Para que se produzcan procesos de proliferación celular, tanto en tejidos normales como tumorales, es necesaria la presencia de vasos sanguíneos. La formación de estos se realiza a través de un complicado proceso denominado angiogénesis y es estimulado fundamentalmente por el factor de crecimiento del endotelio vascular (VEGF), que es el más específico y potente, tanto para la angiogénesis como para la vasculogénesis. La aparición de una neoplasia ocurre como consecuencia de una alteración en el ciclo celular normal que lleva a una proliferación excesiva, y teniendo en cuenta que, uno de los aspectos críticos para el desarrollo de tumores sólidos es la formación de una fuente de irrigación sanguínea, tanto el conocimiento del crecimiento tisular como el de los procesos involucrados en la angiogénesis son fundamentales para enfrentar una de las principales causas de muerte en el hombre y en los animales domésticos, el cáncer. Por otro lado en la regeneración hepática, la formación de nuevos capilares a partir de la microvasculatura existente es un requerimiento fundamental, lo que convierte al hígado remanente a una hepatectomía parcial, en un excelente modelo experimental para estudiar algunos de los mecanismos involucrados en la cronobiología de la angiogénesis. En el presente trabajo de tesis se analiza la evolución de la expresión del VEGF, la síntesis de ADN y la actividad mitótica (AM) a lo largo de un periodo circadiano en los hepatocitos del ratón macho adulto durante la regeneración hepática y el efecto que ejerce la portación de un tumor maligno sobre dichos índices. Se utilizaron 252 ratones machos adultos que fueron sometidos a una hepatectomía parcial, la mitad de los cuales fueron además injertados con un tumor maligno. Estos animales se usaron para la realización de 3 experimentos: 1) estudio de la expresión del VEGF en ratones hepatectomizados portadores y no portadores del tumor; 2) estudio de la ADNs en ratones hepatectomizados portadores y no portadores del tumor y 3) estudio de la AM en ratones hepatectomizados portadores y no portadores del tumor. En cada experimento, los ratones fueron divididos en lotes de 6 a 8 animales y sacrificados cada 4 horas a lo largo de un período circadiano desde las 26 hasta las 50 horas poshepatectomía. En todos los casos se tomaron muestras de la porción triangular del lóbulo derecho del hígado que fueron procesadas con las técnicas histológicas adecuadas según los experimentos diseñados. Tanto para la detección de la expresión del VEGF como para la ADNs se utilizaron técnicas inmunohistoquímicas y para la evaluación de la AM se utilizó H-E. A partir de los registros individuales se estableció el índice de expresión del VEGF, ADNs y AM expresado en porcentaje. A partir de estos índices individuales se calculó la media aritmética ± error estándar de cada lote y de cada grupo. Estos datos se analizaron estadísticamente mediante el análisis de varianza (Anova), en caso de que las diferencias fueran significativas se realizó el Test de Tuckey. Los resultados correspondientes a los ratones hepatectomizados no portadores muestran la existencia de curvas circadianas tanto para la expresión del VEGF con un valor máximo a las 08/46 (hora del día/hora poshepatectomía) de 2.6±0.1; en la ADNs con un valor máximo a las 16/30 de 3.4±0.3 y en la AM cuyo valor máximo se produjo a las 12/50 de 2.3±0.01. La presencia del tumor injertado ES2 genera cambios detectables en las curvas circadianas de la expresión del VEGF, en la ADNs y AM de los animales hepatectomizados, provocando modificaciones en la distribución espacial y temporal de los valores correspondientes a los mismos. El valor máximo de expresión del VEGF se encontró a las 16/30, 8 horas después (00/38) se observó el de ADNs. Por su lado el valor máximo de AM se produjo 8 horas más tarde al mismo a las 08/46. Finalmente, se puede concluir que los factores de crecimiento producidos por las células del tumor maligno ES2, de ratones machos de la cepa C3HS hepatectomizados que son liberados a la circulación general, podrían reconocer al tejido regenerante con una alta tasa proliferativa como una metástasis y serían entonces los responsables de alterar el microambiente del tejido hepático determinando de esta forma cambios en la cronobiología de la síntesis de ADN, de la actividad mitótica y de la expresión del VEGF.

