Includes abstract. Includes bibliographical references.

Infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, where low-and middle-income countries (LMIC) have the highest incidence. Prophylactic HPV vaccines exist but LMIC have limited access. Therefore, alternative preventative measures against HPV infection and cervical cancer progression are needed. Two human proteins have been identified in our laboratory that modulate HPV16 pseudovirus (HPV16-PsVs) infection in vitro, namely surfactant protein A (SP-A) and recombinant human vimentin (rhVim). Previous work suggested SP-A mediated immune recognition of HPV since SP-A-coated HPV16- PsVs enhanced viral uptake by RAW264.7 murine macrophages. These initial observations were confirmed using a murine C57BL/6 cervicovaginal challenge model: pre-incubation of HPV16- PsVs with purified human SP-A significantly reduced the level of HPV16-PsV infection in vivo. Moreover, when isolated cells from female reproductive tracts of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in viral uptake by eosinophils, neutrophils, monocytes and macrophages were observed over time using SP-A-pre-coated virions compared to control particles. Compared to SP-A mediated modulation of HPV infection through activation of innate immune responses, rhVim was suggested to directly interfere with HPV entry into host cells. Indeed, supplementation with non-filamentous rhVim resulted in decreased viral uptake by NIKS cells which was confirmed in vivo using the murine C57BL/6 cervicovaginal HPV16-PsVs challenge model. Co-localisation analysis employing confocal imaging, revealed that rhVim-coated HPV16- PsVs co-localised, to a lesser degree, with surface-expressed heparan sulphate proteoglycans (HSPGs) than control particles. Removal of surface HSPGs on NIKS cells decreased HPV16-PsVs cell surface binding and internalisation, while pre-incubation of HPV16-PsVs with rhVim decreased viral particle binding and internalisation to a greater extent. This indicates that rhVim may modulate HPV16 infection by interfering with its attachment to HSPGs as well as viral engagement with the yet unknown entry receptor(s). In summary, both SP-A and vimentin modulate HPV16-PsVs infection by different mechanisms. These in vivo studies strongly confirm previous in vitro observations, rendering both proteins potentially suitable for further development into possible candidates for use in topical microbicides, which may provide protection against new HPV infections.

Includes abstract. / Includes bibliographical references. / Peanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA).

