Submitted by EDER MARQUES DA SILVA null (biodida@yahoo.com.br) on 2016-05-23T19:54:50Z No. of bitstreams: 1 TESE_Eder30 04.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-25T18:03:50Z (GMT) No. of bitstreams: 1 silva_em_dr_bot.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) / Made available in DSpace on 2016-05-25T18:03:50Z (GMT). No. of bitstreams: 1 silva_em_dr_bot.pdf: 2779899 bytes, checksum: c45da4458d84767b126f9e3b3af87767 (MD5) Previous issue date: 2016-05-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O processo de frutificação é definido como a transição de um ovário em estágio quiescente para estágio de iniciação de formação do fruto. Esta transição é iniciada em resposta aos eventos de polinização e fertilização, os quais induzem o início dos processos fisiológicos e moleculares, que, ao final, irão originar o fruto. A frutificação é fundamental para reprodução nas plantas com flores (Angiospermas). Já foi demonstrado que hormônios (tais como auxina e giberelina) podem atuar em paralelo com fatores de transcrição durante o desenvolvimento do fruto. Alguns desses fatores de transcrição são pós-transcricionalmente regulados por microRNAs. MicroRNAs, são pequenos RNAs (20-22 nt) que regulam pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas.Entretanto, vias genéticas orquestradas por microRNAs associadas com o desenvolvimento de ovário, e consequentemente, do fruto em tomateiro (Solanum lycopersicum L.), permanecem pouco exploradas. Neste trabalho, foi investigada a contribuição da via SlymiR159/GAMYB-like durante o processo de desenvolvimento do ovário e frutificação em tomateiro. O miR159 e seus alvos (SlyGAMYB1 e 2) são dinamicamente expressos durante os estágios de desenvolvimento do ovário e consequentemente do fruto. Eventos transgênicos de tomateiro (cv. Micro-Tom) apresentando super-expressão do precursor SlyMIR159 (denominados OE-159) exibiram frutificação precoce quando comparadas aos controles. Notavelmente, todos os eventos OE-159 apresentaram formação de frutos partenocárpicos. Tal alteração no padrão de frutificação pode estar correlacionada a repressão do gene SlyGAMYB1 em períodos pré-antese de desenvolvimento do ovário, é possível que a atividade do fator de transcrição SlyGAMYB1 seja importante para prevenir o início da frutificação antes da polinização. Além disso, a via regulada pelo miR167/SlyARF8A mostrou-se desregulada em ovários pré-antese de plantas transgênicas OE-159. Adicionalmente, mesmo com o fenótipo de partenocarpia e frutificação precoce, não houve alterações na quantidade de hormônios (e.g. giberelina, ácido abscisico e auxina) relacionados à formação de frutos nas plantas transgênicas OE-159. Contudo, a via miR159/GAMYB-like encontra-se reprimida nos primeiros estágios da frutificação (após polinização e fertilização) e a expressão do SlyARF8A (alvo do miR167) encontra-se induzida na presença de auxina. Em conjunto, nossos dados sugerem que o crosstalk entre as vias moduladas pelos microRNAs miR159 e miR167, integrados com a via de sinalização de auxina, é importante para a frutificação em tomateiro. / Fruit set, defined as the shift from quiescent ovary to a fast-growing young fruit, is a key process for fruit production in flowering plants. It has been shown that hormones (such as auxin and gibberellin) act in parallel with transcription factors during fruit set. Some of these are post-transcriptionally regulated by microRNAs (miRNAs). MiRNAs are a group of small non coding RNAs (21-22 nt) that act by regulating post-transcriptionally genes in plants and animals However, microRNA-regulated genetic pathways associated with tomato (Solanum lycopersicum L.) ovary development and fruit set remain poorly exploited. Here, we investigated the functional role of miRNA159/SlyGAMYBlike in tomato ovary development and fruit set. MiR159 and its targets were dynamically expressed in developing flowers, ovaries and fruit tissues. Transgenic tomato (cv. Micro-Tom) plants over-expressing the SlyMIR159 (termed OE-159) exhibited fruit set earlier when compared with control plants. Strikingly, all transgenic lines presented parthenocarpy fruits. Such developmental modification may be a result of the repression of SlyGAMYB1 in pre-anthesis ovaries, our data suggest that SlyGAMYB1 activity is important to prevent fruit set before pollination. In addition as a consequence of this repression, the miR167 node (miR167/SlyARF8 pathway) was mis-regulated in OE-159 ovaries. In addition, we observed that levels of auxin, gibberellin (GA) and abscisic acid (ABA) in pre-anthesis ovaries of several transgenic lines overexpressing the SlyMIR159 were similar to those of the control. Moreover, our experiments suggested that auxin and GA may act repressing the miR159 node during fruit set initiation, in order to release the miR167-targeted SlyARF8A expression during fruit set. Our data show how two microRNA nodes (miR159 and miR167) integrated with hormone signalling into a circuit that coordinates successive steps along ovary and fruit development.

