Histidine-rich glycoprotein (HRG) is a heparin-binding plasma protein modulating immune, hemostatic and vascular functions. I have studied the antiangiogenic functions of HRG in vitro and in vivo in order to understand the molecular mechanisms of action of HRG as an angiogenesis inhibitor. Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is a central rate-limiting step of tumor development and thus a possible target for cancer therapeutics. Previous studies have shown that HRG has antiangiogenic functions in vivo and that the antiangiogenic effects are mediated via the proteolytically released His/Pro-rich domain of HRG. In this thesis we demonstrate that HRG can inhibit endothelial cell migration by interfering with focal adhesion and cytoskeletal turnover. Moreover we have identified the minimal active domain of HRG, a 35 amino acid peptide derived from the histidine- and proline-rich domain of HRG. Analyzing human tumor tissue samples, we have found that a His/Pro-rich fragment of HRG is bound to the vasculature from cancer patients but not to the vasculature from healthy individuals. The fragment is found in association with platelets, and we show that activated platelets can induce a functional microenvironment for the His/Pro-rich fragment. Cancer patients often display an increased coagulation and our data describe a new mechanism to confer specificity of an angiogenesis inhibitor for situations with enhanced platelet activation, as in the tumor. We have further studied the role of HRG in tumor growth by crossing HRG-deficient mice with a transgenic mouse model of pancreatic insulinoma. We show that mice lacking HRG display an elevated “angiogenic switch” and that the total tumor volume is larger in these mice than in wild type mice. HRG is also involved in regulation of platelet function and platelets can stimulate angiogenesis in various ways. We have depleted mice of platelets to study the possible connection between the function of HRG in angiogenesis and platelet regulation. Our data suggest an involvement of platelets in the antiangiogenic activities of HRG.

Motivation: Hypertension is a fast growing health risk, leading to increased incidences of cardiovascular dysfunction and mortality. However, the prevalence of hypertension is higher in some ethnic populations than others. Several South African studies have found that the African population is more susceptible to the development of hypertension, compared to the Caucasian population. Cardiovascular dysfunction is often accompanied by elevated levels of uric acid (UA) and plasminogen activator inhibitor-I (PAI-1) and both are factors associated with the metabolic syndrome. A lack of data regarding the association of UA and PAL1 with cardiovascular dysfunction in a South African context, serves as a motivation for conducting this study. Objective: To determine the association of UA and PAI-1 with cardiovascular dysfunction in African and Caucasian women from South Africa. Methodology: The manuscript presented in Chapter 2 made use of the data obtained in the POWIRS (Profiles of Obese Women with the Insulin Resistance Syndrome) study. A group of 102 African women and 115 Caucasian women, living in the North West Province of South Africa, were recruited according to their body mass indexes. The groups were divided into lean, overweight and obese according to their body mass index. Anthropometric and cardiovascular measurements were taken and determinations were done of their blood lipid profiles, UA. PAI-1, fasting insulin and glucose levels, HOMA-IR (homeostasis model assessment-insulin resistance) and leptin levels. The subject's total dietary protein intake was determined by means of a dietary questionnaire. Comparisons between the groups were done using an independent t-test as well as a multiple analysis of covariance (MANCOVA) whilst adjusting for certain variables. Each ethnic group was divided into UA and PAI-1 tertiles, for comparison between the 1" and 3' tertiles. Correlation ~0efIi~ientS were determined to show any associations between UA and PAI-1 with cardiovascular variables as well as variables associated with the metabolic syndrome. Forward stepwise multiple regression analyses were performed using UA and PAL1 respectively as dependent variables. The study was approved by the Ethics committee of the North-West University and all the subjects gave informed consent in writing. The reader is referred to the experimental procedure section in Chapter 2 for a more detailed description of the subjects, study design and analytical procedures used in this dissertation. Results and conclusion: Results from the POWIRS-study showed that despite the African women's higher blood pressure, they had significantly lower levels of UA and PAI-I compared to the Caucasian women. Although the Caucasian women had significantly higher circulating levels of UA and PAI-1, they showed no sign of cardiovascular dysfunction. The detrimental effects might, however, become more noticeable with an increase in age. From this study it is concluded that UA and PAL1 is not associated with the increased blood pressure in young African women. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.

