Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-18T12:45:18Z No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-28T18:46:03Z (GMT) No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) / Made available in DSpace on 2017-07-28T18:54:15Z (GMT). No. of bitstreams: 1 FERNANDA_CRISTINA_DE_MESQUITA_TES.pdf: 3705815 bytes, checksum: d4984b596a690199bbc567a00b9a5f63 (MD5) Previous issue date: 2017-03-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Liver fibrosis is the wound healing response to repeated injury of the liver. This process begins with the damage of the parenchymal cells and subsequent inflammation, characterized by the rupture of the hepatic architecture associated to the increase of the expression of the components of the extracellular matrix. The development of hepatic fibrosis is based on the activation of hepatic stellate cells (HSC) that undergo phenotypic changes and are characterized by loss of vitamin A deposition and increased cell proliferation, triggering hepatic microcirculatory dysfunction and fibrogenesis in patients with chronic liver disease (CLD). Liraglutide is a GLP-1 agonist (glucagon-like peptide 1) well established as an antidiabetic drug, but also has anti-inflammatory properties, in addition to the effectiveness for NAFLD (non-alcoholic fatty liver disease). Therefore, the aim of this study was to evaluate the effects of liraglutide on the HSC phenotype and liver microvascular function using diverse pre-clinical models of CLD. The results obtained demonstrate that Liraglutide de-activated human and rat HSC phenotype through a GLP1-Rindependent mechanism. Liraglutide did not affect the HSC viability but decreased cell proliferation. CLD-rats receiving liraglutide exhibited significantly lower portal pressure (-20%) with a consequent reduction in intrahepatic vascular resistance. There was also a marked improvements in hepatic vascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue. In conclusion, this study demonstrates for the first time that liraglutide improves hepatic sinusoidal endothelium in clinically relevant experimental models of cirrhosis, which leads to improvement in fibrosis and portal hypertension, and therefore is valid in the treatment of advanced chronic liver disease. / A fibrose hep?tica ? a resposta cicatricial do f?gado ? les?es repetidas. Este processo inicia com o dano das c?lulas parenquimatosas e consecutiva inflama??o, caracterizado pelo rompimento da arquitetura hep?tica associada ao aumento da express?o dos componentes da matriz extracelular. O desenvolvimento da fibrose hep?tica ? baseado na ativa??o das c?lulas hep?ticas estreladas (HSC) que sofrem mudan?as fenot?picas e se caracterizam pela perda do dep?sito de vitamina A e aumento da prolifera??o celular, desencadeando disfun??o microcirculat?ria hep?tica e fibrog?nese nos pacientes com doen?a hep?tica cr?nica (CLD). A liraglutida ? um an?lago do GLP-1 (glucagon-like peptide 1) bem estabelecido como f?rmaco antidiab?tico, mas que tamb?m possui propriedades antinflamat?rias, al?m da efetividade para NAFLD (doen?a hep?tica gordurosa n?o alco?lica). Por essa raz?o, o objetivo deste estudo foi avaliar os efeitos da liraglutida sobre o fen?tipo das HSC e a fun??o microvascular hep?tica utilizando diversos modelos pr?-cl?nicos de CLD. Os resultados obtidos demonstram que a liraglutida desativou o fen?tipo das HSC humanas e de ratos atrav?s de um mecanismo independente do receptor GLP1. A liraglutida n?o afetou a viabilidade das HSC mas diminuiu a prolifera??o celular. Os ratos com CLD que receberam liraglutida apresentaram press?o portal significativamente menor (-20%) com consequente redu??o da resist?ncia vascular intra-hep?tica. Houve tamb?m uma acentuada melhoria na fun??o vascular hep?tica, fibrose, fen?tipo das HSC e fen?tipo endotelial sinusoidal. Os efeitos anti-fibr?ticos da liraglutida tamb?m foram confirmados em tecido hep?tico humano. Como conclus?o, este estudo demonstra pela primeira vez que a liraglutida melhora o endotelio sinusoidal hep?tico em modelos experimentais clinicamente relevantes de cirrose, o que leva a melhora no quadro fibr?tico e na hipertens?o portal e, portanto, pode ser v?lido no tratamento da doen?a hep?tica cr?nica avan?ada.

