<p>Many blood cell mechanisms in the human body are working all the time to maintain haemostasis in the blood vessels. Once a wound arises platelets are alerted via different substances to cover the wound and prevent loss of blood. Most of the times these mechanisms do stop the blood, and further heal the wound. During other circumstances the platelet-covering continues to form a thrombus, preventing the blood to flow and instead causes myocardial infarction or stroke. There are several risk factors triggering development of circulatory diseases such as obesity, lack of exercise, smoking, infection and stress.</p><p>This thesis describes the interaction between the two platelet thrombin receptors PAR1 and PAR4, together with the interaction of the oral pathogen <em>Porphyromonas gingivalis</em> (with thrombin-like gingipains), and the cross talk with the stress hormone epinephrine and its α_2A adrenergic receptor. Until now PAR1 is thought to be the most important thrombin receptor due to its high affinity for thrombin. From a phylogenetical and patophysiological point of view there must be a reason why platelets express two different thrombin receptors. Today PAR4 is considered less important, but this thesis implies that PAR4 plays an important role in platelet signaling and haemostasis.</p><p>The results show that bacteria pre-stimulated platelets, followed by epinephrine gives a strong and full aggregation and calcium mobilization, in both aspirinated and non-aspirinated human platelets. The amount of bacteria does not itself, or epinephrine alone give aggregation or calcium mobilization. This mechanism is dependent on both Rgp type gingipain released from <em>P. gingivalis</em>, and PARs in an interaction with the α_2A adrenergic receptor.</p><p>Further, results reveal that PAR4 interacts and cross talks with the platelet α_2A-adrenergic receptor in <em>aspirinated </em>platelets. Neither of the two platelet purinergic P2Y-receptors (P2Y₁₂ and P2Y₁) contribute to this action, but the purinergic P2X₁ does. In aggregation studies a low dose of PAR4 activating peptide (AP), but not PAR1-AP, followed by epinephrine results in a strong aggregation and in a calcium mobilization. ATP secretion measurements did reveal that ATP was released during epinephrine stimulation, which indicate that ATP and P2X₁ have a key role in this event. By blocking P2X₁ both aggregation and calcium mobilization were abolished, but not by blocking P2Y₁₂ and P2Y₁. Inhibition of PI3-kinase, both epinephrine-induced calcium mobilization and aggregation were significant reduced. In <em>non-aspirinated</em> platelets PAR1 synergizes with the α_2A adrenergic receptor and P2X₁.</p><p>In conclusion, this thesis suggests that PAR4 plays an intriguing and important role in platelets with inactived cyclooxygenase 1.  The results described in this thesis contribute to an increased knowledge of the platelet thrombin receptors.</p>

One of the most common hallmarks of cancer is dysregulation of cellular apoptotic processes. A comprehensive knowledge of the underlying mechanisms of the apoptotic machinery is vital for the identification of new drug targets and the development of innovative agents that stimulate the cell death process in cancer cells. Studies have shown that the lysosomal protease cathepsin D is important in the extrinsic apoptotic pathway stimulated by the death receptor ligands for TNFR1 and FAS, as well as by oxidative stress and the protein kinase C inhibitor staurosporine. To date, the role of cathepsin D in the chemotherapy-induced apoptotic pathway has not been characterized. This project examined the role of the lysosomal protease cathepsin D in chemotherapy-induced apoptosis of HeLa and U937 cells. The data demonstrated that following stimulation of U937 cells with the chemotherapy drug VP-16, cathepsin D was released into the cytosol approximately 4 hours after drug treatment. This release was selective for cathepsin D, as cathepsin B and the lysosomal markers LAMP and â-hexosaminidase were not released into the cytosol following VP-16 treatment. Inhibitors of caspases and cathepsin D had no effect on cathepsin D release, demonstrating that cathepsin D release occurred independently of caspase and cathepsin D activities. Downregulation of cathepsin D expression in U937 and Hela cells using siRNA was found to inhibit cell death resulting from a variety of stimuli, including death receptor ligands, oxidative stress, PKC inhibitors, and importantly, chemotherapy drugs. In addition, U937 and HeLa cells expressing cathepsin D siRNA exhibited delayed cytochrome c release and caspase-3 activation following VP-16 treatment. Moreover, isolated mitochondria from wild-type U937 cells released cytochrome c in response to cytosolic extracts that were treated with cathepsin D, suggesting that cathepsin D acts on a cytosolic factor to induce cytochrome c release. Inhibition of caspases had no impact on cytochrome c release provoked by cathepsin D-cleaved cytosolic extract, demonstrating that caspases are not mediators of cathepsin D-induced cytochrome c release. Taken together, these results demonstrate that cathepsin D is an important component of the apoptotic pathway and that it acts via an intermediary cytosolic factor to promote cytochrome c release and caspase activation during chemotherapy-induced apoptosis.