Previous studies in our laboratory using microarray gene expression analysis identified members of the nuclear transport protein family as significantly upregulated in cervical cancer biopsies compared to normal cervical epithelial tissues. These results were validated at both mRNA and protein levels, and similar upregulation observed in oesophageal cancer. Recent mass spectrometry (MS) analysis of cancer cell secreted proteins identified elevated levels of 13 members of the nuclear transport protein family in the secretomes of transformed, cervical cancer and oesophageal cancer cell lines. The nuclear transport proteins have functions in many cellular processes including proliferation, mitosis, maturation of RNA, activation of the actin cytoskeleton and restructuring of the nuclear envelope. In addition, they are required for the nuclear import and export of numerous cargo proteins such as transcription factors, oncoproteins and kinases, which often display deregulated activity in cancer cells. The aims of this study were to 1) independently validate the MS data showing elevated levels of the nuclear transport proteins in the secretomes of cervical and oesophageal cancer cell lines, 2) investigate the diagnostic potential of members of the nuclear transport protein family using cervical and oesophageal cancer serum samples and 3) identify the potential binding partners of Kpnβ1, a key member of the nuclear transport protein family, in normal and cancer cells. This study investigated the levels of endogenous expression and secretion of 8 members of the nuclear transport protein family; Kpnβ1, IPO5, IPO7, TNPO1, CRM1, CAS, Kpnα2 and Ran in a normal epithelial cell line (hTERT-RPE1) in comparison to transformed (SVWI38 and CT-1), cervical cancer (HeLa and CaSki) and oesophageal cancer (WHCO5 and KYSE 30) cell lines using Western blot analysis. Our data revealed differential endogenous expression in the cell lines. An analysis of the secretomes of the cell lines showed that all 8 proteins assayed were secreted at elevated levels by the transformed, cervical cancer and oesophageal cancer cell lines compared to the normal cell line. These results validate previous MS data generated in our laboratory. To investigate whether members of this protein family can be detected in the serum of cancer patients, ELISA for Kpnβ1, CRM1, Kpnα2 and CAS proteins were performed using commercially available ELISA kits. The results showed significantly elevated levels of Kpnβ1, CRM1 and CAS in the serum of cervical cancer patients compared to the non-cancer controls. Serum levels of Kpnβ1, CRM1, Kpnα2 and CAS were elevated in the oesophageal cancer patients compared to the non-cancer controls. To investigate the diagnostic potential of these proteins, logistics regression analysis was performed. Our results showed that CAS was the best performing individual candidate biomarker in discriminating between cervical cancer cases and non-cancer controls. It had the highest AUC (0.85±0.03) and highest sensitivity (55%) at 95% specificity compared to those of Kpnβ1 (AUC=0.77±0.04 with 35% sensitivity at 95% specificity), CRM1 (AUC=0.64±0.05 with 20% sensitivity at 95% specificity) and Kpnα2 (AUC=0.51±0.05 with <10% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had an improved AUC of 0.89 with a sensitivity of 100% at 60% specificity. In discriminating oesophageal cancer cases from the non-cancer controls, CAS (AUC=0.86±0.03 with 56% sensitivity at 95% specificity) similarly performed better compared to Kpnβ1 (AUC=0.62±0.05 with 15% sensitivity at 95% specificity), CRM1 (AUC=0.75±0.04 with 32% sensitivity at 95% specificity) and Kpnα2 (AUC=0.73±0.04 with 21% sensitivity at 95% specificity). The combination of Kpnβ1, CRM1, Kpnα2 and CAS as a panel of biomarkers had the highest diagnostic capacity with an AUC of 0.90 and 84% sensitivity at 86% specificity. These results suggest that individual members of the nuclear transport protein family have potential as diagnostic biomarkers for both cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. Our investigation aimed at identifying the binding partners of Kpnβ1 in normal, cervical cancer and oesophageal cancer cell lines using immunoprecipitation coupled to mass spectrometry (IP-MS) identified 100 potential Kpnβ1 binding partners in hTERT-RPE1 normal cell extracts, 179 in HeLa cervical cancer cell extracts, 147 in WHCO5 cell extracts and 176 in KYSE30 oesophageal cancer cell extracts. Venn Dis JavaFX-based Venn and Euler diagram software was used to identify common and unique Kpnβ1 binding partners. 38 proteins were identified as common binding partners of Kpnβ1 in normal and cancer cells and 56 common binding partners of Kpnβ1 in the three cancer cell lines. Of these, 18 proteins were found to be unique to the three cancer cell lines and of these, 10 could be linked via protein-protein interaction mapping using STRING bioinformatic analysis. These include nucleoporin 214 (Nup214), Prem RNA 3'-end-processing factor FIP1 (FIP1L1), cell division cycle and apoptosis regulator 1 (CCAR1), cleavage and polyadenylation specific factor 7 (CPSF7), ribosomal protein L7 (RPL7), ribosomal protein L10 (RPL10), ribosomal protein L13A (RPL13A), ribosomal protein S6 (RPS6), ribosomal protein S4, X isoform (RPS4X) and Ras-related nuclear protein (Ran). Among these, FIP1L1, CCAR1 and CPSF7 have not been previously described as binding partners of Kpnβ1. In conclusion, elevated levels of nuclear transport proteins in the extracellular environment of cancer cells and in cancer patient serum samples suggest that they have potential as diagnostic biomarkers for cervical and oesophageal cancers, with a combination of Kpnβ1, CRM1, Kpnα2 and CAS being the best predictor. In addition, this study shows that Kpnβ1 interacts with several different proteins in normal and cancer cells, with some of the interactions unique to cancer cells presenting as novel binding partners for further investigation.

With a rapidly ageing population, neurological diseases are becoming increasingly relevant in the design of public health policies and strategies in western societies. In the last decades, biochemical research has consistently shown the critical role that neurotransmitters and their associated metabolites play as biomarkers in tracking and diagnosis of different brain disorders and cancers. In particular, dopamine, an organic electrochemical neurotransmitter, has been shown to be paramount for the proper functioning of the neural system. Dopamine's dysfunction, has been shown to underlie the pathogenesis in several neurological disorders such as Parkinson's disease, depression, chronic schizophrenia and psychosis. Unfortunately, currently available diagnostic technologies require expensive equipment and trained personnel, thus slowing the process and increasing overall costs, consequently reducing the access to the general population. Ideal sensing alternatives should offer low-cost, fast, and reliable point-of-care diagnosis. In this work, we present a compact label-free plasmonic biosensors that exhibits sharp optical resonance shifts in response to varying dopamine concentrations. While the nanoimprinting of the optical cavity offers a nanofabrication process fully compatible with large-scale production, its small size ensures the suitability for integration on lab-on-a-chip platforms, hence making it a promising alternative to conventional diagnosis. The plasmonic nanostructures of the chips are functionalized with synthetic single-stranded oligonucleotide to ensure precise sensing selectivity. Particularly, we study the standard 57mer and the novel 44mer configurations, establishing the superior resolution offered by the latter. In contrast to other sensing alternatives, our platforms permit reliable detection with ultralow volumes (~6 µL) and exhibit robustness to interfering species. This work paves the way towards the first generation of low-cost, ultra-low volume, on-chip sensors for in-situ tracking of dopamine.

