Made available in DSpace on 2016-01-26T12:51:47Z (GMT). No. of bitstreams: 1 rodrigocastro_tese.pdf: 1914162 bytes, checksum: b3f5bd6e4ed4e8470c9714a82211ce12 (MD5) Previous issue date: 2013-10-29 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction : The growth and progression of tumors depend on angiogenesis , the process of formation of new blood vessels from a pre-existing endothelium . The vascular endothelial growth factor (VEGF or VEGF - A) is a potent mitogen for endothelial cells and increased expression is associated with tumor growth and metastasis . However, the selection of alternative splicing site in the 3 'end of exon 8 of the VEGF gene results in a sister family of isoforms , VEGF- Axxxb , which are anti- angiogenic and downregulated in tumor tissues . Objectives : To assess quantitatively the expression of isoforms pro- angiogenic and anti- angiogenic VEGF gene into 50 samples of breast cancer and normal adjacent tissue and determining the effect of regulatory proteins to control the event splicing of the VEGF gene regulatory proteins and Splicing , SRPK1 , SRp40 , SRp55 and ASF/SF2 . Methods: The expression of VEGF Axxx, VEGF-A165b isoforms and Splicing regulatory proteins were analyzed by PCR quantitative real-time (PCRq ) using samples from 50 patients with breast cancer and 43 adjacent normal tissue used as controls . Values of Relative Quantification (RQ) were used in association with molecular subtypes and metastasis. The data were tested for normality D' Agostino and Pearson normality test using the program GraphPadPrismv.6 . The values of relative mRNA quantification ( RQ ) of the VEGF-A isoforms and splice regulatory proteins in tumors was analyzed by Wilcoxon Signed Rank Test , since the data is not normal distribution . Spearman correlation was used to evaluate the correlation between the levels of mRNA expression between regulatory proteins and VEGF-Axxx and VEGF-A165b isoforms where P values &#8804; 0.05 were considered significant. Results: Expression significantly increased both isoforms of VEGF- Axxx (median RQ = 7.7, P <0.0001) and VEGF- A165b (median RQ = 2.9 , P < 0.0001) was seen in breast tumors compared to adjacent normal tissues . Comparing the values of expression of VEGF- A165b and VEGF - Axxx by the Mann -Whitney test that showed no significant difference in expression of isoforms in tumors ( P = 0.065 ) . The mRNA expression of SRPK1 protein was significantly elevated in breast tumors compared to normal tissue (P < 0.0001). For ASF/SF2, SRp55 and SRp40 mRNA expression also showed significantly elevated in the tumors (P < 0.0001). Proteins ASF/SF2 , SRp55 , SRp40 and SRPK1 showed positive correlation with both isoforms of VEGF -A . We also found a positive correlation SRPK1 protein with all other SR proteins , mainly ASF/SF2. Conclusion: The concept of alternative splicing of the VEGF-A gene results in isoforms anti- angiogenic and pro- angiogenic research indicates that the quantitative changes in VEGF-A molecule in cancer and other diseases may be insufficient . As shown, the expression of splicing regulatory protein studied here were all positive as compared to the VEGF-A165b and VEGF- Axxx isoforms . / Introdução: O crescimento e progressão de tumores dependem da angiogênese, processo de formação de novos vasos sanguíneos a partir de um endotélio vascular pré-existente. O fator de crescimento endotelial vascular (VEGF ou VEGF-A) é um potente mitógeno de células endoteliais e o aumento de sua expressão é associado com crescimento tumoral e metástase. Entretanto, a seleção do sítio alternativo de splicing na extremidade 3 . do éxon 8 do gene VEGF resulta em uma família-irmã de isoformas, VEGF-Axxxb, que são anti-angiogênicas e downregulated em tecidos tumorais. Objetivo: Analisar quantitativamente a expressão de isoformas pró-angiogênicas e anti-angiogênicas do gene VEGF em 50 amostras de carcinoma mamário e tecidos normais adjacentes