Staphylococcus aureus infections have become a widespread problem. Simvastatin decreases S. aureus invasion. Simvastatin use reduces prenylation of target proteins, including CDC42. Prenylated CDC42 is active at the cell membrane. Our hypothesis is that CDC42 activity at the cell membrane is needed for endocytic S. aureus invasion. The prenylation site on CDC42 was deleted and mutant CDC42 (CDC42C5O7V/V5) was transfected into mammalian cells, which were exposed to S. aureus. Decreased bacterial infection of up to 90% was seen in cells stably expressing CDC42C507V/V5. Mammalian cells were treated with secramine A, an inhibitor of CDC42 activity, and exposed to S. aureus. Decreased bacterial invasion of 70% in these cells was seen. These findings suggest that CDC42 activity at the cell membrane is needed for S. aureus cell invasion. These findings increase understanding of the mechanism of S. aureus cell invasion and could be used to develop new treatment or prevention methods. / Department of Biology

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

The osteoclasts, the bone cells responsible for bone degradation, have a crucial role in the age-related bone loss and post-menopause osteoporosis. Rac1 and Rac2, members of the Rho-family of small GTPases, are known for having a key role in osteoclast formation and activity, which could be translated to bone quality. In this study, we characterize the roles of Rac1 and Rac2 on bone quality using an aged and osteoporotic mouse model. Bones from wild type, Rac1KO and Rac2KO mice were harvested for mechanical tests, bone densitometry, micro-computed tomography and histomorphometric analyses to evaluate bone mineralization and architecture. We observed that the deletion of Rac1 or Rac2 in pre-osteoclasts minimized bone loss in both age-related and post-menopause osteoporosis. These results highlight the importance of the two small GTPases in bone remodeling and identify Rac1 and Rac2 as potential targets for the development of new therapies for the treatment of osteoporosis.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Thesis (Ph.D.)--Ohio University, March, 2009. / Release of full electronic text on OhioLINK has been delayed until April 1, 2011. Includes bibliographical references (leaves 99-108)

Thesis (Ph.D.)--Ohio University, March, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until April 1, 2011. Includes bibliographical references (leaves 99-108)

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 148-158).

