Thèse (M.Sc.)--Université Laval, 2002. / Bibliogr.: f. [87]-99. Publié aussi en version électronique.

Thèse (M.Sc.) -- Univesité Laval, 1997. / Bibliogr.: f. 53-68. Publié aussi en version électronique.

Thèse (Ph.D.)--Université Laval, 2001. / Bibliogr. Publié aussi en version électronique.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed July 9, 2007). PDF text: xv, 147 p. : ill. UMI publication number: AAT 3249674. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2005. / Includes bibliographical references (p. 24-27).

No description available.

Reversible protein phosphorylation, the best studied post-translational modification, regulates most cellular processes, including signaling, migration, cell cycle progression, DNA damage repair, stress response and modulaton of the activities of metabolic enzymes. Therefore, it has emerged as a key therapeutic target in diseases in which these processes are deregulated. Unlike kinases, protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its substrate specificity from a large group of “regulatory subunits” with which it associates to form a range of holoenzyme complexes targeted to specific subcellular localizations and substrates. Inhibition of a specific dephosphorylation event therefore relies on targeting the regulatory rather than the catalytic subunit. The present study uses GFP as a molecular reporter to assess the localization of PP1c and identify the underlying binding events that govern it via a combination of fluorescence imaging, cellular fractionation, affinity purification and quantitative mass spectrometry. While there is some overlap in their targeting and intracellular roles, the three PP1 isoforms show distinct localizations based on relative preferences for particular regulatory subunits. In this study we assembled a comprehensive map of isoform- and compartment- specific phosphatase complexes in three different cultured human cell lines, using the data to extrapolate, with confidence, the distribution of each PP1 isoform between a large pool of known/predicted and novel regulatory subunits. Network analysis also highlighted key multiprotein complexes to which PP1 is targeted by these regulatory subunits, and identified a novel regulatory subunit that links phosphatase activity to regulation of protein degradation. Our work confirmed that Mypt1, the regulatory subunit that targets PP1 activity to the myosin light chain, preferentially associates with the beta isoform of PP1c. We further demonstrated that they are in complex in both the cytoplasm and nucleus, and represent ~30% of the total PP1β holoenzyme complexes in both interaphase and mitotic cells. Further investigation of these complexes led to the discovery of Specc1 and Specc1L, which associate with Mypt1/PP1β via direct binding to Mypt1. Specc1/1L are microtubule binding proteins that can also associate with actin filaments, and we demonstrated that they mediate the distribution of Mypt1/PP1β complexes between these two cytoskeletal networks. Given that disruption of this balance has been implicated in disease states including cancer and hypertension, the Specc1/L family represents a novel therapeutic target for the regulation of Mypt1/PP1 activity. With PP1 now emerging as a promising therapeutic target and the first PP1-targeted therapeutic drug, Sephin 1, in clinical trials, a better understanding of PP1’s in vivo distribution between holoenzyme complexes is essential. Our work establishes an initial “snapshot” of this distribution against which changes can be assessed, as we demonstrated here by showing its re-distribution in mitotic cells. Dynamic redistributions during specific cell processes such as differentiation or in response to perturbations or disease states can be assessed in a similar fashion in future, facilitating both identification of the relevant complexes and the design of specific strategies to target them therapeutically.

Made available in DSpace on 2014-06-11T19:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-12-18Bitstream added on 2014-06-13T19:42:29Z : No. of bitstreams: 1 akinaga_j_dr_botib_parcial.pdf: 68136 bytes, checksum: 63ef0cbc915c8aa41f833dd0c4a86b5b (MD5) Bitstreams deleted on 2014-12-19T18:32:50Z: akinaga_j_dr_botib_parcial.pdf,Bitstream added on 2014-12-19T18:33:36Z : No. of bitstreams: 1 000714063.pdf: 501451 bytes, checksum: 105a66a9ea99849e798ced498018eeef (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As catecolaminas endógenas adrenalina e noradrenalina controlam importantes funções fisiológicas através da ativação de 1A-ARs, que são receptores com 7 domínios transmembrana os quais ativam proteínas G. Muitos agonistas de 1-ARs são utilizados na terapêutica, principalmente por conta de seu efeito vasoconstritor, porém, a taquifilaxia observada com a oximetazolina, um agonista de 1-ARs da classe das imidazolinas, é um efeito adverso importante e pode ser observado principalmente com a utilização de descongestionantes nasais por períodos de tempo prolongado. No presente estudo, investigamos a participação de PKC e GRK2 nos processos de fosforilação, dessensibilização e internalização de 1A-ARs induzidos pelos agonistas noradrenalina e oximetazolina. Segundo os resultados obtidos, a oximetazolina, mas não a fenilefrina, induziu taquifilaxia nas contrações de artéria caudal de rato. Além disso, 1A-ARs ativados pela oximetazolina são fosforilados principalmente pela GRK2, seguida de rápida dessensibilização e internalização. Já 1A-ARs ativados pela noradrenalina são fosforilados principalmente pela PKC, sem dessensibilização nem rápida internalização. Esses resultados em conjunto demonstram que a oximetazolina e a noradrenalina regulam os 1A-ARs através de diferentes mecanismos, envolvendo diferentes quinases / The endogenous cathecolamines epinephrine and norepinephrine control important physiological functions through activation of 1A-ARs, which are 7-transmembrane receptors that activate G proteins. Several 1-ARs agonists are therapeutically useful in reason of its vasoconstrictor effects; however, tachyphylaxis in the vasoconstrictor effects of nasal decongestant containing oxymetazoline, an imidazoline 1-ARs agonist, is an important adverse effect observed after prolonged treatment. On the present study, we investigated the roles of PKC and GRK2 on the 1A-AR phosphorylation, desensitization and internalization process after receptor activation by norepinephrine and oxymetazoline. The results show that oxymetazoline, but not phenylephrine, induce tachyphylaxis in contractions of rat tail artery. Moreover, 1A-ARs activated by oxymetazoline are phosphorylated predominantly by GRK2, followed by rapid desensitization and internalization whereas 1A-ARs activated by norepinephrine are phosphorylated predominantly by PKC and not followed by desensitization or rapid internalization. These results show that norepinephrine and oxymetazoline regulate 1A-ARs through different mechanisms, involving different protein kinases

