Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 137-151).

Techn. University, Diss., 2005--Dresden.

University, Diss., 2006--Giessen. / Zeichendarst. im Sachtitel teilw. nicht vorlagegemäß wiedergegeben.

In title, [beta] is represented by the Greek letter. Copies of author's previously published articles inserted. Errata pages pasted onto back end-paper. Bibliography: leaves 104-137. Studies milk TGF-[beta] and its receptors in the post-natal gut using a rat model to investigate a link between milk TGF-[beta] and the development of the infant gut and gut mucosal immune system. Finds maternal milk may be a major source of TGF-[beta] to the immature gut and may react with receptors on the cells of the mucosal immune system along the gastro-intestinal tract, modulating infant mucosal immune responses in the transition to the post-natal enteral feeding.

BMPs vermitteln ihre zellulären Effekte durch Rekrutierung und Aktivierung von zwei Typen spezifischer, membranständiger Rezeptoren. Die genauen Mechanismen der Rezeptorakivierung und die Komposition eines funktionellen, signalvermittelnden Komplexes auf der Zelloberfläche sind in den letzten Jahren genau untersucht worden. Die dimere Natur aller BMPs, die Promiskuitivität der BMPs sowie der entsprechenden Rezeptoren und die unterschiedlichen Rezeptorkonformationen (PFC, BISC) erschweren jedoch die experimentelle Zugänglichkeit dieser Proteinfamilie. Um den Einfluss der Membranverankerung der Rezeptoren auf deren Affinität zu einzelnen Liganden zu untersuchen, wurden verschiedene Methoden evaluiert, die eine quantitative Kopplung an Plasmamembranen ermöglichten. Die BMP Rezeptorektodomänen wurden u.a. mittels einer lysin-spezifischen Kopplung lipidiert, oder aber als His6-Ektodomänen an membranintegrierte Chelatlipide gekoppelt. / BMPs elicit their cellular functions via recruitment and activation of specific receptor serin/threonine receptor kinases. The precise mechanisms leading to receptor activation and the composition of a functional signal transducing complex on the cell surface has been investigated intensively over the last decades. The dimeric nature of all BMPs, the promiscuity of both, the ligands and the receptors and the different receptor conformations on the cell surface (PFC, BISC) hamper the experimental accessibility of this protein family. To study the membrane anchorage's influence of the receptors on their affinity towards single ligands, different methods were evaluated that enabled us to couple the receptor ectodomains in a quantitative manner to plasma membranes. The BMP receptor ectodomains were, among other techniques, lipidated in a lysine specific way or coupled as hexahistidine fusion proteins to membrane integrated chelating lipids.

Thesis (Ph. D. in Cell Biology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references (p. 98-107).