e determinar o efeito de proteínas reguladoras no controle do evento de splicing do gene VEGF e das proteínas reguladoras de Splicing, SRPK1, SRp40, SRp55 e ASF/SF2. Material e Método: A expressão das isoformas de VEGF-Axxx, VEGF-A165b e das proteínas reguladoras de Splicing foram analisadas em PCR quantitativa em Tempo Real (qPCR) utilizando amostras de 50 pacientes com câncer de mama e 43 amostras de tecido normal adjacente utilizados como controle. Os valores de Relative Quantification (RQ) foram utilizados nas associações com os subtipos moleculares e com metástase. Os dados foram submetidos ao teste da normalidade de D Agostino e Pearson normality test utilizando o programa GraphPadPrismv.6. Os valores de quantificação relativa de RNAm (RQ) das isoformas de VEGF-A e das proteínas reguladoras de splicing em tumores foi analisada por Wilcoxon Signed Rank Test, uma vez que os dados não apresentaram distribuição normal. Correlação de Spearman foi utilizada para avaliar a correlação entre os níveis de expressão de RNAm entre as proteínas reguladoras e as isoformas de VEGF-Axxx e VEGF-A165b, onde valores de P &#8804; 0,05 foram considerados significantes. Resultados: Expressão significativamente elevada de ambas as isoformas VEGF-Axxx (mediana de RQ = 7.7; P < 0,0001) e VEGF-A165b (mediana de RQ = 2.9; P<0,0001) foi observada em tumores de mama em relação aos tecidos normais adjacentes. Quando comparados os valores de expressão de VEGF-Axxx e VEGF-A165b por meio do Mann-Witney Test que não apresentou diferença significativa de expressão das isoformas em tumores (P=0,0.65). A expressão do RNAm da proteína SRPK1 foi significativamente elevada em tumores de mama em relação aos tecidos normais (P<0,0001). Para ASF/SF2, SRp55 e SRp40 o RNAm também apresentou expressão significativamente elevada nos tumores (P<0,0001). As proteínas ASF/SF2, SRp55, SRp40 e SRPK1 apresentaram correlação positiva com ambas as isoformas de VEGF-A. Encontramos também uma correlação positiva da proteína SRPK1 com todas as outras proteínas SR, principalmente com ASF/SF2. Conclusão: O mecanismo de splicing alternativo do gene VEGF-A resultando em isoformas anti-angiogênicas bem como pró-angiogênicas indica que a investigação de mudanças quantitativas da molécula VEGF-A em câncer e outras doenças pode não ser suficiente. Conforme demonstramos, a expressão das proteínas reguladoras de Splicing aqui estudadas, foram todas positivas quando comparadas com as isoformas de VEGF-Axxx e VEGF-A165b.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-10-01T14:53:33Z No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-01T15:33:57Z (GMT) No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-01T15:33:57Z (GMT). No. of bitstreams: 2 Tese - Luciana Ximenes Salustiano - 2013.pdf: 3049558 bytes, checksum: b1f84cfd9b4978f7ecf5dd1b5da12e3d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-12-17 / Retinoblastoma (RB) is the malignant neoplasm of embryonic neural retinal cells and is the most common intraocular tumor of childhood. Early diagnosis associated with modern methods of treatment resulted in increased survival rate of patients with retinoblastoma. However, in those cases whose diagnosis is delayed and in untreated cases, the disease is invariably fatal. The knowledge of the molecular and histopathological features of retinoblastomas can influence the choice of treatment and benefit the prognosis of these tumors. Thus, the objectives of this study are to evaluate the main histopathological and molecular characteristics of retinoblastomas, including the evaluation of p16, Ki67 and VEGF protein expression, as well as the potential role of such molecules as prognostic markers in the retinoblastomas. Fifty-seven cases of RB were evaluated in enucleated eyes at Hospital Araújo Jorge, in Goiânia, in the period of 1998 to 2011. Clinical data were collected from the respective medical files and the cases were reviewed for