Laboratory tests have been performed using a stirred cell where SI and SII gas hydrates have been formed under the presence of the kinetic inhibitor Poly Vinyl Caprolactam (PVCap) and INHIBEX. The latter is a mixture containing 50wt% PVCap 2k and 50wt% butyl glycol. The effect of PVCap is enhanced by the presence of butyl glycol; the latter acts as a synergist for the former. Dissociation temperatures were obtained and compared for hydrates formed 1) in presence of PVCap and 2) in presence of INHIBEX. The effect of INHIBEX concentration on the temperature of dissociation was also investigated. Systems containing INHIBEX dissociated at lower temperatures than the corresponding systems with only PVCap present. Furthermore, 3000 ppm INHIBEX mixtures were found to have higher dissociation temperatures than 1500 ppm INHIBEX mixtures.

The chemical structure of polyether amines (PEA), mainly electron donating multiple oxygen and nitrogen atoms as well as active hydrogen atoms, make such compounds actively participating in the formation of hydrogen bonds with surrounding molecules. Hydrophobic polypropylene glycol functionality gives PEA's properties of multi-headed surfactants having hydrophilic amine groups. These groups have a strong affinity for water molecules, ice and hydrate crystals. Such PEA compounds have been known for several years. However, the hydrate inhibition properties of PEA’s were only discovered about twelve years ago. The first discovery stimulated more research in laboratories and led to practical applications for hydrate inhibition in gas fields. An interesting property of PEAs is their synergistic effect on hydrate inhibition when applied concurrently with polymeric kinetic hydrate inhibitors (KHI) or thermodynamic inhibitors (THI). The combination inhibitors are better inhibitors than a single component one. Quaternized polyether diamines are efficient antiagglomerant (AA) hydrate inhibitors while different derivatization can produce dual functionality compounds, i.e. corrosion inhibitors/gas hydrate inhibitors (CI/GHI). With all of this versatility, PEAs found application for hydrate inhibition in oil and gas fields onshore and offshore in production, flowlines and completion. The PEAs have an excellent record in protecting gas-producing wells from plugging with hydrates. (Final corrected copy of ICGH paper 5347)

The inhibitor and steam injection methods have been examined using a laboratory-prepared methane hydrate bearing sediment. New experimental apparatuses have been designed and constructed. In the case of inhibitor injection, the measurement of gas production vs. time suggested that the inhibitor increased dissociation rate. Core temperature decreased upon the inhibitor injection, in contrast to that in the case of pure water injection. The observed pressure differentials between the inlet and outlet of the core sample suggest that the inhibitor effectively prevented the hydrate reformation within the dissociating core sample. In the case of steam injection coupled with depressurization, it can be seen that the effect of steam (or hot water) injection was clear in the later stage of dissociation, compared with that in the case of depressurization alone. The inner (core) temperature change indicates that the coupling of depressurization and steam injection induces MH dissociation from upstream and downstream to the center of the sample. However, it starts from an upstream region and continues downstream steadily in the case of steam (hot water) injection alone.

Objective: MS-275, a phase trialed histone deacetylase inhibitor will be characterized for its ability reduce neuroblastoma (NB) viability and to target the tumor stem cell (TSC) population in neuroblastoma. Methods: Ability of MS-275 to reduce NB growth is characterized using a tumorigenic NB N-type cell line that has high differentiation potential. TSC enriched side population from NB and a reference teratocarcinoma cell line was analyzed as a model of TSC. The potential of MS-275 to modulate functional characteristics and markers of TSC was also investigated. Results: MS-275 induces a G1 cell cycle arrest, the intrinsic apoptosis pathway in NB and can potentially differentiate NB into a more terminal phenotype. NB TSC-like population is reduced following MS-275 treatment by the targeting of their self-renewal and drug pumping ability. Conclusions: By targeting both the NB and its TSC population, MS-275 has therapeutic potential for neuroblastoma. This warrants further in-vivo investigations.

The objective of this study was to evaluate galvanic corrosion potential under various pH conditions, buffering capacities, and corrosion inhibitors including zinc orthophosphate (ZOP) and orthophosphate (OP). Bench-scale dump-and-fill experiments evaluated metals release from a lead and copper couple under stagnant conditions. Key findings from this study were that increasing DIC from 3 to 7 or 17 mg CaCO3/L significantly reduced lead release with or without corrosion inhibitor; however, the lowest lead concentrations were observed in water conditions with corrosion inhibitor addition. However, addition of 20 mg PO4/L as OP exacerbated lead release in some cases; though dissolved lead release was always below 28 µg/L, particulate lead was as much as 4 times greater compared to no corrosion inhibitor. Overall, this study demonstrated the potential of high dose ZOP and OP for lead corrosion control in drinking water, however, overdosing OP can lead to exacerbated particulate concentrations.