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains xii, 146 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Recent studies show that CaSR suppresses the malignant phenotype through a variety of pathways that inhibits growth and promotes differentiation. CaSR also promotes cytotoxic response to fluorouracil. These studies, taken together, have led me to formulate the following working hypotheses: (i), CaSR is a robust inducer of differentiation by virtue of its ability to activate and integrate diverse growth and differentiation control signals; (ii), loss of CaSR expression enable cellular escape from CaSR control and (iii), loss of CaSR expression is an underlying mechanism of malignant transformation, progression and drug resistance in colon cancer. Previous studies showed that there are endogenous small subpopulations that do not express CaSR in colon carcinoma cell lines. These cells are highly drug resistant. Indeed, immunocytochemical analyses of CaSR showed that the expression of CaSR in both the CBS and HCT116 colon carcinoma cell lines are heterogeneous. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). In order to further test my hypotheses, the isolation and characterization of CaSR null cells are required. Here, I report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional (EMT) molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while the expression of oncogenic micro RNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. I conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer. I discovered that CaSR null cells, cultured in defined human embryonic stem cell culture medium, can be induced to differentiate and acquire CaSR expression when the medium of the null cells was changed to conventional cell culture medium containing fetal bovine serum. I hypothesize that expression of CaSR can alter the phenotype of the CaSR null cells. The objectives of this study were then three folds: (i), determine if induction of CaSR expression could circumvent the molecular phenotype of the CaSR null cells; (ii), determine if CaSR was required in altering the null phenotype and (iii), determine the underlying mechanism of CaSR induction. I hypothesize that if CaSR is a strong promoter of differentiation, then without CaSR, the constraint exerted by CaSR will not be functional and pathways normally inhibited by CaSR will be activated. I found that induction of CaSR expression led to a more indolent phenotype which includes the acquisition of epithelial morphology, down-regulated expression of cancer stem cell markers, down-regulated expression of thymidylate synthase and survivin and increased sensitivity to fluorouracil. Molecular profiling also revealed that the induction of CaSR expression was linked to a down-regulated expression of EMT molecules, EMT associated transcription factors and oncogenic miRNAs with a concurrent up-regulated expression of tumor-suppressive molecules. With the exception of the cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, was directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. I further report that demethylation of the CaSR gene promoter underlie CaSR induction. I conclude that induction of CaSR expression in CaSR null cells resulted in a more indolent phenotype concurrent with a variety of molecular changes and that these changes (with the exception of stem cell markers) are dependent on the expression and function of CaSR. I further conclude that methylation of the CaSR gene promoter is an underlying mechanism of maintaining the CaSR null phenotype while promoter demethylation is an underlying mechanism responsible for CaSR induction. CaSR null is a phenotype of the rapidly proliferating, undifferentiated crypt stem cells at the base of colonic crypts. Differentiation of crypt stem cells toward the apex of a crypt (in the direction of the lumen), on the other hand, is tightly linked to CaSR expression. What induces CaSR expression as the crypt stem cells migrate up the crypts is unknown. I hypothesize that as the colonic crypt stem cells migrate up the crypt, they become increasingly exposed to the colonic fluid in the lumen and components in the colonic fluid can trigger the induction of CaSR expression. Both Ca2+ and vitamin D are good candidates because either Ca2+ or vitamin D can stimulate CaSR expression in the parental CBS and HCT116 human colon carcinoma cells. Certainly, Ca2+ and vitamin D are not the only components involved in regulating CaSR expression. A variety of minerals in the colonic fluid may also serve as good candidates in the induction of CaSR. Of interest is Aquamin, a calcium-rich mineralized extract from the red marine algae, Lithothamnion calcareum, which has been shown to induce differentiation in colon carcinoma cells and possess chemopreventive properties against colon polyp formation in mice fed a high fat diet. CaSR null cells cultured in defined human embryonic stem cell culture medium were used to test this hypothesis because they offer an in vitro model in determining the triggers and the underlying mechanisms of CaSR induction that may resemble that of the colonic crypt stem cells in vivo. I found that all three agonists (Ca2+, vitamin D and Aquamin) induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental cells which express a heterogeneous mixture of cells with different level of CaSR expression. These agonists also induced CaSR mRNA and protein expression and inhibited cellular proliferation in the homogeneous isolated CaSR null cells. In both cases, Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists in the CaSR null cells resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. Induction of CaSR expression resulted in a down-regulated expression of tumor inducers and up-regulated expression of tumor suppressors in the CaSR null cells. Again, Aquamin was found to be most potent in this regard. Taken together, I conclude that nutrients are good candidate in the induction of CaSR and differentiation in colonic epithelia cells. Similar to CaSR, transforming growth factor β (TGFβ) is also a robust promoter of differentiation in the colonic epithelium. The expression profile of both CaSR and TGFβ in the colonic epithelium is tightly linked to differentiation. Both CaSR and TGFβ expression progressively increases as the undifferentiated crypt stem cells migrate and differentiate toward the apex of a crypt in the direction of the lumen. Similar to the loss of CaSR in cancer cells, loss of TGFβ responsiveness has long been considered an underlying mechanism of early colon carcinogenesis. I hypothesize that there is functional linkage between CaSR and TGFβ function. Human colonic epithelial CBS cells originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer an opportunity to determine the functional linkage (if any) between CaSR and TGFβ. I found that knocking down CaSR expression in the CBS cells abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca2+ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca2+ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR, there was no up-regulation of TGFβ receptor. I conclude that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function. In summary, my research has revealed the important role of CaSR in controlling differentiation. CaSR also function as a robust tumor suppressor. My study clearly discerns the multifarious molecular signaling cascades involved in CaSR function and that methylation and demethylation regulates CaSR expression. My work has also established the importance of CaSR in the chemoprevention of colon cancer. My thoughts in regard to future studies and the potential role that CaSR could play in the management of colon cancer are given in the perspective section of this dissertation.

Le remodelage osseux est un phénomène finement régulé selon la balance de formation et de résorption osseuse. La régulation de la résorption osseuse se fait principalement par l'apoptose des ostéoclastes. Nous avons démontré auparavant que plusieurs facteurs comme TRAIL, FASL et TGF-[bêta]1 induisent l'apoptose des ostéoclastes humains. Par contre, nous ne connaissons pas l'effet de l'OPG sur l'apoptose ostéoclastique en plus de ne pas connaître les mécanismes impliqués dans la mort cellulaire induite par le TGF-[bêta]1 chez les ostéoclastes. Afin de vérifier ces effets, nous avons utilisé une culture primaire d'ostéoclastes humains produite à partir de monocytes de sang de cordon ombilical. Ces ostéoclastes sont différenciés à l'aide du M-CSF et du RANKL. L'OPG est un membre de la famille des récepteurs du TNF. L'OPG est un récepteur soluble pour RANKL et pour TRAIL. Nous avons démontré que l'OPG permettait de diminuer l'apoptose des ostéoclastes cultivés dans un milieu sans M-CSF et sans RANKL. Cette baisse surprenante de la mort a été expliquée par une augmentation d'expression de TRAIL par les ostéoclastes lorsque ceux-ci sont cultivés en absence de ses facteurs de survie (M-CSF et RANKL). L'OPG agit sur l'apoptose en séquestrant le TRAIL produit par les ostéclastes [ostéoclastes] empêchant ainsi l'induction de l'apoptose par le TRAIL des ostéoclastes environnants. Ceci en fait un mécanisme de régulation autocrine de la survie des ostéoclastes lors du remodelage osseux. De plus, nous avons aussi démontré un mécanisme expliquant l'apoptose des ostéoclastes par le TGF-[bêta]1. Nous avons premièrement démontré la présence des récepteurs de type I et II à la surface des ostéoclastes proposant une action directe du TGF-[béta]1. Par la suite, nous avons démontré que le TGF[bêta]1 induisait une activation de la caspase 9 objectivant ainsi une apoptose par la voie mitochondriale. Cette activation de l'apoptose par la voie intrinsèque serait le résultat de l'activation de la voie de p38 des MAPK et de la voie des Smad suivie par une augmentation de l'expression des homologues de Bcl-2 pro-apoptotiques Box et Bim. En sachant que le TGF-[béta]1 peut être libéré et activé de la matrice osseuse lors de la résorption, l'induction de l'apoptose des ostéoclastes par ce facteur de croissance en fait un autre mécanisme de régulation du remodelage osseux.