999 resultados para Proteínas celulares Teses
Resumo:
La transferencia de proteínas solubles a la interfase de la membrana lipdica es un paso clave en varios procesos celulares. Esta traslocacin resulta en un importante cambio en el ambiente de la protena con consecuencias sobre su conformacin, estabilidad y actividad biolgica. En este proyecto estudiamos las condiciones que determinan la unin a la membrana, particularmente el balance entre interacciones electrostticas e hidrofbicas, y los factores que determinan la conformacin, estabilidad y dinmica de la protena en interfaces. Particularmente, estudiaremos la interaccin con membranas de la protena transportadora de cido clico de hgado de ave L-BABP, la protena beta-2 glicoprotena humana y la protena asociada a microtbulos SL21. Hemos encontrado que el estado de fase del lpido determina la conformacin y estabilidad de L-BABP unida perifricamente. En este proyecto estudiaremos la naturaleza de las interacciones que determinan esta dependencia. beta-2 glicoprotena humana se une a membranas aninicas e induce cambios estructurales en el lpido cuando ocurre la transicin al estado desplegado de la protena. El proyecto contempla estudiar comparativamente las interacciones del estado nativo y parcialmente desplegado de beta-2 glicoprotena con membranas. Estudiaremos los cambios conformacionales de proteínas y lpidos utilizando espectroscopia infrarroja por transformada de Fourier (FTIR), espectroscopia de emisin de fluorescencia y espectroscopia de dicrosmo circular (CD). Utilizaremos calorimetra diferencial de barrido (DSC) para estudios de estabilidad y espectroscopia de fosforescencia para estudiar dinmica rotacional de proteínas en la membrana.
Resumo:
En la hiptesis de trabajo del presente proyecto se considera la importancia del metabolismo de lpidos y proteínas en los insectos hematfagos, en particular en los vectores de la enfermedad de Chagas, para afrontar exitosamente la demanda energtica de la reproduccin. Las hembras de estas especies pueden ingerir una comida de sangre abundante en lpidos y proteínas, los que son modificados en el intestino para su utilizacin y posterior almacenamiento en estructuras organizadas en el tejido ovrico, sustentando as el rpido crecimiento de los ovocitos. Estos aspectos resultan crticos para el ciclo de vida del insecto y para el mantenimiento de la cadena epidemiolgica de la enfermedad. En estas especies, recientemente hemos caracterizado a nivel bioqumico y celular la interaccin entre lipoproteínas y tejidos [Fruttero y col., Insect Biochem. Mol. Biol. 39: 322-331 (2009); Fruttero y col. Biocel 33 (3): 260 (2009)] y las fases del ciclo reproductivo [Aguirre y col., J. Insect Physiol. 54: 393-402 (2008)]. No obstante, los factores que participan en su regulacin son an escasamente conocidos. En este contexto, el estudio propone emplear dos especies de triatominos con el objeto de: (1) caracterizar los factores involucrados en la formacin y regulacin de reservas nutricionales en los ovocitos; (2) analizar los eventos que participan en la regresin del tejido ovrico: atresia folicular y mecanismos de muerte celular. (3) evaluar el impacto de productos naturales (ureasas vegetales y pptidos derivados) en el desarrollo del tejido ovrico. Para la ejecucin de los objetivos se llevarn a cabo ensayos in vivo e in vitro con trazadores fluorescentes, fraccionamiento subcelular, estudios de expresin de proteínas (mRNA y protena), estudios histo-morfolgicos, ultraestructurales e inmunocitoqumicos, microscopa lser confocalizada, ensayos de actividad enzimtica, ELISA, western-blot, electroforesis bidimensional, espectrometria de masas en tndem, etc. Tambin se evaluarn los mecanismos de muerte celular (apoptosis/autofagia) mediante microscopa electrnica, deteccin de apoptosis in situ (TUNEL), inmunofluorescencia, etc. Los resultados obtenidos permitirn un mejor conocimiento sobre la fisiologa y bioqumica de estos vectores, los que resultan indispensables en el diseo de nuevas estrategias para su control. Debido a la carencia de un tratamiento especfico para la enfermedad y a la falta de mtodos preventivos (vacuna), el control del vector es una de las vas ms importantes para reducir la incidencia de la enfermedad. Actualmente, la situacin socio-econmica que sufren amplios ncleos de nuestra poblacin propicia condiciones de vida que facilitan la reproduccin de los vectores y la transmisin vectorial del parsito. El estudio permitir adems explorar aspectos bioqumicos y celulares bsicos, generando conocimientos que podran ser extensivos a otros insectos de importancia econmica en la ganadera y/o agricultura. The aim of this project is to analyze the biochemical and cellular events involved in the lipid and protein metabolism in Chagas' disease vectors, and to evaluate their impact on the physiology of reproduction, particularly in the formation of nutritional