analysis of histopathological aspects. The expression of Ki67, p16 and VEGF was assessed by immunohistochemistry, using specific antibodies and a detection system associated with polymers. The results were analyzed by descriptive statistics and categorical variables by chi-square test and Fisher exact test, when necessary. Ki67 overexpression was observed in 28% of cases, characterizing the retinoblastomas with high rate of cellular proliferation, while the p16 overexpression was observed in 42% of retinoblastomas evaluated. Significant associations were observed between the overexpression of these proteins and histopathological characteristics of worse prognosis, including invasion of the optic nerve, choroid, sclera and anterior chamber of the eye, as well as to a lower degree of cellular differentiation. VEGF expression was extensively observed in all retinoblastoma cases, independent of the histopathological aspects of the tumors. The associations observed in this study between tumor aggressive histopathological aspects and Ki67 and p16 overexpression, indicate the use of these markers in determining the prognosis of retinoblastomas. On the other hand, the high angiogenic potential of retinoblastomas, translated by diffuse VEGF expression in all cases analyzed in this study, raises new investigations on the use of anti-angiogenic drugs in the treatment of retinoblastomas. / O retinoblastoma (RB) é o tumor maligno das células neurais embrionárias da retina e consiste no tumor intraocular mais comum da infância. O diagnóstico precoce associado a modernos métodos de tratamento resultou em aumento da taxa da sobrevida de pacientes com retinoblastoma. No entanto, nos casos cujo diagnóstico é tardio e nos casos não tratados, a doença é invariavelmente fatal. O conhecimento das características histopatológicas e moleculares dos retinoblastomas pode direcionar a escolha do tratamento e beneficiar o prognóstico desses tumores. Este estudo objetiva avaliar as principais características histopatológicas e moleculares dos retinoblastomas, incluindo a avaliação da expressão das proteínas p16, Ki67 e VEGF e o potencial papel prognóstico desses marcadores que são anticorpos envolvidos na carcinogênese, angiogênese e proliferação celular. Cinquenta e sete casos de RB foram avaliados em olhos enucleados no Hospital Araújo Jorge, em Goiânia, no período de 1998 a 2011. Os dados clínicos foram coletados dos respectivos prontuários e todos os casos foram revistos para análise dos aspectos histopatológicos. A expressão de Ki67, p16 e VEGF foi avaliada por imunoistoquímica, utilizando anticorpos específicos e sistema de detecção associado a polímeros. Os resultados foram analisados por estatística descritiva e as variáveis categóricas pelo teste do qui-quadrado e teste exato de Fisher, quando necessário. A superexpressão de Ki67 foi observada em 28% dos casos, caracterizando os retinoblastomas com alto índice de proliferação celular, enquanto a superexpressão de p16 foi observada em 42% dos retinoblastomas avaliados. Associações significativas foram detectadas entre a superexpressão dessas proteínas e as características histopatológicas de pior prognóstico, incluindo invasão do nervo óptico, esclera, coroide e câmara anterior do olho, bem como ao menor grau de diferenciação celular. A expressão de VEGF foi observada de forma difusa em todos os casos analisados, independente dos aspectos histopatológicos dos tumores. As associações observadas neste estudo, entre os aspectos histopatológicos de maior agressividade tumoral e a superexpressão de Ki67 e p16, indicam que o uso destes marcadores pode influir na determinação do prognóstico dos retinoblastomas. Por outro lado, o alto potencial angiogênico dos retinoblastomas, traduzido pela expressão difusa do VEGF em todos os casos analisados neste estudo, suscita novas investigações sobre o uso de medicamentos anti-angiogênicos no tratamento dos retinoblastomas.