Orientador: Fábio Tebaldi Silveira Nogueira / Resumo: O processo de frutificação é definido como a transição de um ovário em estágio quiescente para estágio de iniciação de formação do fruto. Esta transição é iniciada em resposta aos eventos de polinização e fertilização, os quais induzem o início dos processos fisiológicos e moleculares, que, ao final, irão originar o fruto. A frutificação é fundamental para reprodução nas plantas com flores (Angiospermas). Já foi demonstrado que hormônios (tais como auxina e giberelina) podem atuar em paralelo com fatores de transcrição durante o desenvolvimento do fruto. Alguns desses fatores de transcrição são pós-transcricionalmente regulados por microRNAs. MicroRNAs, são pequenos RNAs (20-22 nt) que regulam pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas.Entretanto, vias genéticas orquestradas por microRNAs associadas com o desenvolvimento de ovário, e consequentemente, do fruto em tomateiro (Solanum lycopersicum L.), permanecem pouco exploradas. Neste trabalho, foi investigada a contribuição da via SlymiR159/GAMYB-like durante o processo de desenvolvimento do ovário e frutificação em tomateiro. O miR159 e seus alvos (SlyGAMYB1 e 2) são dinamicamente expressos durante os estágios de desenvolvimento do ovário e consequentemente do fruto. Eventos transgênicos de tomateiro (cv. Micro-Tom) apresentando super-expressão do precursor SlyMIR159 (denominados OE-159) exibiram frutificação precoce quando comparadas aos controles. Notavelment... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fruit set, defined as the shift from quiescent ovary to a fast-growing young fruit, is a key process for fruit production in flowering plants. It has been shown that hormones (such as auxin and gibberellin) act in parallel with transcription factors during fruit set. Some of these are post-transcriptionally regulated by microRNAs (miRNAs). MiRNAs are a group of small non coding RNAs (21-22 nt) that act by regulating post-transcriptionally genes in plants and animals However, microRNA-regulated genetic pathways associated with tomato (Solanum lycopersicum L.) ovary development and fruit set remain poorly exploited. Here, we investigated the functional role of miRNA159/SlyGAMYBlike in tomato ovary development and fruit set. MiR159 and its targets were dynamically expressed in developing flowers, ovaries and fruit tissues. Transgenic tomato (cv. Micro-Tom) plants over-expressing the SlyMIR159 (termed OE-159) exhibited fruit set earlier when compared with control plants. Strikingly, all transgenic lines presented parthenocarpy fruits. Such developmental modification may be a result of the repression of SlyGAMYB1 in pre-anthesis ovaries, our data suggest that SlyGAMYB1 activity is important to prevent fruit set before pollination. In addition as a consequence of this repression, the miR167 node (miR167/SlyARF8 pathway) was mis-regulated in OE-159 ovaries. In addition, we observed that levels of auxin, gibberellin (GA) and abscisic acid (ABA) in pre-anthesis ovaries of several tran... (Complete abstract click electronic access below) / Doutor

A group of small GTPases consist of over twenty protein families in the super class P-loop. It has a very diverse cell functions. Small GTPases regulate the formation of vesicular follicles, cytoskeleton and nuclear transport. They participate also on cell proliferation and signaling. The aim of my work is to find important amino acids that define family and distinguish each other. I focus on families Arf, Rab, Ran, Ras and Rho. At the Rho family I am also devoted to classes Rho, Rac and Cdc42. Amino acids are identified using bioinformatic programs selected Consurf and Sca5. The objective is also to test P2RANK specialized tool developed at the Charles University in Prague that predict ligand binding sites from protein structure in different families. Founding amino acids can have a big role in the functional divergence of individual families and classes of small GTPases and can be the basis for future study example for the proliferation of cancerous cells. 1.1 Keywords Powered by TCPDF (www.tcpdf.org)