Cancer, a disease of an uncontrolled cell division, growth and metastasis as a result of genetic mutations, environmental factors and host response, is affecting populations worldwide. Etiology, pathogenicity, and genetics related to cancer are not well understood, and treatment has not been as effective as scientists have expected. Continual research is being done to improve current understanding and treatments. Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers (representing >90 % of all head and neck cancers) involving neoplasms of the oral cavity and oropharynx. OSCC is a very pernicious malignancy developed from epithelial cells. There is evidence that a key N-glycosylation gene, DPAGT1, is associated with cancer. Although N-glycosylation of proteins is involved in organ development and homeostasis of tissue, overexpression of DPAGT1 has been implicated in oral cancer initiation and metastasis. Defects in N-glycosylation underlie congenital disorders, while hyper-N-glycosylation has been shown to be a feature of many cancers. The N-glycosylation pathway directs cell adhesion and cytoskeletal dynamics by impacting the function of E-cadherin, a major epithelial cell-cell adhesion receptor. E-cadherin is a tumor suppressor responsible for the organization of multiprotein complexes named adherens junctions (AJs). In epithelial cells, stable AJs are essential for several cellular processes, including inhibition of cell proliferation, reorganization of the actin cytoskeleton, and maintenance of an epithelial phenotype. Indeed, restoration of AJs has been shown to revert cancer cells from a mesenchymal to an epithelial phenotype and to reduce invasiveness. Previous work has shown that upregulation of DPAGT1 plays a pivotal role in driving canonical WNT/β-catenin signaling (also known as canonical Wnt signaling) that represses E-cadherin adhesions and drives tumorigenic phenotypes in oral cancer. This suggests a role in coordinating balance between proliferation and adhesion by DPAGT1. To date, little is known about the molecular and cellular details underlying differences among OSCC cell lines. CAL 27 and HSC-3 are human cancer cell lines commonly used to in laboratory OSCC research. The main differences between these cell lines include capsular tumors formed by CAL27 cells in nude mouse models in contrast to non-capsular and invasive tumors formed by HSC-3 cells. The goal of this study was to characterize biochemical differences between these two cell lines for further research.

This study examines whether recent changes to the mathematics courses offered in the final year of secondary school (Year 12) in the state of Victoria, Australia have affected the learning outcomes of students in terms of then: skill levels in algebra, calculus and problem solving; and in terms of their preparation for a tertiary mathematics unit. The impact of these changes on the transition from secondary to tertiary mathematics is also considered. A comparison is made between students who attempted a first year mathematics unit at the University of Melbourne (U. of M.) having completed the new V.C.E. (Victorian Certificate of Education) mathematics courses and mathematics courses from the previous H.S.C. (Higher School Certificate) system. The comparison involves the use of tests administered upon entrance to a tertiary mathematics unit at the U. of M., and questionnaires. In 1991, V.C.E, students and H.S.C. students attempted the same mathematics test at the U. of M. and their results were compared. In 1992, the tests were attempted by V.C.E. students only. To compare new V.C.E. students and H.S.C. students, questions on the 1991 test were matched with similar questions on the 1992 tests and a panel of experts determined what the H.S.C. students who attempted the 1991 test would have been expected to average on these matched questions on the 1992 tests had they attempted them. These expected average scores were then compared with the actual scores of the new V.C.E. students. The scores of the groups were scaled when necessary. Questionnaires were administered to 1991 U. of M, mathematics students who were part of the V.C.E. pilot group in 1990, secondary mathematics educators, tertiary mathematics educators, and 1991 V.C.E. (1992 U. of M.) students. The mathematical misconceptions exhibited by new V.C.E. students are discussed and their frequencies stated. The research indicates that the new V.C.E. mathematics courses have provided the V.C.E. mathematics students in this study with significantly lower skill levels and a significantly poorer preparation for a tertiary mathematics unit than those which were previously provided by the H.S.C. mathematics courses.

