Members of the Transforming Growth factor-β (TGF-β) superfamily and its subclass Bone Morphogenetic Proteins (BMP) play important roles for nervous system development. In order to study the BMP role for catecholaminergic neurons in vivo, we generated three knock-in mice, expressing the transgenes specifically in the targeting cells. Two genetic modifications result in expression of dominant negative (dn) BMP receptors (BMPRII and ALK2). The tissue-specific expression was achieved by the transgene insertion into 3’- untranslated region of the endogenous gene for tyrosine hydroxylase (TH), the first enzyme in catecholamine biosynthesis. An Internal Ribosome Entry site (IRES) preceded inserted cDNAs, allowing for functional bicistronic mRNA production. While almost no defects in Th-IRES-dnALK2, the Th-IRES-dnBMPRII mouse demonstrated declined levels of catecholamines, including dopamine in the striatum. Losses of midbrain dopaminergic neurons (MDN) might cause the effect. Additionally, intermediate lines of these mice, preserving a neo-cassette, oriented opposite to the locus transcription, demonstrate dramatic decrease of catecholamine level, hence, represent models for rare catecholamine-deficiency diseases, including L-DOPA-responsive dystonia. The third mouse, expressing in the same way Cre-recombinase (Th-IRES-Cre), represents a tool for catecholaminergic cell-limited deletion of any gene, which has to be flanked by loxP sites. Besides TH-positive areas, unexpected sites of Cre-recombination were identified, indicating regions of transient TH expression. Surprising recombination in oocytes opens a possibility to use our mouse as a general Cre-deletor. Using TH-IRES-Cre mouse we generated tissue-specific knockout mice for two BMP signal transducers: Smad1 and Smad4 (also crucial for TGF-β). While no phenotype in Smad1 knockout, TH-IRES-Cre/Smad4 mouse revealed several defects including decreased level of striatal dopamine. These results demonstrate a positive role of BMPs for MDN fate in vivo. Generated mice represent a tool-box for comprehensive study of the BMP function in catecholaminergic neurons. This study is of potential interest for understanding some aspects of Parkinson’s disease.

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.The specific aims proposed were:1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.4- Analyze the regulation of Id genes by BMP signalling in the inner ear.5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

The purpose of the thesis is to explore the role of BMP signaling in developing vertebrate midbrain. BMP signaling plays important roles in various tissues and stages of neural development to regulate cell fate, proliferation, differentiation, morphogenesis and more. We observed that several major BMPs are expressed not only at the roof plate but also the floor plate of the midbrain. This has led us to ask the role of BMP signaling in dorsal and ventral midbrain patterning. Despite ventral experiments, we found that BMP signaling does not regulate ventral cell fate specification in the midbrain. Instead BMPs profoundly influence the shape and early morphogenesis of the midbrain neural plate as it closes into a neural tube. During neural tube closure, one of the early events occurring at the ventral midline is median hinge point (MHP) formation. Failure to form MHP leads to neural tube closure defects, the 2nd most common birth defects in humans. However, the molecular mechanisms underlying MHP formation are not well known. We found that the lowest BMP signaling occurs at the MHP during early neurulation and BMP blockade is necessary and sufficient for MHP formation. Interestingly, we also demonstrated that BMP blockade directs MHP formation by regulating the apicobasal polarity pathway and this regulation may be mediated by biochemical interactions between pSMAD5 and the apical protein, PAR3. Additionally, our time-lapse data suggest that BMP blockade slows cell cycle progression by increasing duration of G1 to S transition and S phase which leads cell nuclei stay at the basal location longer. This mimics basal nuclear migration seen at the MHP where low BMP signaling occurs. Thus, we conclude that BMP signaling regulates neural tube closure via the apicobasal polarity pathway and in a cell cycle dependent manner at the ventral midline. We observed that BMP signaling is necessary and sufficient for the dorsal cell fate specification in a context-dependent manner and ventral BMP signaling affects dorsal cell fates. Taken together, we propose the idea that BMP signaling has distinct roles in different contexts. BMPs regulate tissue morphogenesis in the ventral midbrain and dorsally cell fate specification. / text