Le vascular endothelial growth factor (VEGF) un facteur de croissance essentiel du système vasculaire exerce des fonctions multiples sur les cellules nerveuses en favorisant la neurogenèse, la plasticité synaptique ou encore l'apprentissage et la mémoire. Cependant, les mécanismes impliqués dans son action régulatrice de la transmission et la plasticité synaptiques restent à élucider. Nous avons récemment mis en évidence une nouvelle interaction entre VEGFR2, le récepteur principal du VEGF, et les récepteurs NMDA (NMDAR) au cours de la migration des neurones pendant le développement du cervelet. Comme les NMDAR sont des acteurs clés de la transmission et de la plasticité synaptique, nous avons exploré le rôle du VEGF dans la régulation de l'expression et de la fonction des NMDAR synaptiques dans l'hippocampe. Nos résultats révèlent que le VEGF et son récepteur sont exprimés dans les régions CA1 et CA3 de l'hippocampe et le domaine extracellulaire de VEGFR2 peut se lier à la sous-unité GluN2B des NMDAR. Le VEGF est capable d'augmenter la transmission synaptique dépendant des NMDAR en régulant l'adressage synaptique des récepteurs exprimant la sous-unité GluN2B. Il se produit également une augmentation du nombre de synapses en présence du VEGF. Ces effets du VEGF requièrent la co-activation des récepteurs VEGFR2 et NMDAR et conduisent à un enrichissement synaptique en récepteurs glutamatergiques de type AMPA qui dépend de l'activation de la CaMKII. Nos travaux démontrent pour la première fois un rôle direct de la signalisation VEGF/VEGFR2 dans la fonction de la synapse excitatrice glutamatergique

Cervical cancer is the third most common cancer among women worldwide, and the fourth leading cause of cancer mortality. Despite significant declines in the incidence and mortality rates of cervical cancer in Canada, it remains the 4th most common cancer in women aged 20-29 years. In order to gain novel insights into cervical cancer tumourigenesis and clinical outcome, we investigated and characterized the alterations in microRNA (miRNA) expression in this disease. Firstly, we performed global miRNA expression profiling of cervical cancer cell lines (n=3), and patient specimens (n=79). From this analysis, we identified miR-196b to be significantly down-regulated in cervical cancer, and characterized its role in regulating the HOXB7~VEGF axis. The global miRNA expression data also led to the development of a candidate 9-miRNA signature that was prognostic for disease-free survival in patients with cervical cancer, although we were unable to validate this signature in an independent cohort. This report describes important considerations concerning the development and validation of microRNA signatures for cervical cancer. Our investigations also led us to a comparison of three methods for measuring miRNA abundance: the TaqMan Low Density Array, the NanoString nCounter assay, and single-well quantitative real-time PCR. Our findings demonstrated limited concordance between the TLDA and NanoString platforms, although each platform correlated well with PCR, which is considered the gold standard for nucleic acid quantification. Furthermore, we examined biases created by amplification protocols for microarray studies. Our analysis demonstrated that performing a correction using the LTR-method (linear transformation of replicates) could help mitigate, but not completely eliminate such biases. Overall, this report presents insights into the role of miRNAs in cervical cancer, as well as an evaluation of technical considerations concerning miRNA and mRNA expression profiling studies.