Zinc is a ubiquitous heavy metal that binds to proteins involved in critical cellular processes. Apart from its necessary role, excessive release of intracellular free zinc (Zn) is neurotoxic under stressed conditions characteristic of ischemic or epileptic neuronal injury. Our earlier results indicated that Zn-induced cell death is exacerbated in neurons compared to supporting neuroglia, suggesting that astrocytes have means to upregulate Zn buffering mechanisms, i.e. the Zn-binding protein metallothionein (MT). The first aim of this dissertation sought to address whether MT can effectively maintain Zn levels at a non-toxic minimum. From these studies, we have identified a dichotomous role for MT protective as a Zn buffering agent and detrimental as an oxidant-labile source for toxic Zn. Previous studies demonstrated the role of Zn as a mitochondrial toxin. Although it has been widely speculated that Zn is taken up by the mitochondrial calcium uniporter, the evidence is not entirely convincing. In the second specific aim, we addressed the specific hypothesis that mitochondrial Zn uptake occurs though the uniporter. Using a novel model involving isolated mitochondria pre-incubated with a Zn-selective fluorophore and attached to glass coverslips, we demonstrated for the first time direct visualization of mitochondrial Zn transport. The third specific aim addresses the importance of mitochondria as dynamic intracellular ATP factories, whose intracellular trafficking is critical for neuronal viability. We hypothesized that elevated Zn would attenuate mitochondrial trafficking. Our results revealed that Zn inhibited mitochondrial movement at pathophysiological levels. Intriguingly, acute activation of phosphotidyl inositol 3-kinase was implicated in both Zn-mediated movement inhibition and toxicity, providing a novel role for this traditionally pro-survival signaling pathway. In summary, this dissertation identifies intracellular targets for Zn-mediated neurotoxicity. We specifically emphasize the relevance of mitochondria as a Zn target under two circumstances which are critically dependent on the Zn concentrations established direct mitochondrial interactions that may involve Zn transport, and indirect mitochondrial interactions that affect intracellular mitochondrial trafficking.

The signal transduction field has recently seen a surge of interest in cascade scaffolding proteins. One of these, the Kinase Suppressor of Ras (KSR), has received a great deal of attention as a scaffold for the Ras/ERK signaling pathway. KSR interacts with both MEK and ERK, and possibly binds to Raf-1 as well. Very little is known about the regulation of KSR; however, it has been determined that membrane association is essential for its function in signal augmentation. KSR shares a high degree of sequence homology to Raf-1, including an almost identical phosphatidic acid binding region (PABR). Previous work in the Romero lab has determined the direct interaction of Raf-1 with phosphatidic acid is critical for its membrane recruitment. The PABR is a 35 amino acid sequence consisting of a poly-basic motif (PBM) flanked by two hydrophobic regions. Neutralization of the two arginine residues in the PBM abrogates the binding of Raf-1 to phosphatidic acid (PA), and consequently disrupts its membrane association. This thesis examines lipid-binding properties of the PABR and their potential role in the traffic and function of KSR. Using peptides corresponding to the PABR and tryptophan fluorescence spectroscopy, the data presented in the first section demonstrate that PA induces a blue-shift in the tryptophan emission spectra of WT KSR PABR, and this shift is specific for PA. The second section explores the cellular consequence of KSR PABR mutation. A KSR protein lacking the arginine residues in the PBM expressed in HIRcB fibroblasts retains its membrane-binding ability, but inhibits MEK and ERK phosphorylation in a dominant negative fashion. The data presented here support the conclusion that, although an intact PABR may not be essential for the membrane localization of KSR, it is essential for proper coupling of the pathway.