Throughout the 1980s, there was a trend in Australia towards increased participation rates in post-compulsory education. This study examines factors that influence Year 12 retention rates in four Government secondary schools within the Wollongong Sub-Region. Factors that were thought to influence students to continue to Year 12 and sit for the New South Wales Higher School Certificate Examination included those related to Gender, Socio-economic (relating to occupation of parents, government financial assistance, and language background), Educational and Career. The study involved surveying over 400 Year 10 students in four schools by questionnaires. These schools were chosen for their geographical location and to include two schools with a history of high retention rates and two schools with low retention rates. The questions in the survey were incorporated with a larger survey conducted in 1989 by the Faculty of Education at the University of Wollongong under the coordination of Dr. Noeline Kyle ("Everyone expects you to know; A report on careers advice and industry attitudes towards female students in non-traditional study and work in the Illawarra", 1990). The questionnaire was piloted in 1988 and after seeking recommendations from students, teachers and the NSW Department of School Education Research Group, the survey was administered in 1989. The study used descriptive research methodology, and Chi-square analysis was used to establish significance levels in the data. With regard to gender, the data clearly demonstrated that female students were more inclined than male students to stay on to Year 12, and have positive reasons for their decision. Concerning Socio-Economic factors, the results of this study showed that students whose parents have a professional background are more likely to stay on to Year 12. In addition, the achievement of the Higher School Certificate as a preIV requisite for further study was a strong motivating factor for students staying on to Year 12. However, students in receipt of Austudy did not appear to relate in a statistically significant manner with any of the factors that influence the student to stay on to Year 12. As well, no statistical inference could be drawn from intention to sit for the HSC and the language most used at home by parents. With regard to educational factors, the responses from each of the four schools surveyed showed a high percentage (82 to 85%) of students intending to stay on to Year 12 and sit for the HSC examination. None of these schools had an appreciably higher proportion of students intending to sit the HSC exam. However, it was found that students from one particular school were more likely to undertake further study and students from this school had a high percentage of both parents with a professional occupation than any other school. The findings relating to career factors showed that students who have a professional career in mind are more likely to proceed to Year 12. It was also found that students who had school work experience in a professional occupation were more likely to proceed to Year 12. The study relates the survey findings to the research literature in Australia, and also includes a discussion of the limitations of the survey.

As a teacher of the New South Wales Higher School Certificate Geography Syllabus in the 1970s, I became aware of problems of interpretation and implementation of syllabus documents dealing with models and theories of human aggregate behaviour. A positivistic underpinning allowed explanation in human geography to employ deductive - nomological methodology. This field study investigates a defined literature of academic geography including journals, and both secondary and tertiary documents to identify the extent and quality of nomothetic and idiographic traditions from the late 19th century to the mid 1970s. The literature prior to the late 1950s revealed a dominant regional tradition and idiographic methodology with an emphasis on description of uniqueness of areal phenomena. But underlying currents of a nomothetic nature, running parallel to this regionalidiographic tradition,exerted a noticeable challenge to gain acceptance in geographic circles. This kind of nomothetism was in the form of environmental determinism which held that physical laws operating in nature were also at work to shape and direct human societies. Environmental determinism contained generalised assertions, enjoyed some appeal, but lacked rigorous justification. Even within regional frameworks, authors used environmentally induced determinants to explain the unique character of regions. Not until the 1930s did environmental determinism lose its appeal, after which time the regional - idiographic tradition strengthened as an explanatory mode of human behaviour. Nomothetism emerged in the late 1950s in Australia in the application of models and theories explaining human behaviour. Normative theory was supported by an increased use of quantification and by the growing preference for systematic studies in geography. Neither mode of explanation exists at the total exclusion of the other; so that while nomothetism enjoyed widespread appeal in academic geography from the late 1950s, significant challenges were mounted against it because of its inadequacies as a mode of explaining human aggregate behaviour. Nomothetic explanation in human geography can be seen at the research level and in education circles. Many normative models and theories found their way into senior geography courses to the extent they promoted a systems approach. Teachers would have been aware of normative theory in geography from their university studies and teacher training courses during the late 1950s and throughout the 1960s. The tension between associated explanatory modes in systematic and regional geography becomes apparent in the analysis of the N.S.W. H.S.C. Geography Syllabus in which confusing statements raise problems for teachers interpreting and implementing this prescriptive document. Given these tensions and problems of explanation in human geography, the adoption of a critical rationalist viewpoint as propounded by Karl Popper is suggested as a possible solution for geography teachers when interpreting a syllabus such as that of the N.S.W. H.S.C. Falsification rather than verification should be the node of inquiry towards explanation of human aggregate behaviour.

Le rôle central de VLA-4 et de CXCR4 dans le processus dimplantation médullaire des cellules souches hématopoïétiques (CSH) est bien documenté tandis que la fonction de VLA-5 est plus discutée. Plusieurs études ont rapporté que lamplification ex vivo des CSH en présence de cytokines exogènes réduisait leurs capacités de repeuplement médullaire ; le maintien dune interaction appropriée dans lenvironnement médullaire étant critique pour la conservation dune implantation efficace. Les travaux présentés contribuent à lidentification des changements fonctionnels des récepteurs dadhérence impliqués dans le processus implantatoire des CSH cultivées. En première partie, les interactions statiques et dynamiques de LTC-IC générées ex vivo vis-à-vis de ligands du stroma médullaire ont été comparées aux LTC-IC natives. Lévaluation de ladhérence des LTC-IC in vitro sur les fragments de Fn et VCAM-1 a permis didentifier une altération de létat dactivité de VLA-4 et VLA-5 après expansion. Les adhérences statiques et dynamiques médiées par VLA-4 sur VCAM-1 et Fn étaient compromises contrairement au VLA-5 dont lactivité était augmentée après expansion. Dans un second temps, leffet de lexposition cytokinique sur la contribution respective de VLA-4, VLA-5 et CXCR-4 dans limplantation et le repeuplement médullaire a été examinée chez la souris NOD/SCID et NOD/SCID b2-/-. Il a été montré que limplantation et le repeuplement par des CSH natives sont dépendants de VLA-4, lexpansion ex vivo étant associée à linactivation de VLA-4 ce qui révèle le rôle joué par VLA-5 dans la reconstitution hématopoïétique in vivo. De plus, une diminution de lactivité de CXCR-4 peut induire une perte de sélectivité médullaire lors de limplantation de CSH amplifiées.

