Matrix metalloproteinases (MMP) are zinc- or calcium-dependent proteinases involved in the normal maintenance of the extracellular matrix. When elevated, MMPs degrade matrix components contributing to tissue destruction in infected periodontal sites. The objectives of this study were two-fold: first to assess the ability of Porphyromonas gingivalis hemagglutinin B (HagB) to induce MMP responses in human myeloid dendritic cells and second, to assess the effect of host defense peptide human β defensin 3 (HBD3) to regulate and attenuate the MMP response of HagB treated dendritic cells. HBD3 (0.2, 2.0, or 20.0 µM) was given to primary dendritic cells pre-, co-, or post-treatment to HagB (0.02 or 0.2 µM). At 16 hours, MMP concentrations were determined. There were no significant differences in concentrations for all 3 replications for MMP-2 and -13. There were few significant differences in some of the replications for MMP-3, -7, and -9. There were more pronounced differences in MMP-1, -10, and -12 expression, which were significantly influenced by both the concentration of HBD3 and the timing of administration. Chemokine and cytokine responses were inversely related to MMP production. While MMP responses decreased in a dose related manner, chemokine responses were increased. Concentrations of MIP-1α were high and there were no differences in response to 0.02 and 0.2 M HagB with or without 20.0 M HBD3. However, the MIP-1β and TNFα response to 0.2 M HagB were only attenuated. HagB induces the production of MMPs in dendritic cells and treatment of dendritic cells with HBD3 can alter the profile of HagB-induced MMPs. Such a finding may have importance in the pathogenesis of periodontal disease.

<p>Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function.</p><p>A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression.</p><p>Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice.</p><p>Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice.</p><p>In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.</p>

Effective antiretroviral therapy (ART) is essential for viral suppression (VS) in HIV-infected patients. However, there is a lack of nationally representative data on types of ART regimens used and their impact on VS. This thesis used self-reported interview and abstracted medical record from 2009 Medical Monitoring Project (MMP) to study ART regimen type and related health outcomes. Results showed that 88.6% of HIV-infected adults in care was prescribed ART, and about half took regimens designated as ‘preferred’ according to U.S ART guidelines. Among MMP participants prescribed ART, 62.7% achieved durable VS, 77.8% achieved recent VS, 83.5% were 100% dose-adherent, and 17.1% reported side effects. Multivariate regression analyses revealed that although ART was critical for VS, there were minor differences in health outcomes among the major ART classes in the U.S. ART guidelines or six most-commonly used regimens. This study could be potentially useful for future strategic planning of HIV care.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

SPARC is a highly conserved trimodular Ca2+- and Collagen-binding matricellular protein with diverse functions during development, wound healing and cancer metastasis. Our lab previously generated an embryonic lethal Drosophila SPARC null mutant, Df(3R)nm136, analysis of which revealed that SPARC was required for the deposition of Collagen IV into basal laminae and normal nervous system development during embryogenesis. In contrast to these previous studies, my data revealed that SPARC is not required for the deposition of Collagen IV into embryonic basal laminae or embryonic nervous system development. Further analysis showed that the Df(3R)nm136 chromosome carried a second-site mutation in the Neuralized locus which caused the nervous system defects and embryonic lethality previously associated with a loss of SPARC. Removal of this second site mutation and reanalysis of the SPARC mutant phenotype revealed that SPARC is required for larval development where it appears to play a role in the regulation fat body remodelling. SPARC mutant fat bodies showed changes in cell shape and basal lamina remodelling which resemble the fat body remodelling process that normally occurs during pre-pupal stages via up-regulation of MMP2 in response to the steroid hormone ecdysone. The effects of loss of SPARC on fat body cells were shown to be cell autonomous. Structure-function analysis of SPARC showed that secretion of SPARC is required for its function, whereas Domain1 is dispensable. Together, my studies indicate that SPARC has essential intra and extracellular roles during Drosophila larval fat body development.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

SPARC is a highly conserved trimodular Ca2+- and Collagen-binding matricellular protein with diverse functions during development, wound healing and cancer metastasis. Our lab previously generated an embryonic lethal Drosophila SPARC null mutant, Df(3R)nm136, analysis of which revealed that SPARC was required for the deposition of Collagen IV into basal laminae and normal nervous system development during embryogenesis. In contrast to these previous studies, my data revealed that SPARC is not required for the deposition of Collagen IV into embryonic basal laminae or embryonic nervous system development. Further analysis showed that the Df(3R)nm136 chromosome carried a second-site mutation in the Neuralized locus which caused the nervous system defects and embryonic lethality previously associated with a loss of SPARC. Removal of this second site mutation and reanalysis of the SPARC mutant phenotype revealed that SPARC is required for larval development where it appears to play a role in the regulation fat body remodelling. SPARC mutant fat bodies showed changes in cell shape and basal lamina remodelling which resemble the fat body remodelling process that normally occurs during pre-pupal stages via up-regulation of MMP2 in response to the steroid hormone ecdysone. The effects of loss of SPARC on fat body cells were shown to be cell autonomous. Structure-function analysis of SPARC showed that secretion of SPARC is required for its function, whereas Domain1 is dispensable. Together, my studies indicate that SPARC has essential intra and extracellular roles during Drosophila larval fat body development.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

Leukemia is a hematological neoplasm with abnormal genetic mutation or chromosomal translocation in the myeloblast or lymphoblast, and characterized by accumulation of immature cells and malfunction of lymphocytes and myeloid-derived cells. The prognosis of treatment depends on genetic mutation, chromosomal aberration, disease progression and age of patients. Currently, bone marrow transplantation is a useful therapeutic strategy, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Although chemotherapy improves leukemia treatment, long-term chemotherapy usually leads to the production of drug-resistant cancer cells. Thus, the development of new modality in overcoming drug-resistant should be beneficial for in leukemia therapy. In this thesis, Naja nigricollis toxin £^, piceatannol, caffeine, and Bungarus multicinctus protease inhibitor-like protein 1 (PILP-1) are employed to investigate the molecular mechanisms in regulating apoptosis and invasion of leukemic cell lines K562 and U937. Hopefully, the signaling pathways elicited by these treatments may be aid in identifying new targets in treating leukemia. Toxin £^ inducing cell death is found to evoke p38 MAPK-mediated Bcl-2 down-regulation, which facilitates mitochondria dysfunction, ROS generation and cytiochrome c release. Finally, activation of caspases leads to apoptotic death of toxin £^-treated cells. Piceatannol elicits Ca2+/p38£\ MAPK- mediated c-Jun and ATF-2 phosphorylation, leading to up-regulation of Fas/FasL protein expression and autocrine Fas-mediated death pathway activation. Caffeine treatment down-regulates MMP-2/-9 down-regulation via Ca2+/ROS-mediated inactivation of ERK/c-Fos and activation of p38 MAPK/c-Jun pathway. Consequently, caffeine treatment suppresses invasion of leukemia cells. PILP-1-induced ADAM17 down-regulation suppresses Lyn-mediated Akt phosphorylation, resulting in death of PILP-1-treated leukemia cells. Taken together, the results of the present study elucidate the signaling pathways responsible for apoptosis and invasion of leukemia cells. Moreover, these findings might suggest new targets in developing therapeutic strategy in treating leukemia.