Head and neck squamous cell carcinomas (HNSCC) are characterized by upregulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR as well as p42/44 MAPK activation, in part via extracellular release of transforming growth factor alpha(TGF-alpha) by matrix metalloproteinases (MMP). Src family kinases have been reported to be activated by G-protein-coupled receptors (GPCRs) followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using three different Src-specific tyrosine kinase inhibitors (A-419259, PP2 or PD0180970) decreased GRP-induced EGFR phosphorylation as well as MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src transfected HNSCC cells. Invasion and growth assays demonstrated that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor, Marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation was also inhibited by blockade of Src family kinases. Further investigation showed that TNF-alpha converting enzyme (TACE) underwent Src-dependent phosphorylation and translocation to the plasma membrane in a complex with c-Src and the p85 subunit of PI-3 kinase, where it regulated amphiregulin release. In addition, we identified that PDK1 kinase, a downstream target of PI-3 kinase, directly phosphorylated TACE. Knockdown of PDK1 augmented the anti-tumor effects of the EGFR inhibitor erlotinib. These findings implicate PDK1 as a new target in HNSCC and suggest that therapeutic strategies that block PDK1 may improve the clinical response to EGFR inhibitors. Combined targeting of GRPR and EGFR pathway also showed enhanced anti-tumor efficacy by inhibiting cancer cell proliferation, invasion and promoting apoptosis. Overall, these findings show the promises and benefits of combination therapy when targeting EGFR and GRPR pathways in head and neck cancer.

Mitochondria are the primary generators of ATP and are important regulators of intracellular calcium homeostasis. These organelles are dynamically transported along lengthy neuronal processes, presumably for appropriate distribution to cellular regions of increased need such as synapses. The removal of damaged mitochondria that produce harmful reactive oxygen species and promote apoptosis is also thought to be mediated by mitochondrial transport to autophagosomes. Mitochondrial trafficking is therefore important for maintaining neuronal and mitochondrial health while cessation of movement may lead to neuronal and mitochondrial dysfunctions. The demands for mitochondria differ between developing and mature neurons, and separate mitochondrial recruitment signals have been identified in each case. In the first aim, we examined how mitochondrial dynamics are affected by the development of synaptic connections in cortical neurons. We revealed reduced mitochondrial movement and elongated morphology in mature neurons which probably serve to optimize mitochondrial contact with synaptic sites. Synapses require mitochondria to supply ATP and regulate local [Ca2+]i for neurotransmission. The second aim investigated mitochondrial trafficking patterns relative to synaptic sites on axons and dendrites. We demonstrated that synapses are targets for long-term mitochondrial localization and dynamic recruitment of moving mitochondria, and that trafficking patterns are influenced by changes in synaptic activity. We also found that mitochondrial movement in dendrites is more severely impaired by neurotoxic glutamate and zinc exposures than in axons. These findings suggest a mechanism for postsynaptic dysfunction and dendritic degeneration in excitotoxicity. The third aim examined impaired mitochondrial transport as an early pathogenic mechanism in Huntingtons disease. Recent studies indicate that aggregates composed of mutant huntingtin fragments hinder axonal transport by sequestering wildtype huntingtin, cytoskeletal components and molecular motors. Our studies in cortical neurons demonstrated reduced mitochondrial trafficking specifically to sites of aggregates and impeded passage of moving mitochondria by aggregates resulting in discrete regions of mitochondrial accumulation and immobilization. In summary, this dissertation provides new insight into our understanding of mitochondrial trafficking, morphology and distribution in cortical neurons that are developing, synaptically mature, acutely injured, and diseased. We conclude that mitochondrial movement is dynamic in healthy neurons and that injured neurons exhibit different manifestations of impaired movement.