L’ostéoarthrose (OA) est une maladie articulaire invalidante caractérisée par la perte de l’intégrité du cartilage articulaire. Les recherches tentent de comprendre les mécanismes moléculaires de la maladie afin de trouver des inhibiteurs efficaces pouvant prévenir la dégradation du cartilage articulaire. Les BMPs (bone morphogenic proteins) jouent un rôle dans le processus pathophysiologique de cette maladie. Cette étude cible le rôle d’un antagoniste des BMPs, le gremlin. Nous avons étudié la régulation de l’expression de gremlin par le clonage et la caractérisation de son promoteur et en déterminant si gremlin pouvait jouer un rôle autre qu’antagoniste des BMP, en affectant l’expression d’autres gènes par l’activation d’une cascade de signalisation dans la cellule. Les résultats ont identifié une région importante dans le promoteur de gremlin qui affecte son activité basale et induite, et ont montré que le gremlin ne pouvait pas affecter l’expression génique et l’activation de signalisation intracellulaire indépendamment des BMPs. Cette étude démontre que le rôle de gremlin dans l’OA en est un essentiellement d’antagoniste des BMPs. / Osteoarthritis (OA) is a disease that affects the integrity of the articular cartilage which leads to serious health issues for many individuals. Research is focused on understanding the molecular mechanisms which lead to this loss in integrity in the hopes of finding a way to turn the tide. The bone morphogenetic proteins (BMPs) have been shown to play a role in the progression of this disease and this study focuses on one of their antagonists, gremlin. We therefore decided to study what affects the expression of this protein through the cloning and characterization of its promoter region. We also studied the role of this protein in the disease, can it influence gene expression and can it initiate a signalling cascade within the cell on its own. The results identified a region important for basal and induced activity of its promoter .The results also demonstrated that the main role of this protein in the progression of OA is through BMP antagonism. Gremlin does not initiate a signalling cascade and affect gene expression on its own.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 3 uppsatser.

Chez la brebis, les BMPs inhibent la synthèse et la libération de FSH. La détection des ARNm de certaines BMPs dans l’hypophyse et de leurs récepteurs sur les cellules gonadotropes suggère une action paracrine/autocrine des BMPs sur la production de FSH. L’objectif de cette thèse a visé à déterminer l’importance des BMPs hypophysaires, en particulier BMP-4, dans la régulation de la sécrétion de FSH chez la brebis. Nous montrons que l’expression des ARNm du système BMP-4,analysée par RT-PCR en temps réel, ne varie pas au cours du cycle oestrien ou in vitro quelque soit le temps d’incubation (6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires.Afin de déterminer si les cellules hypophysaires ovines sécrètent des BMPs, des milieux conditionnés(MC) hypophysaires ont été soumis à un test d’activité biologique reposant sur des cellules embryonnaires transfectées avec un élément de réponse BMPs couplé au gène rapporteur luciférase.Aucun effet des MC n’a été observé sur l’activité luciférase comparé au milieu non conditionnésuggérant l’absence ou la très faible activité BMP des MC, et cela quelque soit le temps d’incubation(6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires. Toutefois, nous détectons une activité inhibitrice de BMPs qui est augmentée par la GnRH et l’oestradiol suggérant l’implication d’inhibiteurs de BMPs dans la régulation de la production de FSH. En conclusion,l’ensemble de nos résultats n’est pas en faveur d’un rôle de BMP-4 hypophysaire dans la synthèse de FSH chez l’adulte. Un rôle des BMPs au niveau hypophysaire via la voie endocrine peut s’envisager puisque nous avons mis en évidence une bioactivité de type BMP dans le sérum ovin. Les inhibiteurs BMPs produits par l’hypophyse, qui restent à identifier, pourraient moduler la biodisponibilité des BMPs atteignant l’hypophyse par la voie sanguine. / BMPs inhibit FSH synthesis and release in ewe. The detection of BMP mRNAs in the pituitary as well as the colocalisation of the two types of BMP receptors on gonadotrope cells suggest that these BMPs can exert paracrine/autocrine actions on FSH production. The aim of this thesis work was to determine the importance of pituitary BMPs in the regulation of FSH production in the ewe. We showed that the level of mRNAs for BMP-4 system, analyzed by real-time RT-PCR, did not vary across the oestrous cycle or in vitro whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of pituitary cells. By using a bioactivity test based on embryonic mesenchymal cells transfected with an expression construct containing a BMP-responsive element fused to firefly luciferase reporter gene,we detected no BMP activity within conditioned media from pituitary cells whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of the pituitary cells. However, we detected a BMP inhibitory activity which is increased by GnRH and oestradiol suggesting the implication of BMP inhibitor(s) in FSH regulation. In conclusion, the results are not in favor of a role for pituitary BMP-4 in the regulation of FSH synthesis in adult ewe. An endocrine action of BMPs at pituitary level can be evoked since BMP bioactivity was detected within ovine serum. BMP inhibitors that remain to be identified can modulate the bioavailability of BMPs reaching the pituitary by the blood way.