Chemotherapy is the most common cancer treatment. Traditionally, platinum-based drugs are used in chemotherapy. More recently, researchers have focused on ruthenium based compounds as a substitute for the platinum compounds. Ruthenium-based compounds appear to be less toxic to healthy cells than traditional platinum-based compounds. In this study, 7 ruthenium-based compounds were tested on HT-29 (colon) and MCF-7 (breast) human cancer cell lines with the specific aim of determining whether or not any of the ruthenium-based compounds exhibited cytotoxic properties. In addition, levels of vascular endothelial growth factor (VEGF) production were tested in supernate from the cancer cells treated with various ruthenium-based compounds to determine whether or not the ruthenium-based compounds had an effect their VEGF production. Our results indicate that none of the ruthenium based compounds tested had a cytotoxic effect on the cancer cell lines; however, some of the compounds did exhibit inhibition of cell growth. Results further indicate an initial decrease in VEGF production in the cell lines treated with the ruthenium compounds but that this effect was compound-cell line specific.

Previously, we cloned Heat shock protein A12B (HspA12B), the newest member of a recently defined subfamily of proteins distantly related to the Hsp70 family that are enriched in atherosclerotic lesions. We have found that HspA12B is predominantly expressed in vascular endothelium, and that it is involved in angiogenesis which we probed by in vitro angiogenesis assays (Matrigel), migration assays and Directed In Vivo Angiogenesis Assay (DIVAA). Hsp70s are molecular chaperones that are inducible by stress and have been found to be anti-apoptotic (Li et al. 2000; Nylandsted et al. 2000; Garrido et al. 2001). Because of its homology to Hsp70, we propose that it is the first endothelial-specific chaperone that is required for angiogenesis and interacts with known angiogenesis regulators. To begin to understand the molecular mechanisms underlying the role of HspA12B in angiogenesis, we turned our attention to identifying proteins that are involved in angiogenesis and also interact with HspA12B. Through the use of a yeast two-hybrid (Y2H) system HspA12B was found to interact with a known angiogenesis regulator, A Kinase Anchoring Protein 12 (AKAP12). This interaction was confirmed by co-immunoprecipitation and by colocalization. In primary human umbilical vein endothelial cells (HUVECs), shRNA mediated HspA12B knockdown increased AKAP12 levels and decreased VEGF by more than 75%, whereas HspA12B over-expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-Amino Acid (32-AA) domain in AKAP12 that mediates interaction with HspA12B. Over-expression of this 32-AA domain in HUVECs disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression suggesting the importance of the HspA12B-AKAP12 interaction in regulating VEGF. This is the first evidence that HspA12B promotes angiogenesis resulting in up-regulation of VEGF by suppressing AKAP12. Consistent with the proposed role in angiogenesis, HspA12B was also found to be increased in endothelial cells (ECs) by angiogenic stresses including hypoxia and shearing stress while knockdown of HspA12B abolished hypoxia-induced tubule formation. This work provides new insight into the mechanisms controlling angiogenesis by providing the first example of an EC-specific molecular chaperone that acts as a regulator of angiogenesis and lays the foundation for future studies of HspA12B-derived therapeutics for angiogenesis related diseases.

Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.

