Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Novel therapies and delivery methods directed against malignancies such as melanoma, and particularly metastatic melanoma, are needed. The HIV-1 accessory protein Vpr (viral protein R) has previously been demonstrated to induce G2 cell cycle arrest as well as in vitro growth inhibition/killing of numerous tumor cell lines. In vivo electroporation has been utilized as an effective delivery method for pharmacologic agents as well as DNA plasmids that express "therapeutic" proteins and has been targeted to various tissues including malignant tumors. In this study, we assessed the ability of electroporation-mediated delivery of Vpr plasmid (pVpr) to induce growth attenuation or complete tumor regression in C57BL/6 mice with subcutaneous B16.F10 melanoma lesions. To assess the administration of intratumoral delivery of pVpr with in vivo electroporation, a range of Vpr plasmid dosages, electroporation parameters, and treatment days were evaluated in a subcutaneous B16 murine melanoma model. pVpr was injected directly into the tumors. Immediately following the injection, the subcutaneous tumors were electroporated. Treatment with 25 microgram or 100 microgram of pVpr plus electroporation on days 0 and 4 resulted in complete tumor regressions with long-term survival in 14.3% and 7.1% of the mice, respectively. In order to optimize the treatment regimen, B16 tumors were treated on days 0, 2, and 4 with 100 microgram pVpr plus electroporation which resulted in 50% of the mice with complete tumor regressions and long-term survival. Additional investigations revealed intratumoral Vpr expression and demonstrated that apoptosis was the mechanism by which Vpr caused tumor regression in vivo. This study confirmed that treatment with 100 microgram of pVpr plus electroporation led to durable complete regressions in established murine melanoma lesions. The pVpr plus electroporation treatment regimen has induced complete regressions in mice as well as resistance to tumor challenge in some of the animals. This is the first comprehensive study demonstrating the ability of Vpr, when delivered as a DNA expression plasmid with in vivo electroporation, to induce complete tumor regressions coupled with long- term survival of mice in a highly aggressive and metastatic solid tumor model.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 91-99). Also available online.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 92-115).

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset, progressive neurodegenerative disease. A familial form of ALS, autosomal dominant ALS8, is characterized by a mutation in an ER membrane protein, VAPB. To characterize the role of VAPB in motor neurons, two C. elegans models were generated: one expressing human VAPB-P56S and another with the knockdown of C. elegans VAPB ortholog, VPR-1. Overexpression of human VAPB in DA neurons caused backward locomotion defects, enhanced vulnerability to oxidative stress and premature neuronal death. Knockdown of vpr-1 in C. elegans recapitulated the loss of protein function believed to be associated with human cases of ALS8. It caused backward locomotion defects, such as uncoordination and slowed rates of movement, as well as age-dependent motor neuronal death. In both models, DA6 and DA7 were the most vulnerable motor neurons. Because of the unexpected developmental defects associated with the VAPB transgenic model, the knockdown of vpr-1 may be a better model to recapitulate the human disease. This model provides further support that ALS8 pathogenesis is due to a loss of VAPB protein function and can also be used to test drugs or treatments that may delay the onset of neuronal death.

Human immunodeficiency virus type 1 (HIV-1) is the causative virus for acquired immunodeficiency syndrome (AIDS). AIDS is characterized by chronic inflammation and reduction of CD4+ T-cells in the blood. This leads to the patient becoming immunocompromised and much more susceptible to disease in general. Different phenotypes in the progression of AIDS have been observed in patients in either progressing to AIDS faster as a Rapid Progressor (RP), or slower as a Long-Term Non-Progressor (LTNP). Researching elements that result in the LTNP phenotype is of interest as it has the potential to offer alternative treatments and therapies to those suffering from HIV and improve their quality of life. A separate genome wide association study into a population of LTNP patients had associated the R77Q mutation of viral protein R with the LTNP phenotype. Although this association has been controversial, recent work has shown that the R77Q mutation promotes apoptosis in a variety of cell lines compared to unmutated virus. However, the mechanisms behind the increase in apoptosis remain a place for further research. In this thesis, we attempted to elucidate some of the exact changes in gene expression between cells infected with the R77Q mutation and those without in the induction of apoptosis. We observed that apoptosis could be detected approximately 24 hours after infection via Annexin V staining, but there were no significant differences in the expression of genes within the first 24 hours. Furthermore, we observed 289 genes were then differentially expressed at 72 hours post infection. Analysis through SPIA revealed that the c-myc transcription factor pathway was activated in the R77Q infected cells and further analysis of the individual genes suggested less inflammatory signals in R77Q populations as well as an overall increase in antiapoptotic genes in WT infected cells. Exploration into the ANT1, Bax, and B-cl2 genes revealed that B-cl2 was upregulated in WT HIV infections and provided a degree of protection from apoptotic processes. Differences found in the expression of genes between R77Q and WT infected cells support the observation of increased apoptosis in R77Q infections and identify several targets for further research into the LTNP phenotype.