Members of the Runx family of transcription factors play essential roles in the differentiation and development of several organ systems. Here we address the contribution of the osteoblast-related Runx gene, Runx2, to the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Using a transgenic mouse model, we observe Runx2 transcription through one of its two known promoters (designated P1 in pre-cartilaginous mesenchymal condensations as early as E9.5. Runx2 gene activity is later repressed at the onset of cartilage formation, both in vivo and in vitro, necessitating examination of the regulation and function of Runx2 in mesenchymal stem cells. We demonstrate that Runx2 gene activity is repressed by the direct interaction of the homeodomain transcription factor Nkx3.2 with the proximal Runx2 P1 promoter. This repression was found to be required for the progression of BMP-induced chondrogenesis, thereby identifying Runx2 as a modulator of BMP activity in the chondrogenic as well as osteogenic differentiation program. To further understand the regulation of the Runx2 P1 promoter and to determine the contribution of P1-derived gene product, Runx2 Type II, to the formation of mineralized tissue, we have generated a Runx2 Type II-LacZ gene replacement mouse model in which the initial coding sequences and splice donor sites of the Type II isoform are replaced with the LacZ reporter gene. Activity of the endogenous P1 promoter can therefore be monitored by β-galactosidase production. Analysis of Runx2 Type II-LacZ mice demonstrates that the P1 promoter is transcriptionally most active in mature osteoblasts, but its product, Runx2 Type II is dispensable for embryonic skeletal formation. Lastly, we examine the link between growth control and osteogenic differentiation by tissue-specific deletion of the Mdm2 proto-oncogene in developing skeletal tissues of the mouse embryo. Loss of Mdm2 results in impaired bone formation, with skeletal elements exhibiting lower bone mineral content and higher porosity. Ex vivo cultures of calvarial osteoprogenitor cells exhibit severely decreased osteoblastogenesis and bone nodule formation accompanied by a failure to activate Runx2 gene activity. These findings suggest that Mdm2 is required for inhibition of p53 activity that ultimately allows for post-confluent proliferation and induction of Runx2 during maturation of the osteogenic phenotype. Taken together, our findings suggest that Runx2 modulates the commitment of progenitor cells to the osteogenic and chondrogenic lineages, and that Runx2 activity is inextricably linked to mechanisms that control cellular proliferation.

Made available in DSpace on 2014-12-17T15:32:22Z (GMT). No. of bitstreams: 1 MarceloABN_DISSERT.pdf: 4711000 bytes, checksum: ea3602bfcefe0d8448e9d402030634a2 (MD5) Previous issue date: 2014-02-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / BMPs are components superfamily ligands transformation growth fator-β (TGF-β) secreted into the extracellular environment, with mechanisms of intercellular communication through specific ligands and receptors in various target cells, being recognized for its influence in osteogenic induction, also play an important role in tissue homeostasis, cell proliferation, differentiation control , in addition to being present in the development of various malignancies. The aim of this study was to compare the immunohistochemical expression of BMP-2, BMP-4 and its receptors BMPRIA and BMPRII in cases of ameloblastoma and adenomatoid odontogenic tumor. The sample consisted of 20 cases of solid ameloblastoma (SA), 10 cases of ameloblastoma unicystic (UA) and 16 cases of adenomatoid odontogenic tumor (AOT). The expression of BMPs and their receptors was evaluated in the parenchyma and stroma of lesions, establishing the percentage of immunopositive cells (0 - negative; 1-1 % to 10 % of cells positive; 2 - 11% to 25% of positive cells; 3 - 26% to 50% of cells positive; 4 - 51% to 75 % of positive cells; 5 - more than 75% positive cells). Analysis of the expression of BMP-2 revealed no statistically significant differences in parenchymal (p = 0.925) and stromal component (p = 0.345) between the groups, as well as BMP-4 (p = 0.873 / p = 0.131). In the epithelial component, SA and AOT had a higher frequency of score 5. In turn, all cases of UA were classified as score 5. The