Diamond is a promising material with unique chemical properties. In this thesis, nano-scale diamond quantum size effects were investigated using several chemical property indicators. The results show that the chemical properties are strongly dependent on size for film thicknesses smaller than 1 nm (1D), and for nanodiamond particle diameters less than 2 nm (3D). When the sizes exceed these ranges there are no longer any quantum effects. The influence of surface termination coverage on the surface chemical properties has been calculated for the 2×1 reconstructed diamond (100) surface and for the diamond (111) surface. The terminating species included COOH and NH2 groups, which both are beneficial for the immobilization of biomolecules. The results of the calculations show that it is energetically possible to terminate the diamond surfaces up to 100% with NH2, while it is only possible to cover the surfaces up to 50% with COOH species. The reason for the latter result is most probably the larger sterical hindrance amongst the adsorbates. Both types of termination species were shown to influence the diamond surface electronic properties (e.g., HOMO/LUMO levels). In order to extend the diamond utility for biomedical applications, especially implant design, interactions of various growth factors with the diamond surfaces were also simulated. For non-solvent diamond-biomolecule systems, the results show that adhesion affinities are strongly dependent on biomolecule molecular weights. When including a water based solvent in the systems, the results show good physisorption affinities between proteins and diamond. Proteins structures, before and after physisorption, were visualized, and further investigated with respect to electrostatic properties and functional groups. By comparing the biomolecular structural changes during the adhesion processes, it can be concluded that both the general structures, as well as the binding pocket structures, were kept intact after the adhesion to the diamond surfaces (regardless of the adhesion affinities). In addition, the surface electronic potential distributions were maintained, which indicate preserved biomolecule functionalities. / Vascubone

Shiga toxin-producing E. coli (STEC) comprise a group of pathogenic organisms that elaborate a family of protein exotoxins known as Shiga toxins (Stxs). Intestinal infection with these organisms may lead to hemorrhagic colitis and hemolytic uremic syndrome, a life-threatening condition characterized by thrombocytopenia, non-immune hemolytic anemia, and acute renal failure. Vascular endothelial damage is believed to be a key initiating event in Stx-mediated diseases. At the molecular level, these toxins depurinate human 28S rRNA and inhibit translation. In addition, at concentrations that only minimally affect global protein synthesis, they have been found to alter expression of specific target genes. To better understand the endothelial damage induced by Stx, we investigated the global effects of Stx on endothelial gene expression, and defined a specific group of genes whose expression was altered by the toxin. Of interest, the CXCR4/CXCR7/SDF-1 chemokine pathway, a pathway central to vascular biology, was activated by Stx. In vitro studies demonstrated that Stx enhanced both transcript levels of these molecules, as well as their association with ribosomes. To define the relevance of these findings in vivo, a mouse model was established and key changes were noted in plasma and tissue content of CXCR4/CXCR7/SDF-1 following Stx exposure. Inhibition of CXCR4/SDF-1 interaction decreased indices of endothelial activation and organ injury and improved animal survival. Importantly, in children infected with E. coli O157:H7, plasma SDF-1 levels were significantly elevated in individuals who progressed to hemolytic uremic syndrome. A second pathway critical to endothelial health and function is VEGF signaling. Of interest, our endothelial gene expression analyses revealed changes in this pathway in vitro. VEGF mRNA association with cellular polyribosomes increased following Stx treatment. Further studies in vivo demonstrated decreased cardiac function and blood pressure, and increased vascular permeability in specific tissues. VEGF, an important inducer of vascular permeability, increased in mouse plasma. Additionally, altered mRNA expression was observed in key organs, such as the kidney and heart, following Stx challenge. Inhibition of VEGF significantly improved survival of animals treated with Stx, indicating that VEGF plays a role in Stx-mediated pathogenesis. Moreover, in vitro studies demonstrated that Stx-mediated endothelial permeability was attenuated in the presence of a VEGF inhibitor. Taken together, these data indicate that E. coli-derived Stxs induce pathological changes in two pathways key to vascular biology. These pathways represent novel targets for the development of preventative and therapeutic strategies for complications associated with Shiga toxin-producing E. coli infection.

