No description available.

Introduction: The Rho family of small GTPases, including Rac1 and Rac2, are key regulators of osteoclast differentiation and function; however, little is known about their roles in bone quality and aging. Methods: Male four and nine month old mice (n=10) with Rac1, Rac2 or both (DKO) isoforms deleted in osteoclast precursors were assessed using dual energy x-ray absorptiometry (DXA), scanning electron microscopy (SEM), micro computed tomography (microCT), compression, torsion and three-point bending testing, back scattered electron microscopy (BSE), Goldner’s trichrome and TRAP staining. Results: All Rac null mice demonstrated decreased cortical structural properties and improved trabecular architecture. With age, Rac null mice demonstrated the ability to attenuate age-related bone loss. Conclusions: Using an in vivo model with Rac1, Rac2 or both Rac isoforms deleted in osteoclasts, our findings demonstrate the deletion of Rac1 and Rac2 compromised cortical bone while improving trabecular bone properties and attenuated age-related bone loss.

Introduction: The Rho family of small GTPases, including Rac1 and Rac2, are key regulators of osteoclast differentiation and function; however, little is known about their roles in bone quality and aging. Methods: Male four and nine month old mice (n=10) with Rac1, Rac2 or both (DKO) isoforms deleted in osteoclast precursors were assessed using dual energy x-ray absorptiometry (DXA), scanning electron microscopy (SEM), micro computed tomography (microCT), compression, torsion and three-point bending testing, back scattered electron microscopy (BSE), Goldner’s trichrome and TRAP staining. Results: All Rac null mice demonstrated decreased cortical structural properties and improved trabecular architecture. With age, Rac null mice demonstrated the ability to attenuate age-related bone loss. Conclusions: Using an in vivo model with Rac1, Rac2 or both Rac isoforms deleted in osteoclasts, our findings demonstrate the deletion of Rac1 and Rac2 compromised cortical bone while improving trabecular bone properties and attenuated age-related bone loss.

Indiana University-Purdue University, Indianapolis / This dissertation examines the molecular mechanisms regulating Rac2 gene expression during cell differentiation and identification of a minimal cis-element required for the induction of Rac2 gene expression during K562 cell differentiation. The Rho family GTPase Rac2 is expressed in hematopoietic cell lineages and is further up-regulated upon terminal myeloid cell differentiation. Rac2 plays an important role in many hematopoietic cellular functions, such as neutrophil chemotaxis, superoxide production, cytoskeletal reorganization, and stem cell adhesion. Despite the crucial role of Rac2 in blood cell function, little is known about the mechanisms of Rac2 gene regulation during blood cell differentiation. Previous studies from the Skalnik lab determined that a human Rac2 gene fragment containing the 1.6 kb upstream and 8 kb downstream sequence directs lineage-specific expression of Rac2 in transgenic mice. In addition, epigenetic modifications such as DNA methylation also play important roles in the lineage-specific expression of Rac2. The current study investigated the molecular mechanisms regulating human Rac2 gene expression during cell differentiation using chemically induced megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 as the model system. Phorbol 12-myristate 13-acetate (PMA) stimulation of K562 cells resulted in increased Rac2 mRNA expression as analyzed by real time-polymerase chain reaction (RT-PCR). Luciferase reporter gene assays revealed that increased transcriptional activity of the Rac2 gene is mediated by the Rac2 promoter region. Nested 5’- deletions of the promoter region identified a critical regulatory region between -4223 bp and -4008 bp upstream of the transcription start site. Super shift and chromatin immunoprecipitation assays indicated binding by the transcription factor AP1 to three distinct binding sites within the 135 bp minimal regulatory region. PMA stimulation of K562 cells led to extensive changes in chromatin structure, including increased histone H3 acetylation, within the 135 bp Rac2 cis-element. These findings provide evidence for the interplay between epigenetic modifications, transcription factors and cis-acting regulatory elements within the Rac2 gene promoter region to regulate Rac2 expression during K562 cell differentiation.

No description available.

B cells can be activated by T cell-dependent stimuli, such as CD40 ligation and cytokines, which induce extensive proliferation, class switch recombination and somatic hypermutation. Epstein-Barr virus (EBV) can also induce B cell activation by mimicking T cell help through its main oncoprotein, latent membrane protein 1 (LMP-1). It is regulated by another EBV-encoded protein, EBV nuclear antigen 2 (EBNA-2), which is absent in Hodgkin and Burkitt lymphomas. We have studied LMP-1 induction by cytokines in vitro and shown that LMP-1 is induced through the transcription factor signal transducer and activator of transcription (STAT6) and a newly defined high-affinity STAT6-binding site. When IL-4 is added together with lipopolysaccharide (LPS) or α-CD40 to B cells, it induces homotypic round and tight aggregates in vitro, whereas LPS alone does not induce such morphological changes. I describe here attempts to identify the molecules that regulate these responses. I have shown that the Rho GTPase Cdc42 controls the spreading of B cells, whereas two other molecules in the same family, Rac1 and Rac2, control homotypic adhesion. Further, I have shown by conditional deletion of Cdc42 in B cells that it is important in the humoral immune response.  Dock10 is a guanosine nucleotide exchange factor (GEF) for Cdc42. It is expressed through all differentiation stages of B cell development. However, targeted deletion of Dock10 in B cells does not result in an aberrant phenotype. Furthermore, by studying conditional knockout mice for Dock10, Cdc42, Rac1 and Rac2, I have elucidated the mechanism of cytoskeletal changes during B cell activation, leading to adhesion and motility. My results may lead to a better understanding of normal B cell activation and of EBV infection, which is associated with many human tumours and may help to understand cancer development and progression in B cells. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.