Orientadores: Eduardo Galembeck, Carmen Verissima Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T15:43:32Z (GMT). No. of bitstreams: 1 Milani_Renato_M.pdf: 4434069 bytes, checksum: f6dd6ae86e6b3ac6b79f8c4b28828a91 (MD5) Previous issue date: 2010 / Resumo: A atividade de proteínas quinases é responsável pela regulação de muitos processos biológicos através de cascatas de sinalização que levam a diferentes efeitos celulares. No entanto, a análise da reação de fosforilação reversível catalisada por estas enzimas e prejudicada pela complexidade inerente as interações entre proteínas neste sistema de sinalização. Consequentemente, o foco em uma única proteína quinase pode não ser suficiente para revelar completamente os mecanismos por trás dos fenótipos observados. Nesse sentido, em conjunto com outras técnicas de análise em larga-escala como chips de expressão de mRNA, arranjos de peptídios contendo substratos de proteínas quinases vem sendo cada vez mais utilizados por pesquisadores. No entanto, a falta de uniformidade na análise estatística desses chips tem sido um grande empecilho à obtenção de dados relevantes com o uso dessa técnica. Por conta disso, o objetivo desse trabalho foi desenvolver uma metodologia, chamada de PepMatrix, capaz de aplicar estatística básica de forma automatizada visando a seleção de replicações com baixa variabilidade e a obtenção da anotação das proteínas envolvidas nos eventos de fosforilação ocorridos no chip. Esse novo método foi aplicado em vários conjuntos de dados de diferentes experimentos biológicos e seus resultados revelaram atividades quinásicas significativamente alteradas, muitas das quais tiveram confirmação por Western blot. Alem disso, os resultados ressaltaram a importância da análise sistêmica dos eventos de sinalização celular em conjunto com uma análise crítica das replicações. O alto grau de uniformidade analítica obtido por esse método faz com que ele seja uma poderosa e confiável ferramenta na análise quinômica em larga-escala / Abstract: The activity of protein kinases governs many biological processes through signaling cascades that lead to distinct outputs, from homeostasis to disease. However, analysis of the reversible phosphorylation perpetrated by these enzymes is hindered by the inherent complexity of interactions in this signaling system. Consequently, focusing on a single kinase may not be enough to completely unfold the mechanisms behind observed phenomena. In this sense, together with other omics approaches like mRNA expression analysis, peptide arrays have shown increasing popularity, particularly ones containing kinase substrate sequences. The lack of uniformity in statistical analysis of these chips, though, has been a major issue for the field. In this paper, we propose PepMatrix, a fast and accurate method for selecting so called "reliable replicate spots" and automatically retrieving differential activity and annotation information about phosphorylation events identified in a peptide array of kinase substrates. Here, we present several cases where this new methodology was applied to biological datasets. We successfully identified putative up and down-regulated kinases, many of which were confirmed to have altered activity by Western blot. Moreover, the results emphasized the need for a true systems biology approach to the cellular signaling events alongside a critical replicate selection method. The high degree of analysis uniformity we achieved with this method provides a powerful and reliable addition for high-throughput kinome analysis / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

M.Sc. / Radionuclides exist in the environment because of natural and human activities that are an essential part of our lives. Nuclear processing, medicinal applications (using isotopes) and electric power production by nuclear stations are few examples of human activities that result in production of radioactive waste (RAW). The nuclear power stations in our world have to store their waste in such a manner that the present and future generations are protected from harmful radiations and this is a challenge. Exposure to RAW can result in severe, diverse and irreversible consequences such as damage of the ecosystem, pollution, cancers, birth mutations, to mention just a few. Solvent extraction (SE) technique is currently used to purify large volumes of secondary liquid waste before they are released to the environment or stored. However, even after the SE purification, highly radioactive liquid waste is given off. This highly radioactive liquid waste is solidified in a glass matrix (vitrification). In an attempt to reduce the disposal of large volumes of secondary RAW generated during the purification technology, this study was initiated to investigate the possibility of using multiwalled carbon nanotubes (MWCNTs) as part of the SE technique. As the main nuclear liquid extraction processes involve tributyl phosphate (TBP), the MWCNTs were linked to TBP, polymerised to give a MWCNTs-TBP polymer that was tested in the nuclear environment. This polymer should possess good chelating properties due to the inclusion of the phosphate and should be a good absorbent as MWCNTs are promising absorbent carbon materials. To test the hypothesis of the study MWCNTs-TBP polymer was tested for uranium extraction. The MWCNTs-TBP polymer gave a zero Kd value which indicates that the adsorption capacity of the polymer to remove radionuclides from waste streams was not successful. The MWCNTs were then tested for iodine-131 extraction whereby they were compared with single walled carbon nanotubes (SWCNTs) and double walled carbon nanotubes (DWCNTs). In this test SWCNTs gave a Kd value of 81694 mL/g which proved that they can be used in nuclear waste applications.