No description available.

The HIV protein, Vpr, is a multifunctional accessory protein critical for efficient viral infection of target CD4+ T cells and macrophages. Vpr is incorporated into virus particles and functions to transport the preintegration complex into the nucleus where the process of viral integration into the host genome is completed. This action is particularly important in macrophages, which as a result of their terminal differentiation and non-proliferative status, would be otherwise more refractory to HIV infection. Vpr has several other critical functions including activation of HIV-1 LTR transcription, cell-cycle arrest due to DCAF-1 binding, and both direct and indirect contributions to T-cell dysfunction. The interactions of Vpr with molecular pathways in the context of macrophages, on the other hand, support accumulation of a persistent reservoir of HIV infection in cells of the myeloid lineage. The role of Vpr in the virus life cycle, as well as its effects on immune cells, appears to play an important role in the immune pathogenesis of AIDS and the development of HIV induced end-organ disease. In view of the pivotal functions of Vpr in virus infection, replication, and persistence of infection, this protein represents an attractive target for therapeutic intervention. Numerous studies have reported that Vpr alters NF-kappa B signaling in various cells, however, the findings have so far been largely conflicting with reports both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells and address discrepancies that have been reported in the field. Our results show that Vpr expressed intracellularly is inhibitory to NF-kappa B, while extracelluar Vpr may have some stimulatory effects. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-alpha pathway, but not the LPS pathway, suggesting that multiple targets of Vpr may exist for NF-kappa B regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of I-kappa B-alpha in response to TNF-alpha stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Finally, our findings suggest that NF-kappa B regulation by Vpr is further changed by the presence of other HIV-1 components within the cells, as U1 cells lacking Vpr were unexpectedly less responsive to TNF-alpha than those cells that had normal Vpr expression levels. This data suggests that Vpr may serve an important role in vivo by selectively inhibiting immune activation while stimulating NF-kappa B mediated viral production in HIV-1 infected T-cells and myeloid cells. M-CSF is a cytokine that promotes monocyte differentiation and survival. When over-expressed, M-CSF contributes to pathology in a wide variety of diseases including osteoporosis, obesity, certain human cancers, and in HIV-1 infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1. In this study, our aim is to expand on the current knowledge of M-CSF regulation by NF-kappa B, a prominent transcription factor during inflammation and HIV-1 infection. Our results suggest that TNF-alpha promotes M-CSF secretion in macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-kappa B pathway, diminish this response. We identified four putative NF-kappa B and four C/EBP-beta binding sites within the M-CSF promoter. Our findings using M-CSF promoter constructs mutated at individual NF-kappa B locations suggest these sites are redundant with respect to M-CSF promoter regulation. TNF-alpha treatment promoted NF-kappa B p65 binding to the M-CSF promoter in PMA treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-kappa B response. In conclusion, our findings demonstrate that NF-kappa B induces M-CSF expression on a promoter level via multiple functional NF-kappa B binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages. The oncogenic potential of M-CSF receptor has been has been suggested over thirty years ago, however, few current studies have focused on the role of the receptor in AML. In a clinical trial for AML, Sunitinib was found to hold some efficacy for treating the disease. The authors hypothesized that the primary therapeutic target of Sunitinib in AML is FLT3 kinase. However, FLT3 inhibition alone has not been shown to recapitulate all the effects of Sunitinib in vitro and, furthermore, the drug is also known to have cross reactivity to other potential oncogenic receptors. In this study, we treated three myeloid cell lines, Mono-Mac 1, THP-1 and U937 with Sunitinib and a proprietary cFMS inhibitor from Johnson and Johnson to test the anti-cancer effect in of such treatment. We observed that only Mono-Mac 1 cells had diminished proliferation in vitro. Mono-Mac 1 cells had inhibited ERK as a result of cFMS inhibition and showed a dose dependent increase in cFMS expression with both Sunitinib and J&J cFMS-1 treatment. Our results suggest potential for cFMS as an important target of Sunitinib or other similar drugs AML, either independently or in combination with other targets. Alternatively, cFMS may be a marker for differentiation of AML and may be linked with responsiveness to certain therapeutics. In both cases, the future study of cFMS may produce more targeted therapeutic approaches and may be a suitable tool for the development of personalized medicine for AML. / Biomedical Neuroscience