analysis of the stromal component showed no statistically significant difference between groups with respect to median scores BMPRIA positivity (p = 0.768) and BMPRII (p = 0.779). In the epithelial component of SA and UA, no statistically significant correlations between imunoexpression proteins analyzed were observed. In turn, the group of AOT, statistically significant positive correlations between the scores of expression of all studied proteins were found. In the stromal component, statistically significant positive correlations were found only in the SA group in BMP -4 and BMPRII (r = 0.476; p = .034), in the UA in BMP-4 and BMPRIA (r = 0.709; p = 0.022). The results of this study suggest that the BMPs and their receptors are involved in the development process odontogenic tumors. BMP-4, in turn, besides being present in odontogenic tumors have the capacity to form mineralized material. / As BMPs s?o componentes da superfam?lia de ligantes do fator transformador de crescimento-β (TGF-β), secretados no meio extracelular, com mecanismos de comunica??o intercelular por meio de ligantes e receptores espec?ficos em diversas c?lulas-alvo, sendo reconhecidas por sua influ?ncia na indu??o osteog?nica, tamb?m desempenhando importante papel na homeostase tecidual, prolifera??o celular, no controle de diferencia??o, al?m de estar presente no desenvolvimento de diversas neoplasias. O objetivo deste estudo foi comparar a express?o imuno-histoqu?mica da BMP-2, BMP-4 e seus receptores BMPRIA e BMPRII em casos de Ameloblastoma e Tumor odontog?nico adenomat?ide. A amostra foi constitu?da de 20 casos de Ameloblastoma s?lido (AS), 10 casos de Ameloblastoma unic?stico (AU) e 16 casos de Tumor odontog?nico adenomat?ide (TOA). A express?o das BMPs e seus receptores foi avaliada no par?nquima e estroma das les?es, estabelecendo-se o percentual de c?lulas imunopositivas (0 negativo; 1 - 1% a 10% das c?lulas positivas; 2 - 11% a 25% das c?lulas positivas; 3 - 26% a 50% das c?lulas positivas; 4 - 51% a 75% das c?lulas positivas; 5 - mais 75% de c?lulas positivas). A an?lise da express?o de BMP-2 n?o revelou diferen?as estatisticamente significativas no componente par?nquimatoso (p = 0,925) e estromal (p = 0,345) entre as les?es estudadas, assim como a BMP-4 (p = 0,873 / p = 0,131). No par?nquima, o AS e TOA apresentaram maior frequ?ncia do escore 5. Por sua vez, todos os casos de AU foram classificados como escore 5. A an?lise do componente estromal revelou n?o haver diferen?a estatisticamente significativa entre os grupos em rela??o ?s medianas dos escores de positividade para BMPRIA (p = 0,768) e BMPRII (p = 0,779). No par?nquima do AS e do AU, n?o foram observadas correla??es estatisticamente significativas entre as imunoexpress?es das prote?nas analisadas. Por sua vez, no grupo dos TOAs, foram constatadas correla??es positivas, estatisticamente significativas, entre os escores de express?o de todas as prote?nas avaliadas. No componente estromal, foram constatadas correla??es positivas, estatisticamente significativas, apenas no grupo do AS em BMP-4 e BMPRII (r = 0,476; p = 0,034) e do AU em BMP-4 e BMPRIA (r = 0,709; p = 0,022). Os resultados do presente estudo sugerem que as BMPs e seus receptores est?o envolvidos no processo de desenvolvimento de tumores odontog?nicos. A BMP-4, por sua vez, al?m de estar presente em tumores odontog?nicos possui a capacidade de forma??o de material mineralizado

Made available in DSpace on 2014-12-17T15:32:27Z (GMT). No. of bitstreams: 1 CristinaRFA.pdf: 1625942 bytes, checksum: 99d50db14437c5ea0e3022e4da576321 (MD5) Previous issue date: 2009-03-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Bone morphogenetic proteins (BMPs) are cytokines involved in proliferation and angiogenesis of many kind of human cancer. The present study analyzed the immunohistochemical expression of BMP-2, BMPR-II, BMPR-IA and endoglin (CD105) and their relationship with the biological behavior and local angiogenesis in tongue oral squamous cells carcinoma (SCC). The sample consisted of 25 cases of tongue SCC without metastasis, 25 tongue SCC with metastasis and 25 cases of Inflamatory Fibrous Hyperplasia (IFH).The histological grade of malignancy proposed by Bryne (1998), adapted by Miranda (2002) was used to classify all tongue SCC cases. Score 0 was attributed to absent-weak immunoexpression