Shiga toxin-producing E. coli (STEC) comprise a group of pathogenic organisms that elaborate a family of protein exotoxins known as Shiga toxins (Stxs). Intestinal infection with these organisms may lead to hemorrhagic colitis and hemolytic uremic syndrome, a life-threatening condition characterized by thrombocytopenia, non-immune hemolytic anemia, and acute renal failure. Vascular endothelial damage is believed to be a key initiating event in Stx-mediated diseases. At the molecular level, these toxins depurinate human 28S rRNA and inhibit translation. In addition, at concentrations that only minimally affect global protein synthesis, they have been found to alter expression of specific target genes. To better understand the endothelial damage induced by Stx, we investigated the global effects of Stx on endothelial gene expression, and defined a specific group of genes whose expression was altered by the toxin. Of interest, the CXCR4/CXCR7/SDF-1 chemokine pathway, a pathway central to vascular biology, was activated by Stx. In vitro studies demonstrated that Stx enhanced both transcript levels of these molecules, as well as their association with ribosomes. To define the relevance of these findings in vivo, a mouse model was established and key changes were noted in plasma and tissue content of CXCR4/CXCR7/SDF-1 following Stx exposure. Inhibition of CXCR4/SDF-1 interaction decreased indices of endothelial activation and organ injury and improved animal survival. Importantly, in children infected with E. coli O157:H7, plasma SDF-1 levels were significantly elevated in individuals who progressed to hemolytic uremic syndrome. A second pathway critical to endothelial health and function is VEGF signaling. Of interest, our endothelial gene expression analyses revealed changes in this pathway in vitro. VEGF mRNA association with cellular polyribosomes increased following Stx treatment. Further studies in vivo demonstrated decreased cardiac function and blood pressure, and increased vascular permeability in specific tissues. VEGF, an important inducer of vascular permeability, increased in mouse plasma. Additionally, altered mRNA expression was observed in key organs, such as the kidney and heart, following Stx challenge. Inhibition of VEGF significantly improved survival of animals treated with Stx, indicating that VEGF plays a role in Stx-mediated pathogenesis. Moreover, in vitro studies demonstrated that Stx-mediated endothelial permeability was attenuated in the presence of a VEGF inhibitor. Taken together, these data indicate that E. coli-derived Stxs induce pathological changes in two pathways key to vascular biology. These pathways represent novel targets for the development of preventative and therapeutic strategies for complications associated with Shiga toxin-producing E. coli infection.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Introdução: Ao ser usado o retalho de omento dissecado por videolaparoscopia no tratamento de deformidades da mama, foi constatado um significativo aumento do seu volume nos primeiros meses após a sua transposição, em todas as pacientes operadas, o que não é visto com essa magnitude em nenhum outro retalho adiposo. Métodos: Para se estudar o motivo desse aumento de volume, foram realizados estudos histológicos de amostras de omento coletadas no primeiro tempo cirúrgico, logo após sua transposição da cavidade abdominal para a região mamária e, no segundo tempo cirúrgico, durante a complementação de tratamento para a simetrização das mamas de oito pacientes. Foram documentadas as modificações nas medidas morfométricas dos adipócitos (perímetro, diâmetro e área), na densidade microvascular mediante o marcador endotelial CD31 e na expressão imunohistoquímica do fator de crescimento do endotélio vascular (VEGF). Resultados: O aumento do tamanho dos adipócitos e da densidade microvascular foi estatisticamente significativo (P≤0,012). O valor do VEGF foi menor na segunda amostra em relação com a primeira, redução esta que não atingiu significância considerável (P<0,093). Conclusão: Estes resultados sinalizam um aumento no volume celular que se mostrou consistente quando foram utilizados três diferentes processos de medida: perímetro, diâmetro e área dos adipócitos. Além disso, o aumento do número de vasos na segunda amostra sugere que tenha ocorrido neoangiogênese estimulada pelo aumento inicial dos valores do VEGF. Portanto o aumento do volume do retalho se deve a neovascularização e hipertrofia do adipócito. / Introduction: When laparoscopically harvested omentum flap was used to treat breast deformities, a significant volume increase, which had never been noticed in any other adipose flap, was observed in all the patients in the first months following its transposition. Methods: Histological studies of omentum samples were performed to study the reason for this increase. Samples were harvested at the first surgical time, right after the transposition of the omentum from the abdominal cavity to the breast region, and at the second surgical time, during treatment complementation for breast symmetrization of eight patients submitted to the transposition of the omentum flap. Modifications in the morphometric measurements of the adipocytes (perimeter, diameter, and area), in the microvascular density by the CD31 endothelial marker and in the imunohistochemic expression of VEGF were documented. Results: the increase in adipocyte size and microvascular density was statistically significant (P≤0.012). The value of VEGF was lower in the second sample, which was not significant (P<0.093). Conclusion: These results suggest an increase in cellular volume that was consistent when three different measurement procedures were used: adipocyte perimeter, diameter, and area. Moreover, the increase in the number of vessels in the second sample suggests neoangiogenesis stimulated by the initial increase in VEGF values obtained in the first sample. The flap increase was probably caused by adipocyte hypertrophy, resulting from the neoangiogenesis.