The calpain family of intracellular Ca2+-dependent cysteine proteases is involved in a number of intracellular signaling processes. Calpain hyperactivity has also been implicated in ischemic injury, neurodegenerative diseases and cataract formation. However, the specific function of calpains in these normal and diseased states remains unclear. Competitive inhibition of calpain is useful for studying their functions and can lead to pharmacological treatments, while monitoring their activity with activity-based probes (ABPs) can reveal how calpain is regulated and be applied to screen for inhibitors in vivo. But these strategies are complicated by the similarity of the calpain active-site when compared to other intracellular cysteine proteases. Therefore, there is a need to design inhibitors and ABPs that selectively target calpain. Using X-ray crystallography, the interactions between the calpain active-site and each of two reversible inhibitors was studied. This led to the discovery of novel non-covalent aromatic stacking and hydrogen bonding interactions between the primed-side adenine group of one inhibitor and indole ring of an active-site Trp residue in μ-calpain. A substrate-based competition assay later confirmed that these interactions provided this compound with an inhibitory advantage over the other, which lacked any primed-side interactions, thereby providing insight into the development of new, more specific reversible calpain inhibitors. Next, a fluorescent ABP, containing features borrowed from an irreversible and presumably calpain-specific inhibitor, was evaluated for its ability to detect calpain activitiy. Although this probe appropriately targeted the calpain active site in its Ca2+-activated form, it was unable to detect calpain activity in a cell extract. Nevertheless, the results of this study have yielded insights into ways of improving the calpain detecting ability of this ABP. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-01 15:39:07.023

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), and is a frequent event in sporadic thyroid carcinomas. Multiple endocrine neoplasia 2B (MEN 2B), a subtype of MEN 2, is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET), however the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10 fold increase in ATP binding affinity, and leads to a more stable receptor-ATP complex, relative to the wildtype receptor. We also show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease. In the second part of the study, we identified a novel β-catenin-RET kinase signaling pathway which is a critical contributor to the development and metastasis of human thyroid carcinoma. We show that RET binds to, and tyrosine phosphorylates, β-catenin and demonstrate that the interaction between RET and β-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate β-catenin-specific transcriptional programs in a RET-dependent fashion. We show that downregulation of β-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumour growth in nude mice. Finally, we used a structure guided approach to identify and characterize a novel, non-ATP competitive, RET inhibitor; SW-01. We show that SW-01 provides significant RET inhibition in an in vitro kinase assay using purified RET. Moreover, RET phosphorylation is blocked, or dramatically reduced, in vivo in cells overexpressing active RET. We observe a significant decrease in cell proliferation and colony formation in RET-expressing cells in the presence of SW-01. Together, our data suggest that SW-01 has potential as a novel RET kinase inhibitor with clinical utility. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-09-15 16:20:59.976

Calpains are a family of intracellular cysteine proteases activated by calcium. They participate in many processes including cell motility, cell cycle progression and cell death, in response to calcium signaling. Because calpain over-activation as a result of calcium dysregulation is a contributing factor to many disease states, these enzymes are important therapeutic targets. Within the cell, calpains 1 and 2 are regulated by the protein inhibitor calpastatin. This unstructured protein is specific for calpain, binds tightly, and recognizes only the activated form of the enzyme. Detailed kinetic data obtained using surface plasmon resonance allowed the association and dissociation rates of each of the four calpastatin inhibitory domains to be measured. Based on this, inhibitory domain 4 was selected to be co-crystallized bound to calpain 2. The X-ray crystal structure of this complex provided both the first view of the active enzyme, as well as the first view of how it is inhibited. Calpastatin wraps around the enzyme making contact with each domain. It lies in the active site as a contiguous polypeptide chain and escapes cleavage by forming a loop away from the catalytic cysteine. In addition to inhibiting substrate cleavage, calpastatin protects calpain in two ways; it prevents autoproteolysis, and it prevents calcium-dependent aggregation. The crystal structure of the calpastatin:calpain complex revealed no obvious reason for this stabilization. To elucidate how this protection occurs, peptides were synthesized corresponding to conserved subdomains of calpastatin. Surprisingly, each peptide alone was capable of preventing aggregation in vitro, by blocking hydrophobic patches exposed upon activation. The increased hydrophobic surface of the activated enzyme may alter calpain’s affinity for other proteins such as substrates. By binding across many domains of calpain, calpastatin could act to block protein-protein interactions. These studies have characterized calpastatin’s interaction with calpain, which will further our understanding of the enzyme’s regulation and aid in the development of better calpain inhibitors. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-04-29 15:27:16.208