resources in developing oocytes. At present, little is known about these critical aspects for the life cycle of the insect and for the epidemiology of the disease. The experimental approaches, which will be carried out using two species of triatomines, were designed: (1) to characterize factors involved in the formation and regulation of nutritional resources in developing oocytes; (2) to analyze the biochemical and cellular events that play a role during the regression of ovarian tissue, including the processes of oocyte resorption and programmed cell death. (3) to evaluate the impact of natural products (ureases from jackbean and related peptides) in the development of ovarian tissue. Methods and techniques involved in the project are: in vivo and in vitro assays with fluorescent tracers, ELISA, chemical assays, enzyme activities, western-blot; protein expression (mRNA), histological techniques, immunohistochemical and ultrastructural studies. Cell death will be analyzed by detection of apoptosis in situ (TUNEL), immunofluorescence (for autophagy), among others. The results obtained from the study will offer the opportunity to explore important aspects in the biology and physiology of Chagas' disease vectors that could be of potential utility in designing alternative strategies for the control of the insect.
Resumo:
El estudio del trfico intracelular en neuronas ha despertado gran inters en los ltimos aos, debido a que un gran nmero de enfermedades neurodegenerativas y neuropsiquitricas parecen tener origen en en el transporte defectuoso de proteínas en estos tipos celulares. Mediante el uso de tcnicas de biologa celular y molecular, fuimos capaces de describir una de las vas que regula la fisin de las vesculas que llevan su cargo desde la ltima cisterna del Aparato de Golgi hacia la superficie celular en clulas epiteliales no polarizadas. Uno de los componentes clave de esa va result ser la Proteina Kinasa D1 (PKD1), cuya actividad en el Aparato de Golgi es esencial para un normal transporte intracelular. Sorprendentemente, observamos que la PKD1 en neuronas con polaridad establecida no regula la fisin en el Golgi, pero si estara involucrada en la selectividad y distribucin (sorting) de vesculas cuyo cargo debe ser especficamente dirigido a las membranas dendrticas. El bloqueo de la actividad de la PKD1 no solamente cambia el destino final de estos cargos, que son enviados de esta forma a la membrana terminal del axn, sino que tambin es capaz de inducir defectos en el desarrollo y crecimiento de los procesos dendrticos a largo plazo. En este proyecto estudiaremos de que manera influye la perturbacin del sorting, en ausencia de PKD1 activa y de otros componentes que la regulan, en la distribucin de receptores de factores neurotrficos y de neurotransmisores glutamatrgicos, y cmo estos cambios en su distribucin afectan el nmero, tamao, y funcionalidad de los procesos neuronales (axones y dendritas). Estos resultados contribuirn a adquirir mayores conocimientos de los mecanismos dependientes del transporte y sorting de proteínas de membrana que participan en la regulacin del crecimiento neuronal, los cuales a su vez aportarn informacin valiosa en la comprensin de un gran nmero de enfermedades neurolgicas. The study of intracellular trafficking in neurons has arisen a great deal of interest in the last years, since a great number of neurodegenerative and neuropsychiatric disorders seem to be originated in abnormal protein transport in these type of cells. Using cell and molecular biology methodologies, we have been capable of describe one of the pathways that regulate the fission of vesicles that carry their cargo from the last Golgi Apparatus cisternae to the cell surface in non-polarized epithelial cells. One of the key components in this pathway is the Protein Kinase D1 (PKD1), whose activity in the Golgi Apparatus is essential for a normal intracelular transport. Surprisingly, we have observed that PKD1 does not regulate fission in neurons with established polarity, but it would be involved in vesicles' sorting at Golgi, particularly of those that carry specific dendritic cargo. Blocking PKD1 activity changes the final destination of these cargoes, which is now sent to the axons' terminal membranes, and also produces late dendritic development and growing defects. In this project we will study how sorting perturbation in absence of PKD1 and its regulators activities influences selectivity and distribution of neurotrophic and neurotransmitter receptors, and how these sorting changes affect number, size and functionality of neuronal processes (axons and dendrites). These results will help to acquire greater knowledge about transport and sorting mechanisms of neuronal growth regulatory membrane proteins. In addition, these studies will contribute with new valuable information necessary to understand numerous neurological diseases.