Introdução: Anormalidades nos supressores tumorais p14, p16 e p53 são relatadas em diversos tipos de câncer em humanos. Na carcinogênese ovariana, p16 e p53 foram extensivamente estudados, mas p14 foi analisado somente em carcinomas. Objetivo: O estudo visa determinar a expressão imuno-histoquímica de p14, p16 e p53 em tumores ovarianos epiteliais benignos, borderline e malignos. Método: Estudo transversal utilizando imuno-histoquímica em amostras de tumores epiteliais ovarianos emblocados em parafina do Hospital de Clínicas de Porto Alegre. Resultados: p14 foi positivo em 93% dos tumores benignos, 94% dos borderline e 60% dos malignos. A perda de expressão foi estatisticamente associada com carcinomas. p16 foi positivo em 94,6% dos carcinomas, 75% dos tumores borderline e 45,7% dos benignos. p53 foi positivo em 29,7%, 16,7% e 2,9% dos tumores malignos, borderline e benignos, respectivamente. Os subtipos de carcinoma não mostraram diferenças de expressão. Conclusão: Nosso estudo foi o primeiro a descrever a expressão de p14 em tumores benignos e borderline. Ela permanece estável nos benignos e borderline, enquanto os carcinomas exibem uma perda de expressão significativa. Isso pode indicar que anormalidades de p14 acontecem tardiamente na carcinogênese. As taxas de expressão de p16 e p53 foram semelhantes a estudos anteriores. Estudos futuros devem investigar anormalidades genéticas nas sequencias codificadoras de p14 e incluir todos os tipos de tumor epitelial ovariano. / Background: Abnormalities in tumor suppressors p14, p16 and p53 are reported in several human cancers. In ovarian carcinogenesis, p16 and p53 have been extensively studied, but p14 was only analyzed in carcinomas. Aim: This study seeks to determine p14, p16 and p53 immunohistochemical expression in benign, borderline and malignant ovarian epithelial tumors and correlate them with survival and clinical variables. Methods: Cross-sectional study utilizing immunohistochemical staining of p14, p16 and p53 in paraffin-embedded tissue samples from ovarian epithelial tumors obtained from Hospital de Clinicas de Porto Alegre. Results: p14 was positive in 93% of benign, 94% of borderline and 60% of malignant tumors. Loss of expression was statistically associated with carcinomas. p16 was positive in 94.6% of carcinomas, 75% of borderline and 45.7% of benign tumors. p53 was positive in 29.7%, 16.7% and 2.9% of malignant, borderline and benign tumors, respectively. Carcinoma subtypes showed no difference in expression. Conclusions: To our knowledge, this is the first description of p14 expression in benign and borderline tumors. It remains stable in benign and borderline tumors, while carcinomas show a significant absence of staining. This may indicate p14 abnormalities occur later in carcinogenesis. p16 and p53 expression rates show similar results to previous reports. Future studies should investigate genetic abnormalities in p14 coding sequences and include all types of ovarian epithelial tumors.

No description available.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.

Coat color and patterning phenotypes have been extensively studied as a model for advancing our understanding of the relationship between genetic and phenotypic variation. In this thesis, genes of relevance for pigment cell biology were investigated. The dissertation is divided in two parts. Forward genetics was used in the first part (Paper I and II) to identify the genes controlling the Silver and Sex-linked barring loci in chicken. In the second part, reverse genetics was employed to create a mouse line in which the PMEL17 protein is inactivated (Paper III). In Paper I, we report five mutations in SLC45A2 causing plumage color variants in both chicken and Japanese quail. Normal function of the SLC45A2 gene has previously been shown to be essential for the synthesis of both red/yellow pigment (pheomelanin) and brown/black pigment (eumelanin) in numerous species, including humans. The major discovery in this paper is the specific inhibition of pheomelanin in Silver chickens, whilst null mutations at this locus cause an almost complete absence of both pheomelanin and eumelanin. In Paper II, we report that Sex-linked barring in chickens is controlled by the CDKN2A/B tumor suppressor locus. The locus encodes two proteins, INK4B and ARF. The genetic analysis indicates that missense mutations in ARF or mutations in the promoter region of the ARF transcript are causing Sex-linked barring. In previous studies, mutations inactivating the CDKN2A/B tumor suppressor locus, have been shown to be responsible for familiar forms of human melanoma. Here we propose that these mutations in chicken CDKN2A/B cause the premature cell death of melanocytes as opposed to the cell proliferation and tumor growth associated with loss-of-function alleles in humans. In Paper III, we created a mouse line in which the PMEL17 protein is inactivated. Missense mutations in the gene encoding PMEL17 have previously been shown to be associated with reduced levels of eumelanin in epidermal tissues in several vertebrate species. The knockout mice are viable, fertile, and display no obvious developmental defects. The eumelanosomes within the melanocytes of these mice are spherical in contrast to the cigar-like shaped eumelanosomes present in wild-type animals. PMEL17 protein inactivation has only a subtle diluting effect on the coat color phenotype in four different genetic backgrounds. This suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations.