Ovarian cancer is the leading cause of gynecological cancer death in the United States. Even though no single genetic alteration can be attributed to all ovarian cancers, 90% of ovarian tumors express telomerase, a ribonucleoprotein that elongates telomeric (TTAGGG)n repeats de novo. In normal somatic cells, telomerase is absent. In cancer cells, the re-expression of telomerase allows senescence to be bypassed contributing to cellular immortalization, a key step for cellular transformation, making telomerase a potentially important target for therapeutic intervention. Ovarian cancer cells secrete vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) that feedback through their receptors present on ovarian cancer cells to promote cell growth. Since telomerase can be regulated by growth factors, I examined VEGF regulation of telomerase activity and the possible contribution of LPA as an upstream regulator of VEGF-mediated telomerase activity in ovarian cancer. My data reveal that both VEGF and LPA upregulate telomerase activity by ERK 1/2-dependent transcriptional activation within the -976 to the -378 bp hTERT promoter regions where Sp1 is one of the major mediators of VEGF- and LPA-induced transactivation of hTERT. It also identifies telomerase as a novel molecular target of LPA as well as a target of VEGF in non-endothelial cells. In addition I found that, vitamin E, a dietary supplement able to degrade and suppress LPA activity, consistently abrogrates LPA-mediated telomerase activity through transcriptional inhibition of the hTERT -976 to -578 bp promoter regions. Lastly, since epidermal growth factor (EGF) promotes ovarian surface epithelial (OSE) cell growth and EGF receptors are frequently constitutively activated in ovarian cancers, the potential contribution of EGF in the regulation of telomerase activity was also examined. While none of the ovarian cancer cell lines examined produced large amounts of EGF, EGF stimulation of telomerase activity was mediated by Sp1 and c-Myc transcription factors within the hTERT core promoter in an ERK 1/2 /Pyk2-dependent manner. In conclusion, my research shows differential regulation of telomerase activity by growth factor and/or anti-oxidant nutraceuticals. In the future, these factors may be exploited as adjuvant therapy for improved chemotherapeutic benefit to decrease the mortality associated with ovarian cancer.

The field of microfluidics is expanding into what is known as paper microfluidics. This uses a paper platform rather than materials (i.e. PDMS, PMMA) that are commonly used in microfluidics research. Current devices require an expensive manufacturing process and external sources to power the device. Such devices are not practical in low resource environments. As a consequence, it is the goal of this Thesis to develop a three-dimensional, multiplexed assay chip using nitrocellulose membranes. This device comprises of multiple layers of nitrocellulose membranes with defined fluidic channels. The multiple layers are bound together using double backed tape, and imbedded between the layers are conjugate reagents. In the detection region both antibodies and aptamers were evaluated. The fiberglass pad where conjugate reagents would be contained, were initially saturated in dye. As sample was inputted into the three-dimensional chip, the fluid path could be visualized. Without the use of the conjugate pad the chip’s four detection regions showed detection within one minute of one another. However, the addition of this fibrous pad skewed time points dramatically. The hypothesis that a three-dimensional chip could be designed to detect different biomarkers in a multi-analyte sample was satisfied. However, simultaneous detection was only possible if the conjugate pad was either neglected or, possibly, a different material was used. Additionally, current lateral flow assay technologies, another research area that paper microfluidics spawns from, use antibodies in order to capture biomarkers in sample and provide visual signal to the user. However, antibodies are sensitive to denaturation with pH and temperature, whereas aptamers can withstand much more extreme environmental conditions. A two-dimensional nitrocellulose chip was designed to compare antibodies and aptamers as capture reagents to detect VEGF, using colloidal gold as a particle to visualize detection. Both monoclonal and polyclonal anti-VEGF antibodies were used and showed no signal. On the other hand, the anti-VEGF aptamer produced a visual signal when conjugated to biotin on its 5’ end. This data was further validated by a separate project analyzing the binding kinetics of the antibody and the aptamer using Surface Plasmon Resonance. Therefore, the hypothesis that aptamers could be used as a possible capture reagent in a paper microfluidic chip for the detection of VEGF was satisfied.

