Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains x, 185 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May, 2005" Includes bibliographical references.

No description available.

Natriuretic peptides are a family of hormones released by several different tissues and exert various physiological functions by coupling with cell surface receptors and increasing intracellular cyclic gyanylyl monophosphate (cGMP). Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP) are released in response to mechanical stretch of the atrial or ventricular myocardium, respectively and their plasma level is markedly elevated during myocardial infarction and heart failure. Heart failure in turn is associated with symptoms suggestive of perturbed gastrointestinal function such as nausea, indigestion and malabsorption. Intragastric pressure was monitored using a balloon catheter in anesthetized mice. The pressure before and after treatment with a 10 ng/g intravenous dose of ANP, BNP, CNP or vehicle was compared and analyzed. All the natriuretic peptides significantly decreased intragastric pressure compared to vehicle. These effects were attenuated or absent in natriuretic peptide receptor type-A (NPR-A) knockout mice. Furthermore, the effect of BNP on gastric emptying and intestinal absorption was examined using a meal consisting of fluorescence labeled dextran gavage fed to awake mice. BNP significantly decreased gastric emptying and absorption as compared to vehicle control. Using a cryoinfarction acute myocardial injury model, our investigation showed that mice with acute cryoinfarction had a significantly lower gastric emptying and absorption of a gavage fed meal compared to sham. Circulating BNP levels were significantly higher in the infarcted mice compared to controls. Immunostaining showed amplified distribution of the non-muscle myosin type-II (MCH-II) in BNP treated mice. MCH-II is involved in movement of intestinal villi. In summary, natriuretic peptides in general and BNP in particular, have gastrointestinal effects including reduced gastric contractility, emptying and absorption. In addition to their effect on smooth muscle relaxation mediated by cGMP, natriuretic peptides appear to have an effect on distribution of MHC-II in cells of the intestinal villi. We postulate that these effects are aimed at mediating a 'communication' between the cardiovascular and gastrointestinal systems. Further characterization of such a link will not only add a dimension to the understanding of the pathophysiology of heart failure but also enhances the search for further therapeutic targets.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vii, 114 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Orientador: Mário Tyago Murakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T22:14:14Z (GMT). No. of bitstreams: 1 Pinto_AlineSampaio_M.pdf: 3074349 bytes, checksum: 0fdb140ae2fd1c1975ba6cde0cf00bca (MD5) Previous issue date: 2012 / Resumo: As miosinas formam uma superfamília de proteínas de alto peso molecular com atividade mecanoquímica capaz de hidrolisar a molécula de ATP e de interagir com os filamentos de actina. A estrutura das miosinas pode ser divida de modo geral em cabeça motora, pescoço e cauda. São conhecidas 35 classes de miosinas em eucariotos sendo a classe II de miosinas denominada miosinas convencionais e as demais chamadas de não convencionais. Em plantas são somente encontradas as miosinas não convencionais de classe VIII e XI. A classe VIII caracteriza-se por sua alta processividade sobre os filamentos de actina e a classe XI é a maior classe em número de genes, tendo uma estrutura muito semelhante às miosinas de classe V. Uma terceira classe, a classe XIII, foi posteriormente descoberta e somente foi encontrada no gênero Acetabularia apresentando dois genes, porém essa classificação é controversa havendo aqueles que dizem que as miosinas da classe XIII possuem tanta afinidade filogenética com as da classe XI que elas deveriam compor uma única classe. As miosinas VIII e XI desempenham papéis chave no transporte direcional de componentes intracelulares em plantas, principalmente devido às grandes dimensões das células de plantas que não sobrevivem utilizando somente a difusão como mecanismo de transporte intracelular. Neste trabalho, buscamos selecionar os melhores representantes de cada classe a partir de análises in silico para desenvolvermos os testes de expressão, purificação e análises biofísicas. Os domínios selecionados, cauda globular (GT), dilute e SH3, foram clonados em pET28a e pET28aSUMO, os testes de expressão com diversas cepas de E coli, mostraram que o domínio dilute expressa em grande quantidade na fração solúvel, porém forma agregados impedindo as análises biofísicas e os ensaios de cristalização. O domínio SH3 também foi obtido na forma solúvel, porém em pouca quantidade e apresentou migração anômala no gel, sendo identificado a partir de análises de espectrometria de massas. Foram realizados testes iniciais de cristalização para o domínio SH3, mas não resultou na formação de cristais adequados a difração de raios X. A cauda globular foi obtida apenas na fração insolúvel e submetida a procedimentos de refolding para sua solubilização. Mesmo conseguindo o reenovelamento da construção, a mesma se manteve agregada inviabilizando os testes de cristalização. Por outro lado foram analisadas as interações da cauda globular da miosina XIh com presas identificadas por duplo-híbrido realizado pelo nosso grupo com a miosina humana Va. Três das proteínas que interagiram com a miosina Va humana também mostraram sinais de interação com a miosina XIh de Arabidopsis, indicando que mesmo havendo diferenças nas sequências polipeptídicas entre as classes de miosina, a estrutura terciária se mantém permitindo que ambas apresentem interações com algumas proteínas em comum / Abstract: Myosins belong to a superfamily of high molecular weight proteins, presenting mechanochemical activity by hydrolyzing ATP molecule and ability to interact with actin filaments. The myosin structure can be divided into motor head, neck and tail. 35 myosin classes are known in eukaryotic cells, where class II is known as conventional myosins and all others, as unconventional myosins. Plants possess only unconventional myosins including classes VIII and XI. The class VIII, is characterized by its high processivity on actin filaments while class XI possesses multiple genes and its protein structure is very similar to class V myosins. A third class, XIII, was later discovered and only found in Acetabularia genome presenting two genes. However this classification is controversial, because some studies shown that class XIII myosins share such high phylogenetic similarity with class XI that they should form the same class. Myosins VIII and XI play key roles on directional intracellular components transport in plants, mainly due to the large plant cell size which would not survive only by the diffusion mechanism for intracellular transport. In this work, we have selected the best representative targets of each class from in silico analyses to develop the protein expression, purification and biophysical characterization. The selected domains (globular tail (GT), dilute and SH3) were cloned into pET28a and pET28aSUMO expression vectors. Results of expression tests with several E coli strains, showed that dilute domain is expressed in large amounts in the soluble fraction; however, it aggregates preventing biophysical analysis and crystallization trials. The SH3 domain, expressed in low soluble concentration, presented an abnormal migration in SDS-PAGE and its identity was confirmed by mass spectrometry analysis. Initial crystallization tests were conducted with SH3 domain, but owing to the low protein concentration only clear drops have appeared, and no crystals or aggregates were observed. The globular tail domain, obtained only in insoluble fraction, was subjected to refolding procedures/ techniques in order to recover its native-like state; however, even the refolded protein displayed secondary structure, the protein remained aggregated. Furthermore, we analyzed the interactions between of myosin XIh globular tail with pre-identified proteins by yeast two-hybrid conducted by our group with human myosin Va. Three proteins that interacted with human myosin Va also showed interaction with Arabidopsis myosin XIh, indicating that despite of differences in polypeptide sequences between the classes XI and V, the tertiary structure may be maintained allowing both of them to have interactions with common proteins / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.