Introdução: Anormalidades nos supressores tumorais p14, p16 e p53 são relatadas em diversos tipos de câncer em humanos. Na carcinogênese ovariana, p16 e p53 foram extensivamente estudados, mas p14 foi analisado somente em carcinomas. Objetivo: O estudo visa determinar a expressão imuno-histoquímica de p14, p16 e p53 em tumores ovarianos epiteliais benignos, borderline e malignos. Método: Estudo transversal utilizando imuno-histoquímica em amostras de tumores epiteliais ovarianos emblocados em parafina do Hospital de Clínicas de Porto Alegre. Resultados: p14 foi positivo em 93% dos tumores benignos, 94% dos borderline e 60% dos malignos. A perda de expressão foi estatisticamente associada com carcinomas. p16 foi positivo em 94,6% dos carcinomas, 75% dos tumores borderline e 45,7% dos benignos. p53 foi positivo em 29,7%, 16,7% e 2,9% dos tumores malignos, borderline e benignos, respectivamente. Os subtipos de carcinoma não mostraram diferenças de expressão. Conclusão: Nosso estudo foi o primeiro a descrever a expressão de p14 em tumores benignos e borderline. Ela permanece estável nos benignos e borderline, enquanto os carcinomas exibem uma perda de expressão significativa. Isso pode indicar que anormalidades de p14 acontecem tardiamente na carcinogênese. As taxas de expressão de p16 e p53 foram semelhantes a estudos anteriores. Estudos futuros devem investigar anormalidades genéticas nas sequencias codificadoras de p14 e incluir todos os tipos de tumor epitelial ovariano. / Background: Abnormalities in tumor suppressors p14, p16 and p53 are reported in several human cancers. In ovarian carcinogenesis, p16 and p53 have been extensively studied, but p14 was only analyzed in carcinomas. Aim: This study seeks to determine p14, p16 and p53 immunohistochemical expression in benign, borderline and malignant ovarian epithelial tumors and correlate them with survival and clinical variables. Methods: Cross-sectional study utilizing immunohistochemical staining of p14, p16 and p53 in paraffin-embedded tissue samples from ovarian epithelial tumors obtained from Hospital de Clinicas de Porto Alegre. Results: p14 was positive in 93% of benign, 94% of borderline and 60% of malignant tumors. Loss of expression was statistically associated with carcinomas. p16 was positive in 94.6% of carcinomas, 75% of borderline and 45.7% of benign tumors. p53 was positive in 29.7%, 16.7% and 2.9% of malignant, borderline and benign tumors, respectively. Carcinoma subtypes showed no difference in expression. Conclusions: To our knowledge, this is the first description of p14 expression in benign and borderline tumors. It remains stable in benign and borderline tumors, while carcinomas show a significant absence of staining. This may indicate p14 abnormalities occur later in carcinogenesis. p16 and p53 expression rates show similar results to previous reports. Future studies should investigate genetic abnormalities in p14 coding sequences and include all types of ovarian epithelial tumors.