Made available in DSpace on 2016-06-02T20:20:29Z (GMT). No. of bitstreams: 1 2721.pdf: 2622918 bytes, checksum: 770cc0646b8e50e47027cdfd43e54fbd (MD5) Previous issue date: 2009-11-17 / Universidade Federal de Minas Gerais / Disintegrins are small cysteine-rich proteins with an RGD motif isolated from viperidae snake venoms. DisBa-01, a recombinant RGD-disintegrin from Bothrops alternatus snake venom, inhibits melanoma cell metastasis and also platelet aggregation in murine models by interactions with &#946;3 integrin cell receptors. Also, this RGD-disintegrin inhibits human microvascular endothelial cell (HMEC-1) proliferation in vitro and angiogenesis in vivo. Alternagin-C (ALT-C), a disintegrin-like protein from Bothrops alternatus, is a potent inhibitor of the collagen binding to &#945;2&#946;1 integrin which plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Also, this protein can modulate angiogenesis where low concentrations of ALT-C positively regulate the formation of new blood vessels, whereas high ALT-C concentrations negatively modulates this process. This work presents the effects of DisBa-01 and ALT-C: 1) on the adhesion of breast cancer invasive MDA-MB-231 cells to the ECM proteins and to the subendothelial matrix produced by HUVEC (human vascular endothelial cells) under static and dynamic conditions; and 2) on the VEGFRs expression on endothealial cells. ALT-C inhibited tumoral cell adeshion to almost ECM proteins individually and also to ECM. Only 10000nM ALT-C inhibited tumoral cell adhesion to the HUVEC matrix under static conditions. Under flow, tumoral adhesion were inhibited by 100nM and 1.000nM ALT-C. Flow induces &#945;2&#946;1 activation in endothelial cells and MDA-MB- 231 cells expressed high levels of &#945;2&#946;1. Thus, a possible activation of this integrin by the shear stress could enhance its interactions with ALT-C. DisBa-01 had no effect on the tumoral cell adhesion to HUVEC matrix under static conditions. On the other hand, under flow the RGD-disintegrin strongly inhibited tumoral and platelet adhesion. Thus, a possible activation of &#945;V&#946;3 by flow could enhance its interactions with DisBa-01. About VEGFR expression, DisBa-01 down-regulated VEGFR-1 and VEGFR-2 expression in almost all tested concentrations. ALT-C up-regulates VEGFR-2 expression at lower concentration. These results can explain at least in part the effects of DisBa-01 and ALT-C in angiogenesis. The anti-adhesives and antiangiogenics ALT-C and DisBa-01 properties may be helpful for the study of tumoral metastasis as well as in the design of new therapeutic agents targeting tumor cell adhesion on extravasation and invasion steps. / As desintegrinas são pequenas proteínas isoladas de veneno de serpente, ricas em cisteína e com um motivo RGD. DisBa-01, uma desintegrina-RGD recombinante de do veneno de Bothrops alternatus, inibe metástase de melanoma e a agregação plaquetária em modelos murinos pela interação com integrinas &#946;3. Além disso, a desintegrina-RGD inibe a proliferação endotelial in vitro e a angiogênese in vivo. Alternagina-C (ALT-C), uma desintegrina-like de Bothrops alternatus inibe a ligação ao colágeno através da integrina &#945;2&#946;1, a qual desempenha um papel essencial na adesão normal e tumoral a MEC. Esta proteína do veneno também pode modular a angiogênese, onde doses baixas de ALT-C regulam positivamente, ao passo que doses maiores inibem o processo. Este trabalho apresenta os efeitos de DisBa-01 e ALT-C: 