Resumo:
Tese de mestrado em Biologia Humana e Ambiente, apresentada Universidade de Lisboa, atravs da Faculdade de Cincias, 2015
Resumo:
Considerando que as doenas cardiovasculares representam a maior causa de mortalidade e morbidade em pases ocidentais, a aterosclerose se destaca pelo fato de predispor os pacientes ao infarto do miocrdio, a acidentes vasculares cerebrais e a doenas vasculares perifricas. Neste contexto, a oxidao de lipoproteínas do plasma, particularmente LDL, um dos fatores de risco para eventos cardiovasculares, pois reconhecida e internalizada por macrfagos, ocasionando a sua diferenciao em foam cells. Diversos fatores participam deste processo de diferenciao, como a expresso de receptores de scavenger CD 36, proporcionando aumento na captao de LDL oxidada, aumento na sntese endgena de colesterol e ativao de fatores nucleares que iniciam a transcrio de proteínas especficas e fatores de crescimento que disparam a aterognese. Os fenmenos celulares relacionados apoptose tambm so de especial importncia, tanto no desenvolvimento da leso aterosclertica como na estabilidade da placa e formao de trombos. As prostaglandinas (PGs) ciclopentennicas (CP-PGs), em particular a PGA2 e a 15-desxi-12,14-PGJ2 so uma classe especial de PGs que, em diminutas concentraes, disparam a expresso das proteínas de choque trmico (hsp), que so citoprotetoras. Alm disso, CP-PGs bloqueiam a ativao do fator nuclear pr-inflamatrio NF-B tornando-as potentes agentes antiinflamatrios. Embora as PGs das famlias A e J guardem uma srie de caractersticas em comum, a 15-desxi-12,14- PGJ2 o ligante fisiolgico do fator nuclear pr-aterognico PPAR-, enquanto as PGs da famlia A ativam apenas a via citoprotetora das hsp. Este trabalho teve como objetivo avaliar os efeitos das CP-PGs sobre a expresso gnica de fatores relacionados diferenciao de macrfagos em foam cells, bem como proteínas reguladoras do processo de apoptose, em clulas da linhagem pr-monoctica humana U937. Para tal, as clulas foram tratadas com CPPGs em presena e/ou ausncia de LDL nat e LDL ox, o RNA foi extrado para a realizao de RT-PCR para PPAR-, CD 36, HMG-CoA redutase e proteínas de apoptose Caspase 3, p53 e Bcl-xL. O tratamento estatstico utilizado foi anlise de varincia (ANOVA one-way) e teste t de student, com resultados expressos como mdias + desvios-padro da mdia, com P<0,05. Os resultados obtidos demontraram que as CP-PGs PGA2 (20M-24h) e PGJ2 (1,5M-24h) inibiram a expresso gnica do fator nuclear PPAR- (64 % (PGA2), 88 % (15- d-PGJ2)) nas clulas U937, em presena de LDL oxidada, quando comparado ao controle. PGA2 inibiu a expresso de HMG-CoA redutase (33 %), enzima chave da sntese de colesterol intracelular, e o tratamento com as CP-PGs tambm inibiu a apoptose nas clulas tratadas em presena de LDL oxidada. Os dados sugerem que as CP-PGs apresentam grande potencial para o tratamento da aterosclerose, j que, alm de apresentarem efeito antiinflamatrio, inibem a expresso do fator nuclear pr-aterognico PPAR-, do receptor de scavenger CD36 (apenas a 15-desxi-12,14-PGJ2) e da enzima HMG-CoA redutase. O bloqueio da apoptose nas clulas estudadas pode estar relacionado citoproteo oferecida por estas PGs. Embora investigaes in vivo deste laboratrio tenham mostrado a eficcia do tratamento com CP-PGs em camundongos portadores de aterosclerose, estudos adicionais so necessrios para esclarecer-se o efeito antiaterognico das mesmas.