Among 18 family members in the myosin superfamily, myosin IX is unique by possessing a GTPase activating protein (GAP) for Rho. It is also attention-grabbing since it is a single-headed processive motor, as well as a minus-end directed motor. Although many biochemical properties have been revealed, its physiological function is largely unknown. As an initial step to address this question, I attempted to find the binding partner of myosin IXb using the yeast two-hybrid screen. Through the screen using the tail domain of myosin IXb as bait I found BIG1, a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor (Arfl), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney (NRK) cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by addition of BIG1 with IC50 of 0.06 μM. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with a concentration similar to that which inhibit the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the ArfGEF, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. Together, the results imply that the RhoGAP activity of myosin IXb is down-regulated by BIG1 at the Golgi, where myosin IXb could be involved in the regulation of actin cytoskeleton through the Rho-signaling pathway.

Type II 5'-deiodinase (D2) catalyzes the conversion of T4 to the transcriptionally active T3. When T4 levels are high, D2 activity levels are low. Conversely when T4 levels are low, D2 catalytic activity is high. Immunocytochemistry and biochemical data from cultured rat astrocytes revealed that physiological concentration of T4 and the non-transcriptionally active metabolite rT3, but not T3, initiates the budding of D2 containing endosomes and their subsequent translocation to the perinuclear space. Further analysis showed that this process required a polymerized actin cytoskeleton but not cellular transcription or translation; however the precise mechanism remained unknown. In this present investigation, we characterized the requirement of an unconventional myosin motor protein, myosin 5a, in the actin-based endocytosis of D2 containing vesicles. We developed an in vitro actin binding assay that exploited the T4 dependent binding of D2 containing vesicles to F-actin, and showed that D2p29:F-actin interactions are calcium, magnesium and ATP-dependent suggesting that a calmodulin (CaM) regulated myosin ATPase is required. Introduction of in vitro transcribed and translated vesicle-binding tail, which lacked the actin binding head, of myosin 5a to the in vitro actin binding assay created a dominant negative inhibitor of D2 binding to the actin cytoskeleton by competing with the native myosin 5a. A replication deficient adenoviral vector expressing the fusion protein of the 29 kDa substrate binding subunit of D2 with a green fluorescent protein reporter molecule enabled us to directly examine T4 dependent regulation of D2 in vitro as well as in living cells. Using immunoprecipitation we showed a T4 dependent association between the vesicle binding tail of myosin 5a and D2 containing vesicles. Biochemical analysis of the interaction of the myosin 5a tail with D2 containing vesicles revealed that the last 21 amino acids of myosin 5a were both necessary and sufficient for the attachment of D2 containing vesicles to the F-actin cytoskeleton. Using rapid acquisition time-lapse digital microscopy in p29GFP expressing rat astrocytes, we showed directed T4 dependent p29GFP movement from the plasma membrane to the perinuclear region. This hormone dependent vesicle movement was not observed in cells treated with T3 or no hormone. Time lapse motion studies allowed for the calculation of the velocity and of the distance traveled for individual fusion protein containing vesicles. The velocity for cells treated with T4 or rT3 was identical to that reported for vesicle-laden myosin 5a in mouse melanophores. In contrast cells treated with T3 or those receiving no hormone treatment had velocities similar to diffusion of proteins within the plasma membrane. Astrocytes constitutively expressing both p29GFP and dominant negative myosin 5a inhibitors failed to show hormone induced centripetal movement. These data demonstrate that myosin 5a is the molecular motor responsible for thyroid hormone dependent actin based endocytosis in astrocytes.