1) na adesão de células de adenocarcinoma mamário MA-MB-231 a proteínas da MEC e MEC subendotelial, sobre condições estáticas e dinâmicas; e na expressão de VEGFR em células endoteliais. ALT-C inibiu a adesão das células tumorais a quase todos os componentes de MEC testados individualmente e também a MEC. Entetanto, somente uma dose alta da proteina, 10.000nM, teve efeito sobre a MEC em condições estáticas. Sob estresse de cisalhamento, a adesão tumoral foi totalmente inibida por ALT-C 100 e 1.000nM. O fluxo induz a ativação de &#945;2&#946;1. Assim, uma possível ativação das células por estresse de cisalhamento pode ter reforçado a interação com ALT-C. DisBa-01 não teve efeito na adesão tumoral sob condições estáticas. Por outro lado, sob fluxo a desintegrina-RGD inibiu fortemente a adesão tumoral. Assim, uma possível ativação de &#945;V&#946;3 por fluxo pode ter reforçado as interações com DisBa-01. Sobre a expressão dos VEGFRs, DisBa-01 diminuiu o nivel de ambos em quase todas as concentrações testadas. ALT-C em baixas doses, por sua vez, aumentou a expressão do VEGFR-2. Tais resultados podem explicar, ao menos em parte, os efeitos de DisBa-01 e ALT-C na modulação da angiogênese. Os efeitos anti-adesivos e anti-angiogênicos de ALT-C e DisBa-01 podem ser úteis para o estudo de metástase tumoral, bem como para o desenho de novos agentes terapêuticos que ajam na adesão tumoral nas etapas de extravasamento e invasão.

Made available in DSpace on 2016-06-02T20:21:34Z (GMT). No. of bitstreams: 1 DissJUR.pdf: 1290933 bytes, checksum: 72ad2925663cff469dc6dc50316dbf22 (MD5) Previous issue date: 2005-02-17 / Universidade Federal de Minas Gerais / Disintegrins are snake venom proteins used in the study of integrin-mediated cellular adhesion through integrins. Recombinant disintegrin production may notable supply faster, larger and most homogeneous protein samples than bruit venom processing. This work describes optimization of the recombinant ACLD-C expression technique. ACLD-C is a disintegrin-like protein from Agkistrodon contortrix laticinctus, that have a disintegrin-like domain with DCD motif, and a cysteine-rich domain. This protein was subcloned from the ACLD clone (GenBank U86634), a metallopeptidase. A protocol of expression was established in our laboratory, where recombinant ACLD-C is produced in E. coli, using the expression vector pMal-p. This allows protein target expression in form of N-terminal fusion with MBP (maltose-binding protein), that facilitate purification, correct folding and production of disintegrin in soluble form. In fact, MBP/ACLD-C was was expressed in soluble form and purified in two stages: affinity comatography in amilose resin and cromatography of gel-filtration in superdex-200 resin. Modifications in expression protocol optimized MBP/ACLD-C expression in culture of 0,4 mg/L of culture of 1,2 mg/L (12,6% total of protein in periplasmic space of E.coli). MBP/ACLD-C imobilizated in amilose resin was subjected to digestion with enzyme factor Xa to separate ACLD-C of MBP. The separation was confirmed through positive reaction with anti-ALT-C antibody. ALT-C is a disintegrin-like protein from Bothrops alternatus. ALT-C induced both expression of VEGF in human fibroblasts and proliferation of endothelial cells and angiogenesis. In second part of this work, we investigated if MBP/ACLD-C was also able to induce the expression of this growth factor in this cells. MBP/ACLD-C and two other disintegrins, echistatina, a