Resumo:
Uma significativa quantidade de proteínas vegetais apresenta-se compartimentalizada nas diversas estruturas celulares. A sua localizao pode conduzir elucidao do funcionamento dos processos biossintticos e catablicos e auxiliar na identificao de genes importantes. A fim de localizar produtos gnicos relacionados resistncia, foi utilizada a fuso de cDNAs de arroz (Oryza sativa L.) ao gene da protena verde fluorescente (GFP). Os cDNAs foram obtidos a partir de uma biblioteca supressiva subtrativa de genes de arroz durante uma interao incompatvel com o fungo Magnaporthe grisea. Estes cDNAs foram fusionados a uma verso intensificada de gfp e usados para transformar 500 plantas de Arabidopsis thaliana. Outras 50 plantas foram transformadas com o mesmo vetor, porm sem a fuso (vetor vazio). Foram obtidas aproximadamente 25.500 sementes oriundas das plantas transformadas com as fuses EGFP::cDNAs e 35.000 sementes das transformadas com o vetor vazio, produzindo, respectivamente, 750 e 800 plantas tolerantes ao herbicida glufosinato de amnio. Aps a seleo, segmentos foliares das plantas foram analisados por microscopia de fluorescncia, visando o estabelecimento do padro de localizao de EGFP. Foram observadas 18 plantas transformadas com a fuso EGFP::cDNAs e 16 plantas transformadas com o vetor vazio apresentando expresso detectvel de GFP. Uma planta transformada com uma fuso EGFP::cDNA apresentou localizao diferenciada da fluorescncia, notadamente nas clulas guarda dos estmatos e nos tricomas. Aps seqenciamento do cDNA fusionado, foi verificado que esta planta apresentava uma insero similar a uma seqncia codificante de uma quinase, uma classe de enzimas envolvidas na transduo de sinais em resposta infeco por patgenos.
Resumo:
studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins
Resumo:
Reactive oxygen species (ROS) are continuously generated and can be derived from cellular metabolism or induced by exogenous factors, in addition, have the capacity to damage molecules like DNA and proteins. BER is considered the main route of DNA damage oxidative repair, however, several studies have demonstrated the importance of the proteins participation of other ways to correct these injuries. NER enzymes deficiency, such as CSB and XPC, acting in the damage recognition step in the two subways this system influences the effectiveness of oxidative damage repair. However, the mechanisms by which cells deficient in these enzymes respond to oxidative stress and its consequences still need to be better understood. Thus, the aim of this study was to perform a proteomic analysis of cell lines proficient and deficient in NER, exposed to oxidative stress, in order to identify proteins involved, directly or not, in response to oxidative stress and DNA repair. For this, three strains of human fibroblasts, MRC5-SV, CS1AN (CSBdeficient) and XP4PA (XPC-deficient) were treated with photosensitized riboflavin and then carried out the differentially expressed proteins identification by mass spectrometry. From the results, it was observed in MRC5-SV increase expression in most of the proteins involved in cellular defense, an expected response to a normal cell line subjected to stress. CS1AN showed a response disjointed, it is not possible to establish many interactions between the proteins identified, may be one explanation for their sensitivity to treatment with riboflavin and other oxidants and increased cell death probably by induction of pro-apoptotic pathways. Already XP4PA showed higher expression of apoptosis-blocking proteins, as there was inhibition or reduced expression of others involved with the activation of this process, suggesting the activation of an anti-apoptotic mechanism in this lineage, which may help explain the high susceptibility to develop cancers in XPC individuals. These results also contribute to elucidate action mechanisms of NER in oxidative damage and the understanding of important routes in the oxidative stress correlation, repair and malignant tumors formation
Resumo:
Coordenao de Aperfeioamento de Pessoal de Nvel Superior
Resumo:
Receptor ativador nuclear κappa B (RANK), ligante do receptor ativador nuclear κappa B (RANKL) e osteoprotegerina (OPG) so membros da famlia do fator de necrose tumoral relacionados com o metabolismo sseo. A formao, diferenciao e atividade dos osteoclastos so reguladas por estas trs proteínas. RANK um receptor transmembrana presente em diversos tipos celulares, principalmente em clulas de linhagem macrofgica, linfcitos, clulas dendrticas e fibroblastos e quando ativado pelo seu ligante, RANKL, promove a diferenciao e ativao de clulas osteoclsticas responsveis pelo processo de reabsoro ssea. A OPG impede a ligao RANK/RANKL atuando como um receptor inibitrio para a atividade osteoltica. O objetivo deste estudo foi comparar a expresso imuno-histoqumica destes biomarcadores em cistos radiculares (n=20) e cistos dentgeros (n=20). A expresso imuno-histoqumica destes marcadores foi avaliada no epitlio e na cpsula dos cistos por escores e percentuais mdios de imunomarcao. Para o epitlio, a anlise semi-quantitativa revelou um padro similar dos escores de imunomarcao de RANK, RANKL e OPG nas leses, no havendo diferena estatstica significante (p=0.589, p=0.688, p=0.709, respectivamente). Para a cpsula cstica a anlise quantitativa, mostrou diferena estatstica significante entre os percentuais mdios de imunomarcao do RANK e RANKL (p=0,001 e p=0,005, respectivamente) nos cistos. A correlao dos escores de imunomarcao de RANKL e OPG no epitlio do CR e do CD revelou diferena estatstica significante (p=0,029, p=0,003, respectivamente). No epitlio dos CRs e dos CDs observou-se uma maior imunoexpresso da OPG comparada a do RANKL. Os resultados apontam a presena de RANK, RANKL e OPG nos cistos radiculares e cistos dentgeros, sugerindo a atuao destas proteínas no desenvolvimento e expanso das leses no osso adjacente
Resumo:
Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy in the oral cavity and reach a large number of individuals, has become an important public health problem. Studies have demonstrated changes in pathway components BMP in various types of cancers as prostate, colon, breast, gastric and OSCCs. Is the current knowledge that these proteins may exert pro-tumor effect in more advanced stages of neoplastic development coming to favor progression and invasion tumor. The inhibition of the signaling pathway BMP-2 through its antagonists, have shown positive results of antitumor activity and use of Noggin may be a novel therapeutic target for cancer. Given this evidence and the few studies with BMP-2, Noggin and OSCC, the objective of this research was to evaluate the effect of BMP-2 and its antagonist Noggin on proliferation and migration cell in line of cell cultures of human tongue squamous cell carcinoma (SCC25). The study was divided in three groups, a control group, where SCC25 cells suffered no treatment, a BMP-2 group, in which cells were treated with 100ng/ml of BMP-2 and a group of cells that were treated with 100ng/ml of Noggin. For the proliferation assay and cell cycle were established three time intervals (24, 48 and 72 hours). Proliferative activity was investigated by trypan blue and cell cycle analysis by staining with propidium iodide flow cytometry. The potential for migration / invasion of SCC25 cells was performing by a cell invasion assay using Matrigel in a 48-hour interval. The proliferation curve showed a higher proliferation in cells treated with BMP-2 in 72 hours (p < 0.05), and lower overgrowth and cell viability in Noggin group. Recombinant proteins favored a greater percentage of cells in cell cycle phase Go/G1 with a statistically significant difference in the interval of 24 hours (p < 0.05). BMP- 2 produced a greater invasion of cells studied as well as its antagonist Noggin inhibits invasion of cells (p < 0.05). Thus, these results indicate that BMP-2 promotes malignant phenotype, dues stimulates proliferation and invasion of SCC25 cells and, its antagonist Noggin may be an alternative treatment, due to inhibit the tumor progression
Resumo:
Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP)
Resumo:
Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP)
Resumo:
Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP)
Resumo:
Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP)