The incidence rate of chronic hepatopathy in Taiwan is high, which afflicts the patients by progressively developing irreversible cirrhosis. Hepatic fibrosis is the intermediate and crucial stage of this process, characterized by reversibility. If treated properly in this stage, cirrhosis can be successfully prevented. In the liver, activated stellate cells are the key mediators of fibrosis. Transforming growth factor-

Galveston Bay is the second largest estuary in the Gulf of Mexico. The bay’s watershed and shoreline contains one of the largest concentrations of petroleum and chemical industries in the world, with the greatest concentration within the lower 15 km of the San Jacinto River/Houston Ship Channel (SJR/HSC). Extensive groundwater has been withdrawn to support these industries and an expanding population has resulted elevated land subsidence, with the highest land subsidence in the lower SJR/HSC, of over 3 m (3 cm yr^-1) and has decreased seaward throughout the bay to 0.6 cm yr^-1 near Galveston Island. Mercury (Hg) contamination is well documented throughout the bay’s sediments. Sediment vibra-cores were collected throughout the bay systems. 210Pb and 137Cs geochronologies from these cores was used to determine sedimentation rates and correlated to Hg profiles to estimate input histories. Relative Sea Level Rise (RSLR) is the sum of eustatic sea level rise and land subsidence. The results show sedimentation rates are high in areas with high rates of RSLR and the rates are of the same order of magnitude, however, in general, sedimentation rates are as much as 50% of RSLR, indicating that sedimentation has not kept pace with land subsidence, although they have the same relative order. Hg core profiles were correlated with radioisotope geochronologies and show significant input of Hg beginning around 1940, with a peak around 1971, and a dramatic drop off in concentration afterwards, demonstrating it to be a valuable geochronology tool. Hg concentrations were found to be dramatically higher proximal to the SJR/HSC and progressively decreasing seaward and to distal parts of the bay.

<p>Hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation are regulated by signaling through protein tyrosine kinases (PTK) such as c-kit, Flt-3 and Tie2. PTKs work in concert with receptor protein tyrosine phosphatases (PTPs) to maintain cellular equilibrium. The functions of PTPs in counterbalancing PTK signaling in HSCs however remain incompletely understood. Our laboratory has demonstrated that a heparin binding growth factor, Pleiotrophin (PTN), promotes the expansion of murine long-term (LT)-HSCs via binding to a PTP, protein tyrosine phosphatase receptor type Z (PTPRZ). The addition of PTN to murine PTPRZ-/- c-Kit+Sca-1+Lineage- (KSL) cells caused no expansion of HSCs in culture, suggesting that PTPRZ mediates PTN effects on HSC growth. We subsequently screened for the expression of other receptor PTPs in murine HSCs. Among 21 different receptor PTPs, we found that protein tyrosine phosphatase receptor type S (PTPRS) was significantly overexpressed in mouse and human HSCs compared to more mature hematopoietic cells. Ptprs-/- mice displayed no difference in mature blood counts or phenotypic HSC frequency compared to Ptprs+/+ mice. However, competitive transplantation of bone marrow (BM) cells from Ptprs-/- mice resulted in more than 8-fold increased multilineage hematopoietic repopulation in primary and secondary recipient mice compared to mice transplanted with BM cells from Ptprs+/+ mice. While Ptprs-/- mice displayed no differences in cell cycle status, HSC survival or homing capability compared to Ptprs+/+ mice, PTPRS-/- BM cells expressed significantly increased levels of activated Rac1, a RhoGTPase which regulates HSC engraftment capacity, compared to PTPRS+/+ BM cells. PTPRS-/- BM cells displayed significantly increased transendothelial migration capacity and cobblestone area forming cells (CAFC), consistent with increased Rac1 activation. Furthermore, inhibition of Rac1 abrogated the increased transendothelial migration capacity of PTPRS-/- BM cells, suggesting that the augmented engraftment capacity of PTPRS-/- BM cells was mediated via Rac1. Translationally, we demonstrated that negative selection of human cord blood Lin-CD34+CD38-CD45RA- cells for PTPRS expression yielded a 15-fold enrichment for human long term HSCs compared to Lin-CD34+CD38-CD45RA- cells or Lin-CD34+CD38-CD45RA- PTPRS+ cells. These data suggest that PTPRS regulates HSC repopulating capacity via inhibition of Rac1 and selection of human PTPRS - negative HSCs is a translatable strategy to significantly enrich human cord blood HSCs for transplantation.</p> / Dissertation