Introdução: Anormalidades nos supressores tumorais p14, p16 e p53 são relatadas em diversos tipos de câncer em humanos. Na carcinogênese ovariana, p16 e p53 foram extensivamente estudados, mas p14 foi analisado somente em carcinomas. Objetivo: O estudo visa determinar a expressão imuno-histoquímica de p14, p16 e p53 em tumores ovarianos epiteliais benignos, borderline e malignos. Método: Estudo transversal utilizando imuno-histoquímica em amostras de tumores epiteliais ovarianos emblocados em parafina do Hospital de Clínicas de Porto Alegre. Resultados: p14 foi positivo em 93% dos tumores benignos, 94% dos borderline e 60% dos malignos. A perda de expressão foi estatisticamente associada com carcinomas. p16 foi positivo em 94,6% dos carcinomas, 75% dos tumores borderline e 45,7% dos benignos. p53 foi positivo em 29,7%, 16,7% e 2,9% dos tumores malignos, borderline e benignos, respectivamente. Os subtipos de carcinoma não mostraram diferenças de expressão. Conclusão: Nosso estudo foi o primeiro a descrever a expressão de p14 em tumores benignos e borderline. Ela permanece estável nos benignos e borderline, enquanto os carcinomas exibem uma perda de expressão significativa. Isso pode indicar que anormalidades de p14 acontecem tardiamente na carcinogênese. As taxas de expressão de p16 e p53 foram semelhantes a estudos anteriores. Estudos futuros devem investigar anormalidades genéticas nas sequencias codificadoras de p14 e incluir todos os tipos de tumor epitelial ovariano. / Background: Abnormalities in tumor suppressors p14, p16 and p53 are reported in several human cancers. In ovarian carcinogenesis, p16 and p53 have been extensively studied, but p14 was only analyzed in carcinomas. Aim: This study seeks to determine p14, p16 and p53 immunohistochemical expression in benign, borderline and malignant ovarian epithelial tumors and correlate them with survival and clinical variables. Methods: Cross-sectional study utilizing immunohistochemical staining of p14, p16 and p53 in paraffin-embedded tissue samples from ovarian epithelial tumors obtained from Hospital de Clinicas de Porto Alegre. Results: p14 was positive in 93% of benign, 94% of borderline and 60% of malignant tumors. Loss of expression was statistically associated with carcinomas. p16 was positive in 94.6% of carcinomas, 75% of borderline and 45.7% of benign tumors. p53 was positive in 29.7%, 16.7% and 2.9% of malignant, borderline and benign tumors, respectively. Carcinoma subtypes showed no difference in expression. Conclusions: To our knowledge, this is the first description of p14 expression in benign and borderline tumors. It remains stable in benign and borderline tumors, while carcinomas show a significant absence of staining. This may indicate p14 abnormalities occur later in carcinogenesis. p16 and p53 expression rates show similar results to previous reports. Future studies should investigate genetic abnormalities in p14 coding sequences and include all types of ovarian epithelial tumors.

Philosophiae Doctor - PhD / This dissertation documents the attempt to address one of the major problems in nursing education i.e. the existing gap between the educational philosophy of nursing and nursing education practices, by means of an action research project during the period 1991-1996. The research in this dissertation is recorded in three phases. Phase One elaborates on the biographical and professional background of the researcher and the reasons why action research was selected for the purpose of this particular project Since action research provides opportunities for teachers to change and transform their own teaching practices, it was obviously the best choice for the research. The emphasis was on collaboration and participation and the researcher was morally bound to consider and observe all internal and external factors which influence and limit her own teaching practice, in order to initiate change and transformation in teaching. In order to define and contextualize the problem and to describe the situation in which this particular problem has been identified, the role of the statutory body, the South African Nursing Council which governs the profession and basic professional nursing education were explored. The problem is formulated as the existing gap between the educational philosophy on which existing nursing and nursing education practices are theoretically grounded and the way in which both nursing and nursing education practices appear in reality. Phase One also deals with the historical and philosophical foundations and development of nursing and nursing education. In an attempt to describe the researchers's teaching practice appropriately, as a social practice, it was essential to consider not only the professional and social boundaries of nursing education, but also the current situation regarding national education, the existing health system and all factors related to education and health. The dissertation then draws the attention to the essential features and historical context of a progressive and critical pedagogy, as a foundation for action research. In this regard it was especially the contributions of Dewey, Habermas, Freire, Giroux and McLaren, which guided the research to approach nursing education from a critical perspective. Phase Two deals with the research methodology. For this particular research project John Elliotts's Action Research Framework for Self-Evaluation in Schools was used. Within this framework of Elliott the dissertation then describes the research methodology of this particular project: Ideal and Reality in Nursing Education and Nursing Practices in South Africa: An Action Research Approach. The rationale and the development of the project is first described, whereafter action research is discussed as a process which enables nursing .practitioners and tutors to become empowered and to initiate change and establish transformation within their own practices. A major part of the dissertation is dedicated to the project in action with two groups of participants during two action research cycles. Finally Phase Three of this dissertation draws the attention to the conclusions based on the outcomes of the project. with the emphasis on the urgent need for change and transformation within the nursing profession in order to lessen the extensive gap between nursing theory and nursing practices. The existing gap between the philosophy on which nursing practices are based and how existing nursing practices appear in reality, seems to be the major cause of the prevailing discontent in the nursing profession.