disintegrin-RGD, and EC6, a heterodimeric disintegrin, were incubated with fibroblasts. The influency of these proteins in VEGF expression was evaluated by ELISA. MBP/ACLD-C was able to strongly induce the expression of VEGF until 4 hours, when the level decreased until 24 hours. Experiment in period of 24 48 hours was not done due to problems of contaminations. Echistatin did not induced VEGF expression, but induced fibroblasts dettachment from plastic. The latter event was not observed in MBP/ACLD-C or EC6 incubated cells. / As desintegrinas são proteínas de veneno de serpente usadas no estudo de processos biológicos envolvendo a adesão celular mediada por integrinas. A produção de desintegrinas recombinantes é de grande importância para tais estudos, pois podem ser obtidas de forma mais rápida, com maior rendimento e qualidade mais constante quando comparada com a obtenção a partir de veneno bruto. Esta dissertação descreve a otimização da expressão da ACLD-C recombinante. ACLD-C é uma proteína tipo desintegrina da serpente Agkistrodon contortrix laticinctus, que possui um domínio desintegrina-like com um motivo DCD e um domínio rico em cisteína. Esta proteína foi subclonada a partir do clone da ACLD (GenBank U86634), uma metalopeptidase. Um protocolo de expressão foi estabelecido em nosso laboratório, onde ACLD-C recombinante é produzida em E. coli, utilizando-se o vetor de expressão pMal-p. Este vetor possibilita a expressão da proteína-alvo na forma de fusão Nterminal com a proteína MBP (maltose-binding protein), a qual facilita a purificação, o enovelamento correto e a produção da desintegrina na forma solúvel. De fato, MBP/ACLD-C foi expressa na forma solúvel e purificada em duas etapas: cromatografia de afinidade em resina de amilose e cromatografia de gel-filtração em resina superdex-200. Modificações no protocolo de expressão permitiram a otimização da expressão da MBP/ACLD-C de 0,4mg/L de cultura para 1,2mg/L de cultura O rendimento final da MBP/ACLD-C foi de 1,2mg/L de cultura (12,6% do total de proteínas no espaço periplásmico de E. coli). MBP/ACLD-C imobilizada em resina de amilose foi submetida à reação de clivagem com a enzima fator Xa para separar ACLD-C da MBP. A separação foi confirmada através da reação positiva ao anticorpo anti-ALT-C, uma desintegrina-like de Bothrops alternatus. Estudos com ALT-C mostraram que ela foi capaz de induzir a expressão de VEGF (vascular endothelial growth factor) em fibroblastos humanos e induzir a proliferação de células endoteliais e a angiogênese. Assim, resolveu-se investigar se MBP/ACLD-C também é capaz de induzir a expressão deste fator de crescimento nestas células, o que constitui a segunda parte deste trabalho. MBP/ACLD-C e outras duas desintegrinas, echistatina, uma desintegrina-RGD, e EC6, uma desintegrina heterodimérica, foram incubadas com os fibroblastos para verificar a influência dessas proteínas na expressão de VEGF através de ensaios de ELISA. MBP/ACLD-C foi capaz de induzir fortemente a expressão de VEGF até 4h, quando o nível da citocina decresceu até 24 e novamente aumentou até 48h. EC6 também foi capaz de induzir a expressão do fator de crescimento até 4h, quando o nível também decresceu até 24h. O experimento no período de 24h-48h não foi realizado devido a problemas de contaminação. Echistatina, por sua vez, não apresentou indução de VEGF em relação ao controle, e as células incubadas com essa desintegrina desaderiram do plástico, o que não ocorreu com as outras duas proteínas.