La protéine E2F-1 est un facteur de transcription qui participe au contrôle de la prolifération cellulaire en stimulant le passage des cellules en phase S du cycle cellulaire et est aussi capable d'induire l'apoptose. Ayant préalablement décrit son profil d'expression différentielle dans les tumeurs bronchiques, nous avons étudié son rôle potentiel dans ces tumeurs.<br>L'épissage alternatif des pré-ARNm joue un rôle important dans la régulation de l'apoptose et les protéines de la famille SR sont des régulateurs principaux des événements de l'épissage alternatif et constitutif. Actuellement très peu de données existent concernant leurs activateurs en amont ainsi que leurs gènes cibles impliqués au cours du processus apoptotique. Dans ce travail, nous avons identifié la protéine SC35, un membre de la famille des protéines SR, comme une cible transcriptionnelle directe de E2F1. Nous avons montré que les deux protéines interagissent et coopèrent pour induire l'apoptose, notamment en réponse aux agents génotoxiques, en modifiant les patrons d'épissage de certains gènes apoptotiques dont Bcl-x, ou caspase-9, en faveur des transcrits codant pour les isoformes pro-apoptotiques. Ces résultats démontrent la capacité de E2F1 à réguler les profils d'épissage de transcrits contrôlant l'apoptose via la protéine SC35. Finalement, nous avons obtenu des résultats impliquant SC35 dans le contrôle des fonctions transactivatrices de E2F1 au niveau de gènes contrôlant la division cellulaire, et notamment la synthèse d'ADN. La protéine VEGF-A existe sous la forme de multiples isoformes protéiques résultant de l'épissage alternatif de son ARNm pré-messager et dénommées de façon générique VEGFxxx (formes pro-angiogéniques) et VEGFxxxb (formes anti-angiogéniques). L'expression des différentes isoformes de VEGF-A est fortement dérégulée dans les tumeurs. Dans un deuxième axe de ce travail, nous avons montré que E2F1 réprime l'activité du promoteur du VEGF-A dans nos lignées dépourvues de p53 fonctionnelle et dans des conditions normoxiques. De plus, nous avons montré que E2F1 n'affecte pas négativement le niveau d'expression des isoformes VEGFxxxb suggérant que E2F1 stimule l'inclusion de l'exon 8b du VEGF-A par un mécanisme restant à identifier. Nos résultats montrent qu'E2F1 affecte la balance des isoformes du VEGFA en faveur de ses formes anti-angiogeniques et suggèrent que le facteur d'épissage SC35 contribue à ces effets. Nos travaux ouvrent des perspectives quand aux conséquences biologiques de la dérégulation de l'expression de E2F1 dans les cancers bronchiques via une modification des facteurs d'épissage et un rôle de E2F1 dans le processus de néoangiogénèse.

En 2002, notre équipe a montré que la stimulation de cellules primaires cortico-surrénaliennes bovines en culture primaire par l'hormone hypophysaire ACTH induisait l'expression du VEGF de manière indépendante de sa transcription. En effet, en réponse à l'ACTH, l'expression du VEGF est régulée au niveau post-transcriptionnel par la stabilisation/déstabilisation de son transcrit via des séquences riches en AU (ARE) situées dans la région 3‘ non traduite (3'UTR) de son ARNm. Le laboratoire a mis en évidence une protéine, TIS11b, exprimée également en réponse à une stimulation des cellules par l'ACTH, mais de manière concomitante avec la phase de déstabilisation de l'ARNm du VEGF. TIS11b appartient à une famille de protéines déstabilisatrices des ARNm via les séquences ARE. L'interaction de TIS11b avec l'ARNm du VEGF a été confirmée par la suite, et le site de liaison de la protéine à l'ARNm a également été identifié.