SCREENING ASSAY FOR SELECTIVE ESTROGEN RECEPTOR MODULATORS Elena Sirbu, BS University of Pittsburgh, 2006 Estrogen influences the development and progression of breast cancer and of other types of cancer, such as ovarian and lung cancer. The best strategy for prevention and treatment of estrogen dependent cancers is to selectively block estrogen activity in the affected estrogen dependent tissues. The beneficial role of estrogen in the other tissues should be preserved. One of the most common methods to prevent the harmful effects of estrogen is to block the estrogen receptor signaling. The intense research in the breast cancer treatment and prevention field produced a number of estrogen related compounds. The existing screening assays to test the selectivity and potency of these compounds have major limitations. I propose here the development and validation of a rapid screening assay for selective estrogen receptor modulators. This assay is based on the use of an ERE (estrogen response elements) to drive expression of a fluorescent protein that can be visualized directly in living cells. I presented here the first step in developing the screening assay, the generation and evaluation of two fluorescent clones, ERE-GFP and ERE-DsRed. The clones were introduced in CV-1 cells, together with ER, using transient transfection in order to test whether they are under tight estrogenic control. The cells were further treated with know ER ligands. These results predict that the clones function as expected. A robust signal resulted in the presence of estradiol, while with a pure antiestrogen such as ICI 182,780 resulted in very little red/green fluorescence. The vehicle control (ethanol) also elicited very little response (fluorescence). Further, these clones can be stably integrated in CV-1 cells together with either ER alpha or ER beta in order to develop a high content screening assay for SERMs. The new SERMs identified using this assay can be used eventually in therapy of breast or lung cancers or as hormone replacement. In addition, compounds that differentiate ER¦Á and ER¦Â will be valuable tools to further dissect ER signaling pathways. It is important to know more about coactivator recruitment, gene expression profile or about the response with ER mutations. This will lead to a better understanding of estrogen related cancers and will help designing new therapeutic approaches.

The G protein-coupled receptors (GPCRs) and solute carriers (SLC) are two large families of membrane-bound proteins. The aim of this study was to characterize these two families in terms of evolution and function. The melanocortin (MC) receptors belong to the Rhodopsin family of GPCRs and we cloned the MC4 and MC5 receptors from the rainbow trout, MC3 and MC5 from the spiny dogfish and MCa and MCb from the river lamprey. Pharmacological characterization of the cloned MC receptors demonstrated higher affinity for adrenocorticotropic hormone (ACTH) compared to melanocyte stimulating hormone (MSH) peptides (alpha-, beta- and gamma-MSH). We performed expression analysis with reverse transcription PCR, which showed that the MC4 and MC5 receptors in the rainbow trout are expressed centrally as well as in peripheral tissues. The dogfish MC3 and MC5 receptors were expressed in the brain, while the lamprey MCa and MCb receptors were expressed in the periphery. An extensive tissue localization analysis was performed for the entire family of Adhesion GPCRs in the rat and mouse. Using quantitative real-time PCR (qRT-PCR) we discovered that the majority of GPCRs were expressed either specifically in the CNS or ubiquitously in the CNS and peripheral tissues. We identified all non-olfactory GPCRs in the dog and classified them into Adhesion, Frizzled, Glutamate, Rhodopsin and Secretin families. The dog GPCR repertoire seemed to be more similar to the human repertoire than to the repertoires in rodents. Solute carrier family 25 includes mitochondrial membrane transporters. Using bioinformatics techniques we identified 14 novel members of the SLC25 family, which now has 46 members. We identified orthologs of the novel SLC25 family members in yeast and performed expression analysis of 9 of them with qRT-PCR on a panel containing 30 central and peripheral tissues from the rat. To conclude, this study has expanded our knowledge of the repertoire of genes coding for membrane-bound proteins and provided information about their functional roles.