and score 1 for strong immunostaning and pattern of distribution was focal or diffuse. Microvessel counts (MVC) was established for CD105. Most of the patients with tongue SCC was male. The principal age in tongue SCC without metastasis was over 65 years and in tongue SCC with metastasis was between 45-65 years. There were predominance of stage II in TNM and in the specimens with high-grade, independent of studied group. For BMP-2, 56% of tongue SCC without metastasis and 72% tongue SCC with metastasis exhibited score 1 while the IFH showed secore 0 in 72% of the cases, with statistical association (p=0,007). Considering the BMPR-II, 52% of tongue SCC without metastasis exhibited score 0; 56% tongue SCC with metastasis and 60% IFH showed score 1. The majority cases of BMPR-IA demonstrated score 1 and 100% of CD105 exhibited strong immunoexpression in tongue SCC. Regarding the pattern distribution, it was noted a tendency to diffuse pattern for the proteins in all groups. The means of MVC were similar in tongue SCC without metastasis (32,91) and in tongue SCC with metastasis (32,05), however existed statistical difference with IFH (p<0,001). There was statistical association of BMP-2 expression with BMPR-II (p=0,008), BMPR-IA (p=0,006) and CD105 (p=0,046). An association between TNM and BMP-2 immunoexpression and their receptors was not detected, nevertheless this association was found with MVC (p=0,047) whose averages were higher for the stages II (35,97) e IV (35,69). No association between histological grading and these proteins was observed. This study suggests that the superexpression of BMP-2 signaling pathways acts on cell proliferation in tongue SCC and can be implicated with more invasive potential. Additionaly, the CD105 is a potent biological marker of neovascularization in this neoplasm and their association with BMP-2 and BMPR-IA receptor, showed that this type of cancer in BMP-2 is presented as pro-angiogenic in the metastatic process / As BMPs (prote?nas morfogen?ticas ?sseas) s?o citocinas relacionadas com a prolifera??o e angiog?nese em diversos tipos de c?ncer humano. Com este trabalho foi analisada a express?o imunoistoqu?mica das prote?nas BMP-2, BMPR-IA, BMPR-II e endoglina (CD105), correlacionando-a com o comportamento biol?gico e a angiog?nese local nos carcinomas epiderm?ides de l?ngua (CEL). A amostra foi composta de 25 casos de CEL sem met?stase (CELSM), 25 CEL com met?stase (CELCM) graduados segundo Bryne (1998) e adaptado por Miranda (2002), al?m de 25 casos de hiperplasia fibrosa inflamat?ria (HFI), utilizado como grupo controle. Foi utilizado escore 0 para marca??o ausente-fraca e 1 para forte; tipo de distribui??o focal ou difuso. Adicionalmente, para o CD105 foi realizada a contagem microvascular (MVC). A maior parte dos pacientes com CEL foi do sexo masculino, no grupo CELSM a faixa et?ria foi maior que 65 anos e o CELCM se encontrou entre 45-65 anos; houve predom?nio do est?gio II do TNM, assim como de esp?cimes de alto grau, independente do grupo estudado. Para BMP-2, 56% dos CELSM e 72% dos CELCM exibiram escore 1, enquanto a HFI exibiu 72% de escore 0, apresentando associa??o estat?stica (p=0,007). Para BMPR-II 52% dos CELSM exibiram escore 0; 56% CELCM e 60% da HFI escore 1 e no BMPR-IA ocorreu uma predomin?ncia de escore 1 e para o CD105 100% de marca??o forte nos CEL. Quanto ao tipo de distribui??o notou-se tend?ncia de distribui??o difusa de todas as prote?nas, em todos os grupos. Observaram-se, para MVC, m?dias muito semelhantes entre os CELSM (32,91) e os CELCM (32,05) exibindo, contudo, diferen?a estat?stica com as HFI (p<0,001).Observa-se uma associa??o estat?stica da BMP-2 com a BMPR-II (P=0,008), BMPR-IA (p=0,006) e o CD105 (0,046). N?o se observou associa??o entre o TNM e a imunoexpress?o da BMP-2 e seus receptores, por?m foi encontrada com a MVC (p=0,047), cujas maiores m?dias foram para os est?gios II (35,97) e IV (35,69), tal como n?o ocorreu associa??o entre a grada??o histol?gica e as prote?nas. Conclui-se que a superexpress?o da via de sinaliza??o da BMP-2 atua na prolifera??o celular, contribuindo para maior invasividade do CEL. O CD105 ? um potente marcador de neovasculariza??o deste neoplasma e sua associa??o com a BMP-2 e o receptor BMPR-IA, mostra que neste tipo de neoplasia a BMP-2 se apresenta como pr?-angiog?nico no processo metast?tico