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Ma thèse porte sur l'étude des rôles régulateurs des GTPases Rho et de leurs effecteurs ROCK sur l'expression de ligands du système immunitaire, sur des cellules de mélanomes murins et humains ainsi que les conséquences sur le développement tumoral de modulateurs de la voie RhoA/ROCK. A l'heure actuelle, les traitements du mélanome métastatique ont une efficacité limitée, c'est pourquoi les nouvelles stratégies s'orientent vers l'immunothérapie notamment en recherchant de nouvelles molécules pharmacologiques capables d'amplifier les réponses immunes anti mélanome. Mon travail a porté sur l'étude de trois ligands de l'immunité modulés par la voie RhoA/ROCK : - Nous avons étudié la régulation du ligand MICA qui est exprimé sur des mélanomes humains, mais qui est reconnu par les cellules NK humaines et murines du système immun inné. En utilisant des statines, qui sont des inhibiteurs de l'activité des Rho, nous avons induit une surexpression membranaire de MICA sans toxicité cellulaire. Cette surexpression s'accompagne d'une sensibilisation des mélanomes à la lyse par les cellules NK. Elle induit également un ralentissement de leur croissance tumorale sous-cutanée en souris NMRI nu/nu et une diminution de l'implantation des métastases pulmonaires. Nous avons aussi montré que cette régulation de MICA induite par les statines ne dépendait pas de l'inhibition des GTPases Ras ou Rho mais de la voie de PPAR?. - Nous avons ensuite étudié la régulation de la molécule de costimulation CD70 par les GTPases Rho sur des mélanomes humains et son rôle dans ces tumeurs. Nous avons montré que les mélanomes primitifs expriment CD70, que cette expression diminue au cours de la maladie et que la GTPase RhoA et la voie des MAPK contrôlent positivement l'expression de CD70 sur nos lignées de mélanome humain. De façon surprenante, nous avons aussi montré que CD70 possède une fonction non immunologique dans ces tumeurs. En effet, la trimérisation de CD70 favorise l'invasion tumorale et l'apparition de métastases en activant la voie de signalisation BRAF/MEK/ERK/RhoE et en inhibant les fibres de stress d'actine et des points focaux d'adhésion. - Enfin, nous nous sommes intéressés aux conséquences de la modulation de FasL sur le développement tumoral du mélanome murin B16F10. Des travaux précédents de l'équipe ont montré que la protéine RhoA et ses effecteurs ROCK régulent de façon négative l'expression de FasL à la membrane des cellules B16F10. Nous avons étudié le rôle in vivo de la surexpression de FasL induite par l'inhibition de ROCK par le H1152. Nous avons mis en évidence un ralentissement de la croissance tumorale in vivo chez les souris immunocompétentes. Ce contrôle du développement tumoral est dépendant de la voie Fas/FasL et de l'activité des lymphocytes TCD8+ et de l'IFN-?. De plus, l'inhibition de ROCK réduit le nombre de métastases pulmonaires sans intervention de la réponse immune adaptative. L'ensemble de mes travaux montre que le ciblage de la voie des GTPases Rho et de leurs effecteurs ROCK constitue une approche nouvelle pour amplifier les réponses immunes protectrices innées et adaptatives anti mélanome, suggérant que des inhibiteurs de cette voie pourraient être envisagés dans de nouveaux protocoles d'immunothérapie du mélanome / My thesis focuses on the study of the regulatory roles of Rho GTPases and their effectors ROCK on the expression of immune system ligands in murine and human melanoma cell lines and the impact on tumor development of modulators of the RhoA/ROCK pathway. Current therapies for metastatic melanoma have poor efficiency. It is the reason why new immunotherapeutic strategies are developed for to find new pharmacological molecules that could improve anti-melanoma immune responses. My work is based on the study of three immune ligands: - We studied the regulation by Rho GTPases of MICA ligand expression in human melanoma cell lines and their recognition by NK cells. Using statins, inhibitors of Rho GTPases activity, we have induced MICA over-expression without any cell toxicity. This MICA over-expression enhanced melanoma cells sensitivity to NK cells lysis, then reduced subcutaneous tumor growth in NMRI nu/nu mice and also decreased pulmonary metastases implantation. We also showed that statins-induced MICA over-expression was not linked to Ras or Rho GTPases inhibition but to PPAR? pathway. - Then, we studied the expression and the function of a co-stimulatory molecule, CD70, and its regulation by the Rho pathway in human melanomas. We demonstrated that the RhoA GTPase and MAPK pathway positively regulate CD70 expression in our melanoma cell lines. Surprisingly, we observed a non-immunological function of CD70 in melanoma. Indeed, CD70 trimerization enhanced melanomas invasion and metastatic capacities through an activation of BRAF/MEK/ERK/RhoE pathway, which inhibited stress fibers and focal adhesions. - Finally, we analyzed the consequences of FasL over-expression on B16F10 murine melanoma development in vivo. Our previous studies have showed that RhoA/ROCK pathway negatively regulates membrane FasL expression on B16F10 cells. We studied in vivo the role of this FasL over-expression induced by ROCK inhibitor H1152, on melanoma cells. We showed tumor growth shrinkage in immunocompetent mice, when B16F10 cells were pretreated with H1152. The Fas/FasL pathway and the activity of TCD8+ cells and IFN- ? induced this tumor slowing down. Moreover, ROCK inhibition induced a reduction of pulmonary metastases implantation independently of T lymphocytes response. Altogether, my work showed that targeting Rho GTPases/ROCK pathway could be interesting in order to improve innate and adaptative anti melanoma immune responses, suggesting that inhibitors of this pathway could be envisaged in new melanoma immunotherapy protocols

RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol (DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras ERK pathway in response to B cell receptor (BCR) signaling. By introducing point mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺- interaction loop in the first EF-hand is required for RasGRP1 translocation and the consequential activation of the Ras-ERK pathway in response to BCR signaling. However, RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not required for Cl domain-mediated membrane localization, but were needed for PT-mediated plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane localization by repressing the SuPT domain. The REM and GEF domains, which co ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras, the REM-GEF domain complex suppressed both plasma membrane and endomembrane targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed in systemic lupus erythematosus (SEE) patients, was examined. This splice variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane localization that was partially overridden by deletion of SuPT, while membrane localization control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via alternative splicing appears to functionally disable the first EF-hand of RasGRP1. / Medicine, Faculty of / Medical Genetics, Department of / Graduate