ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

Dans l'industrie, les poudres de carbure de silicium (SiC) sont élaborées principalement par le procédé Acheson. Durant ce procédé multi-étapes, les poudres subissent diverses opérations pouvant être à l'origine de la dissémination de poussières inhalables dans l'environnement de travail. Jusqu'à présent, la toxicité des poudres de SiC n'a été que très peu étudiée avec des résultats contradictoires soulignant l'intérêt d'évaluer l'activité biologique de poudres de SiC par une approche pluridisciplinaire. Dans une première partie, l'activité biologique de poudres de SiC produites industriellement par le procédé Acheson a été évaluée. Par suite, des modifications de la surface de ces particules par le biais de traitements thermiques oxydants ont permis de mettre en évidence l'influence de la surface des particules dans la réponse cellulaire. Pour finir et compléter l'étude, nous avons étendu le sujet au cas des nanoparticules de SiC, synthétisées par pyrolyse laser et voie sol-gel.<br />Pour évaluer l'activité biologique des particules de SiC, des tests In Vitro sur des macrophages de culture (RAW 264.7) ont été réalisés en étudiant différents domaines de la réponse cellulaire (état d'inflammation, mort cellulaire, stress oxydant) en relation avec les propriétés physico-chimiques des particules (taille, surface, morphologie, structure cristallographique, chimie, état de surface, activité radicalaire).<br />Les résultats, illustrés suivant un modèle vecteur, soulignent que les particules industrielles de SiC sont principalement caractérisées par une activité modérée de l'état inflammatoire, aucun effet cytotoxique et un impact significatif sur le stress oxydant. Des différences en fonction de la taille des particules ont été observées ainsi qu'une forte capacité des particules à générer directement des radicaux libres (HO•, COO•-). Après modification de la surface des particules par traitement thermique oxydant, la réponse cellulaire se caractérise par une forte augmentation de l'état d'inflammation et de la cytotoxicité. Enfin, un stress oxydant significatif est observé avec les nanoparticules de SiC, bien supérieur à celui observé avec les particules industrielles de SiC.

<p>All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. </p><p>We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K.</p><p>The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway.</p><p>ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation.</p><p>Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.</p>

Adipose tissue (AT) expansion and remodeling that maintains healthy function relies on stromal preadipocytes capable of differentiating into new adipocytes (adipogenesis). During chronic positive energy balance, a relative deficit in adipogenesis, from either a decrease in preadipocyte number or their capacity to differentiate, leads to excessive adipocyte hypertrophy and AT dysfunction. AT contains macrophages whose number and activation state is dynamically regulated with changes in AT mass. This study aims to investigate the effect of macrophage-secreted factors on preadipocyte survival. To assess the effect of macrophage-secreted factors on preadipocytes, murine 3T3-L1 preadipocytes or human primary preadipocytes were incubated with macrophage-conditioned medium (MacCM), prepared from either murine (J774A.1, RAW264.7, bone marrow-derived) or human (THP-1, monocyte-derived) macrophage models, respectively. MacCM inhibited preadipocyte apoptosis and activated pro-survival signaling in both preadipocyte models. Inhibition of PDGFR, Akt, or ERK1/2 reduced the pro-survival effect of MacCM in 3T3-L1 preadipocytes. Inhibition of reactive oxygen species (ROS) generation, or enhancement of ROS clearance, reduced MacCM-dependent 3T3-L1 preadipocyte survival. Whereas anti-inflammatory activated macrophages retained the ability to prevent preadipocyte apoptosis, pro-inflammatory activated macrophages did not. TNF-α immunoneutralization restored the survival activity of pro-inflammatory MacCM on 3T3-L1 preadipocytes. These studies reveal a novel pro-survival effect of MacCM on preadipocytes, and identify signaling molecules (PDGF, Akt, ERK1/2, and ROS) that underlie this action. Macrophage activation was found to regulate the pro-survival activity of MacCM. These in vitro cell culture studies are consistent with a model in which the extent of preadipocyte apoptosis in vivo may determine preadipocyte number and the ability of AT to expand while maintaining healthy function during chronic positive energy balance.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K. The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway. ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation. Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.