<br /><br />Dans ce contexte, le premier objectif de ma thèse était d'évaluer la possibilité d'utiliser les propriétés déstabilisatrices de TIS11b sur l'ARNm du VEGF pour une thérapie anti-angiogène et anti-tumorale. Pour cela, la protéine a été vectorisée par la fusion de petits peptides ou PTD (protein transduction domain, Tat issu du VIH ou les polyarginines R7 et R9) lui permettant de traverser les membranes et d'atteindre sa cible intracellulaire, l'ARNm du VEGF. Nous avons pu montrer dans cette étude que 100 nM de protéines de fusion Flag-Tat-, Flag-R7- et Flag-R9-TIS11b sont capables d'induire une diminution du taux d'ARNm du VEGF ainsi que de la production de la protéine VEGF, dans des cellules en culture après 24 h d'incubation. De plus, nous avons pu montrer que l'injection de 100 nM de la protéine de fusion Flag-R9-TIS11b dans la glande corticosurrénalienne de souris, induisait une diminution d'environ 50 % de l'expression du VEGF par cette glande et que cette diminution était maintenue à 48 h. Des résultats préliminaires encourageants d'inhibition de croissance tumorale ont été obtenus avec l'injection de cette protéine de fusion dans des tumeurs pré-établies chez la souris nude, et doivent être confirmés.<br /><br />L'ACTH étant une hormone qui active la voie de l'AMPc et de la protéine kinase A (PKA), le deuxième objectif de ma thèse était de caractériser la phosphorylation de TIS11b par la PKA en réponse à une stimulation par l'ACTH et d'étudier les conséquences de cette phosphorylation sur son activité déstabilisatrice des ARNm. Nous avons pu montrer pour la première fois, que la PKA phosphoryle la protéine TIS11b, in vitro, au niveau de la sérine 54. Un deuxième site de phosphorylation en réponse à une stimulation par l'ACTH, via probablement une autre kinase que la PKA, a été mis en évidence au niveau de la sérine 334. Il semblerait que la protéine TIS11b comporte un domaine d'activation et un domaine d'inhibition susceptibles d'être régulés en réponse à une stimulation par l'ACTH. L'attribution de ces domaines aux sérines 54 et 334 nécessite des expériences complémentaires.

Les cellules osseuses évoluent in vivo sur des matrices extracellulaires principalement formées de collagène de type I, dont le degré de minéralisation varie au cours du remodelage osseux. Le minéral de l'os, de structure apatitique, a été montré comme potentialisant les activités et modifiant la forme des cellules ostéoblastiques. Dans le but de comprendre les effets du micro-environnement matriciel sur les évènements précurseurs à la phase de formation, nous avons émis l'hypothèse que ces modifications morphologiques pouvaient expliquer en elles-mêmes l'augmentation de l'activité descellules ostéoblastiques, par augmentation de leur mécano-sensibilité, et que ce changement de préhension environnementale pouvait moduler la réponse aux stimulations mécaniques les plus fréquemment observées in vivo, à savoir les micro-vibrations. Nous avons montré que sur les matériaux de collagène minéralisé ACC (Apatite Collagen Complex), les pré-ostéoblastes de la lignée MC3T3-E1 synthétisaient une matrice riche en ostéopontine, fibronectine et facteurs angiogéniques, de façon concomitante à une augmentation dépendante de la quantité de minéral de leur adhérence et de leur migration. Nous avons de plus observé une augmentation de la mécano-sensibilité (expression et turn-over augmentés des adhésions focales) des pré-ostéoblastes sur ACC. Finalement, nous avons établi que la réponse aux stimuli vibratoires était positive sur des matériaux non minéralisés (information) et négative sur ACC (stress) par rapports aux supports non stimulés, ce que nous avons interprété comme une hypersensibilité mécanique cellulairelors de la culture sur ACC. L'ensemble de ces données nous a montré que les modifications de mécanique cellulaire de préostéoblastes cultivés sur ACC engendraient une fonctionalisation spécifique ressemblant à celle observée in vivo dans la ligne cémentante, indispensable à la formation osseuse. D'autre part, les modifications de mécano-sensibilité observées sur ACC, en faisant un support mécano-mimétique et nous amenant à la comparaison du comportement cellulaire observé avec les ostéocytes, pourraient en elles-mêmes expliquer le dépôt matriciel spécifique et la réception modifiée aux signaux vibratoires. Dans notre but ultime de création d'un modèle de remodelage osseux in vitro, les paramètres physicochimiques matriciels osseux et l'établissement de cocultures seront à prendre en compte