The aim of this thesis was to determine the role of nucleoside analog activating and deactivating enzymes in nucleoside analog metabolism and resistance development. Nucleoside analogs are anti-cancer drogs and are often used to treat different leukemias, attributably to presence of high levels of nucleoside analog activating enzymes in hematopoietic cells. More recently some of the newer analogs have been used  successfully to treat solid tumors as well. We have used human leukemic cell lines, and isolated cells from patients with leukemia, to investigate the nucleoside analog activating enzymes deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) and some of the deactivating enzymes called 5'nucleotidases (5'-NTs). We have measured mRNA expressions and enzymatic activities and correlated them with the cytotoxic response to nuc1eoside analogs and changes in cell cycle progression. We optimized and evaluated a siRNA-transfection method and decreased the activities of dCK and dGK in two different cell lines in order to find out more about their respective contribution to activation of these drogs. An expression microarray analysis of a nucleoside analog resistant cell line was also performed in order to clarify which genes are involved in development of resistance. We found that expressions and activities of dCK and dGK were not correlated. The enzyme activities of activating and deactivating enzymes changed during cell cycle progression, giving actively proliferating cells a more favorable enzymatic profile with regard to nucleoside analog cytotoxicity. The activities of dCK and dGK could be reduced transiently in leukemic and solid tumor cell lines, thereby confer either resistance or increased sensitivity to nucleoside analogs to variable degrees. Expression microarray analysis was used to evaluate the effect of the transfection method and the specificity of siRNA. We concluded that cells tolerated the transfection weIl without major effects on gene expression, and considered the siRNA used to be specific to its target. An expression microarray experiment on a nucleoside analog-induced resistant cell line revealed a hypomethylating capacity of the drog and induction of fetal hemoglobin and a multidrog resistance efflux pump as a result of the hypomethylation. This pump should not be affected by nucleoside analoges since they are not a substrate of it, and upregulation of the pump unfortunately renders the cells highly cross-resistant to different types of drogs. Our preliminary data supports our theory that it may be upregulated in order to help excrete hemoglobin that otherwise would be toxic to the cells.

($-$)Deprenyl is an irreversible inhibitor of monoamine oxidase B (MAO-B) frequently used as an adjuvant therapy in the treatments of Parkinson's and Alzheimer's Disease (PD and AD). Recent evidence, however, has found that certain deprenyl metabolites are associated with an anti-apoptotic action within some neuronal populations. Interestingly, deprenyl's anti-apoptotic actions do not depend upon the inhibition of MAO-B. Due to a paucity of information surrounding ($-$)deprenyl's ability to spare neurons in vivo, a series of studies were conducted to further investigate this phenomenon in two diverse models of apoptotic neuronal death: hypoxia/ischemia and kainic acid induced excitotoxicity. Results indicated that ($-$)deprenyl reduced delayed neuronal death in a model of unilateral global ischemia/hypoxia within the CA1 CA3 and CA4 subfields of the hippocampus as compared to saline-treated subjects, and that the observed parameters were consistent with an anti-apoptotic action of ($-$)deprenyl, but inconsistent with MAO-B inhibition. Similarly, within the kainic acid model of apoptotic neuronal death, data revealed that ($-$)deprenyl increased hippocampal neuronal numbers compared to saline-matched controls. Furthermore, it was discovered that ($-$)deprenyl treatment could be stopped 14 days following CNS insult by kainate, with evidence of neuronal sparing still present by day 28, and that ($-$)deprenyl treatment can produce functional recovery of normal exploratory behaviour within an open-field paradigm following kainate administration. Given deprenyl's anti-apoptotic actions, it is proposed that ($-$)deprenyl may be beneficial in the treatment of a variety of neurodegenerative diseases where evidence of apoptosis exists, such as PD and AD, by slowing the disease process itself.