Candida albicans, a commensal of gastrointestinal and uro-vaginal tract can cause superficial as well as life threatening disseminated infections under conditions of lowered immunity of the host such as HIV infection, drug induced immune suppression [given during organ transplantation to prevent rejection] and radiation therapy [head and neck cancer patients] (Odds, 1988; Fidel and Sobel, 1996). Candida albicans shows a range of morphologies, it can switch from budding yeast morphology to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and hyphae (linear filaments without visible constrictions at the septa) (Mitchell, 1998). The various factors that contribute to its virulence include its ability to undergo yeast to hyphal transition, formation of biofilms, adhesion and secretion of aspartyl proteinases. Hyphae are considered to be involved in invasive growth as they are frequently identified in infected tissues and strains defective in morphological transition (yeast to hyphal) are avirulent (Leberer et al., 1996; Lo et al., 1997; Stoldt et al., 1997). Morphological switching is not only necessary for successful establishment of infection but important for evading components host defense system like macrophages or dendritic cells. A network of signaling pathways that operate in C. albicans continuously assess the nutrient availability, cell density and other environmental conditions. The integrated output of these pathways determine the response of C. albicans under given set of environmental/media conditions and eventually determines the gene expression and morphogenic transition (Liu., 2001). C. albicans utilizes at least two major signaling pathways besides others for regulating the morphological transition. One of these two pathways uses Cph1 as transcription factor and is the homolog of Ste12 in S. cerevisiae which is shown to be involved in Pseudohyphal growth and mating. The other pathway includes Efg1 (homolog of Phd1 in S. cerevisiae) as transcription factor. Biofilm formation by Candida species is an important virulence factor and has gained considerable interest recently as these specialized survival structures are found in implanted devices such as indwelling catheters and prosthetic heart valves (Hawser and Douglas, 1994; Douglas, 2003). These biofilms lead to the failure of implants besides providing multiple drug resistance (Baillie and Douglas, 1999). A better understanding of the C. albicans interaction with the host at the site of infection and with the components of immune system will help in identifying new potential drug targets. (a) Genome wide expression profile of Candida albicans from patient samples and characterization of CaRPB4/7: To get a better insight in C. albicans response at the site of infection we were interested in mapping the expression profile of Candida albicans in active state of human infections. Patients suffering from head and neck cancer undergoing radiation therapy have high risk of C. albicans infection. We identified five such patients with heavy oral thrush infections and C. albicans samples were collected from them. Candida albicans was confirmed in these samples by various microbiological tests following which the samples were used for RNA isolation. The whole genome expression analysis leads to the identification of 188 up regulated and 88 down regulated genes in patient samples. Our data analysis revealed that Protein Kinase A pathway and many downstream genes of the same were differentially expressed. Analysis of saliva (saliva is known for antifungal and antibacterial activity) from these patients showed that unlike healthy individuals, the patient saliva favours yeast to hyphal transition of C. albicans cells. This might be a reason for high risk of infection. A major class of upregulated genes is found to be functionally involved in transcription which includes some RNA polymeraseII and III subunits. CaRPB4, the forth largest subunit of RNA polymeraseII, was found to be upregulated in patient samples. RPB4 has been shown to form sub complex with RPB7, the seventh largest subunit of RNA polymeraseII, and both subunits are known to play a role in a variety of stress conditions and pseudohyphal development in Saccharomyces cerevisiae. We characterized the CaRPB4 and CaRPB7 (homolog in Candida albicans) for their ability to complement their S. cerevisiae counterparts. CaRPB4 and CaRPB7 were able to complement majority of the phenotypes associated with these subunits in S. cerevisiae. Overexpression of CaRPB7 in S. cerevisiae enhances pseudohyphal growth. Considering the high degree of conservation of signaling pathways between S. cerevisiae and C. albicans it can be speculated that CaRPB7 might be involved in pseudohyphal development in C. albicans. We found that over expression of CaRPB4 in Candida albicans shows enhanced agar invasive growth which can be thought analogous to tissue invasion in host and hence might contribute for establishment of infection. This suggests that both the RNA polII subunits have a role to play in the virulence of C. albicans. (b) Characterization of UDP-Galactose 4-Epimerase (GAL10) from Candida albicans and their role in virulence. Enzyme UDP-Galactose-4-Epimerase [GAL10] is responsible for conversion of UDP-galactose to UDP-glucose which then gets metabolized by the cells through glycolysis and TCA cycle. The enzyme catalyzes a reversible reaction and can convert glucose to galactose in the absence of galactose as shown in Trypanosoma brucei and also involved in its virulence. In this study, we have identified the functional homolog of GAL10 in Candida albicans. S. cerevisiae and C. albicans GAL10 homologs are similar in their domainal organization as the proteins have a mutarotase and an epimerase domain. The former is responsible for conversion of ﾟ-D-galactose to a-D-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. The synteny of galactose metabolizing structural genes is conserved among some fungi. To study the importance of CaGAL10 we generated deletion mutant of the gene in C. albicans. Our studies show that CaGAL10 [C. albicans GAL10] is involved in cell wall organization and in oxidative stress response. The mutant strain of GAL10 is hyperfilamentous in Lee’s and spider medium and the biofilm formed is morphologically different from the wild type strain. These set of results suggests that CaGAL10 plays an important role in organization/integrity of cell wall in C. albicans and speculate that it might be involved in virulence. (c) Study of Candida albicans-macrophage interaction and identification of transcriptional regulator of genes encoding proteins of translation machinery: Macrophages serve as the effector cells of cell mediated immunity in the control of infections. They are considered to be important for resistance to muco-cutaneous and systemic candidiasis. Our studies were aimed at understanding the response of Candida albicans cells to the presence of macrophages for extended period of time. The response was monitored using microarrays. Specifically genes involved in galactose, protein and lipid metabolism and stress response undergo concerted changes in their transcript levels. We analyzed the promoters of coregulated genes to identify common DNA elements present in them which might be involved in their transcriptional regulation. Promoter analysis of differentially expressed genes revealed presence of CPH1 and EFG1 transcription factor binding sites. Besides identifying CPH1 and EFG1 Binding sites, we identified two novel DNA elements in promoters of coregulated gene. A conserved motif TGAAAAGGAAG was identified in the promoters of genes involved in energy generation. Another 18 mer consensus palindromic sequence TAGGGCTNTAGCCCTAAT was identified in the promoters of about 48 genes. Majority of these genes encode ribosomal proteins. With the help of techniques like EMSA (Electophoretic Mobility Shift Assay) and south-western we had shown the presence of a protein of ~66 KDa molecular weight binding to the sequence with high specificity.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 139-154).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.