apoptotic and autophagic markers during non-ischemic (control), ischemic (ISCH) and ischemia/reperfusion (IR) conditions. Rat cardiomyocytes are exposed to medium containing fatty acids conjugated with bovine serum albumin for 24 hours. VA and EA have no significant effect on biomarkers of apoptosis or cell death. Interestingly, a similar effect is observed with autophagic and apoptotic markers of LDLr-/- mice whose diets were supplemented with VA or EA. Cells pre-treated with EA prior to 60 minutes of simulated ISCH and 120 minutes of IR increased cell death compared to control through augmented apoptosis. VA decreases the number of dead cells during ISCH and IR. However, the apoptotic parameters remain unchanged. We also observe that VA decreases oxidized phospholipid content in non-ischemic conditions. We conclude that not all TFAs are deleterious to the heart. EA is toxic to cardiomyocytes with or without ISCH or IR whereas VA is cardioprotective during IR and ISCH conditions. We believe VA decreases oxidized phospholipid content to produce this cardioprotective effect.
For the purposes of comparison, we examined the effects of α-linolenic acid (ALA), an essential polyunsaturated fatty acid found in foods like flaxseed. Omega-3 fatty acids have been associated with improved cardiovascular outcomes. Here, isolated adult rat cardiomyocytes from male Sprague Dawley rats were exposed to medium containing ALA for 24 hours and then exposed control, ISCH or IR conditions. Cell death increases during ISCH and IR. An increase in DNA fragmentation and caspase-3 activity was observed in both the ISCH and IR conditions. Pre-treatment of the cells with ALA subsequently inhibits cell death during ISCH and IR challenge and significantly reduced both DNA fragmentation and caspase-3 cleavage during ISCH and IR. Cardiomyocyte resting Ca2+ increased and Ca2+ transients decreased during ISCH or I/R but ALA pre-treatment did not improve either parameter significantly. We hypothesize that apoptosis is initiated through phosphatidylcholine oxidation within the cardiomyocytes. Pre-treatment of cells with ALA resulted in a significant incorporation of ALA within cardiomyocyte phosphatidylcholine. Two pro-apoptotic oxidized phosphatidylcholine (OxPC) species, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) were significantly increased during both ISCH and IR. ALA pre-treatment significantly decreased the production of POVPC and PGPC during ISCH and I/R. It is concluded that ALA protects the cardiomyocyte from apoptotic cell death during simulated ISCH and IR by inhibiting the production of specific pro-apoptotic OxPC species. In summary, we observe a differential effect of ALA, VA and EA on parameters of cardiomyocyte viability during ISCH or IR.
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Étude de l'impact des acides gras trans de source naturelle sur les lipoprotéines plasmatiques /Motard-Bélanger, Annie. January 2008 (has links) (PDF)
Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.
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