La hiperplasia suprarrenal congénita (HSC) comprende un conjunto de trastornos hereditarios de la esteroidogénesis, causados por la deficiencia de alguna de las enzimas necesarias para la conversión del colesterol en cortisol. El más frecuente es el déficit en 21-hidroxilasa, responsable de cerca del 90% de los casos de HSC. El déficit en 21-hidroxilasa es una enfermedad hereditaria autosómica recesiva, causado por una alteración genética en el gen CYP21B que codifica la enzima.Esta enfermedad presenta un espectro muy amplio de manifestaciones clínicas; desde formas severas que se presentan al nacimiento, en las que la virilización puede ir o no acompañada de un síndrome pierde sal, hasta formas más leves en que los signos de virilización se presentan más tardíamente. La incidencia de las formas clásicas de este déficit, oscila alrededor de 1:10.000-15.000 nacimientos, mientras la de las formas no clásicas se ha estimado en 1:1.000 en la población blanca, con marcadas variaciones étnicas.El objetivo ha sido la caracterización clínica, biológica y genética de la HSC por déficit en 21-hidroxilasa de una población de la Comunitat Autònoma de Catalunya, con el fin de poder realizar un diagnóstico etiológico concluyente y establecer el consejo genético familiar completo.Han colaborado 53 familias (106 cromosomas no emparentados) con al menos un hijo diagnosticado de HSC por déficit en 21-hidroxilasa, siendo el total de individuos estudiados de 214. De este total, 71 son pacientes que según los síntomas clínicos fueron clasificados en: 13 SW; 5 SV y 53 NC; 102 son progenitores y 41 son hermanos de dichos pacientes, ninguno de ellos clínicamente afecto.Para la determinación de la función hormonal se practicó una prueba de estimulación con corticotropina (test de ACTH), con valoración de las concentraciones plasmáticas de 17-hidroxiprogesterona antes y tras 30 minutos de la inyección. La determinación de los antígenos de Clase I y Clase II del Sistema HLA, se realizó mediante la técnica de Microlinfocitotoxicidad. El análisis genético consistió en la detección de grandes reordenamientos mediante la técnica de "Southern blot", y la detección de nueve de las mutaciones puntuales más comunes mediante el diseño de una nueva metodología en la que se emplean dos estrategias según la mutación a detectar: ganancia o pérdida de al menos una diana de restricción en el producto de PCR amplificado a partir de DNA genómico o creación de una diana de restricción por amplificación (ACRS-PCR) y posterior digestión con una enzima de restricción apropiada.El test de ACTH se confirma como fiable, ya que no se observaron falsos positivos ni falsos negativos en los valores obtenidos de 17-hidroxiprogesterona post estimulación. El nomograma de 17-hidroxiprogesterona proporciona un patrón hormonal que facilita, en la mayoría de los casos, el diagnóstico de las distintas formas de presentación clínica del déficit. No se observó la presencia del antígeno HLA-Bw47 en ninguno de los grupos analizados. Las formas no clásicas se encuentran significativamente asociadas al haplotipo HLA-B14-DR1, mientras que las formas clásicas al antígeno HLA-B5(w51).La metodología utilizada para el análisis molecular del gen ha permitido detectar la mutación causal en el 89,62% de los cromosomas afectos, evaluados globalmente, consolidándose ésta como altamente efectiva en el diagnóstico de esta patología. Las mutaciones más frecuentes observadas han sido: Intrón 2 (50%) en SW; Intrón 2 (50%) y I172N (16,6%) en SV y V281L (61,5%) en NC. El análisis de los pedigrees ha permitido diagnosticar veintiséis formas crípticas y revelar en dos casos la presencia de mutaciones originadas de novo. La concordancia hallada entre el genotipo y el fenotipo ha sido del 97,2%, de ahí, que las predicciones desde el genotipo han de ser hechas todavía con gran cautela.