The differential expression of the a2(I) procollagen gene in normal and transformed human fibroblasts has been correlated with differential in vitro DNA-protein interactions on the basal promoter region between -100 and -67. A 23 bp region of the a.2(1) procollagen promoter encompassing the G/CBE (CCTCCATTGG) and the Ctv'IE (GGAGGCCCTTTT) has previously been shown to engage in specific DNA protein interactions that determined the transcriptional activity of the promoter. The CME forms two distinct DNA-protein complexes that might be crucial in the regulation of the a2(I) procollagen gene in a cell specific manner. The hypothesis was, therefore, that depending on the protein that participates in complex formation with the CME, the gene would be activated or repressed. The objective of this study was to investigate the role of this 23 bp region in the regulation of expression. of the a2(I) procollagen gene in transformed fibroblasts. In addition, the study sought to establish the role of the proto-oncogene c-fos-in the regulation of expression of the a2(I) procollagen gene. In contrast to previous observations, this study demonstrated that only one DNA protein complex is formed on the CME and the second complex is a specific proteolytic cleavage of the product of the larger complex. Preparation of nuclear extracts in the absence of protease inhibitors, specifically leupeptin, resulted in the formation of a smaller complex, previously shown to bind the CME. The importance of this proteolytic fragment that still retains DNA binding activity is yet to be determined. In addition, the CME binding proteins were fairly ubiquitously expressed in both a.2(1) collagen producing and non-producing cells. CT-1 fibroblasts (transformed by y-irradiation) synthesise over 80% of total a2(I) collagen produced by its untransformed counterpart (WI-38 fibroblasts), whereas the gene, is down regulated in the human embryonic lung fibroblasts transformed with SV40 (SVWI-38 fibroblasts). These cell lines are therefore ideal for studying regulation of a.2(I) procollagen gene. To analyse the importance of the G/CBE and CME regions of the a.2(1) procollagen gene promoter, point mutations were introduced by site-directed mutagenesis. Mutated promoter DNA was cloned into a p8CAT reporter vector, and the activity of the promoter determined in transient transfection experiments. Mutations introduced in the G/CBE region of the a.2(1) procollagen promoter resulted in a 3-12-fold decrease in the activity of the promoter. The decrease was observed with both proximal (-343 bp) and basal (-107 bp) promoter constructs~ a significant reduction in promoter activity was observed in both CT-1 and SVWI-38 fibroblasts. These results imply that the G/CBE region of the promoter is required for the activation of transcription of the a.2(1) procollagen gene and therefore the factor that interacts with the G/CBE functions as a transcriptional activator. Previously, this factor was shown to complex with antibodies raised against the mouse CCAAT binding factor (CBF), suggesting that the protein belongs to the CBF family of transcription factors. Furthermore, these results demonstrate that the adjacent, upstream inverted GGAGG sequence is crucial for activation of the gene through the CCAAT binding element. The inhibition of promoter activity in constructs with a mutated G/CBE element was correlated with lack of protein binding to the mutated sequence as confirmed by electrophoretic mobility shift assays. Transfection of a.2(1) procollagen promoter constructs containing mutations in the CME region, however, resulted in a significant increase in promoter activity in both CT-1 and SVWI-38 fibroblasts. A much higher increase, 3-fold, was observed for the SVWI-38 cell line compared to a 1.5-fold increase observed for CT-1 fibroblasts. These results suggested that the factor that interacts with the CME functions as a repressor of the a.2(1) procollagen gene. Interestingly, the promoter activity in SVWI38 fibroblasts transfected with mutated CME constructs was similar to that observed in CT-1 fibroblasts transfected with the wild type promoter construct. An interesting observation was that repression of the a.2(1) procollagen gene via the CME required upstream elements since transfection of the basal mutated promoter did not result in increased promoter activity. From these results, it can be concluded that the CME binding protein is involved in cell-specific repression of the a2(I) procollagen gene and that the mechanism of repression appears to be dependent on the presence of upstream elements. Mutations in the G/CBE and CME pointed out the significance of these elements in the expression of the a2(I) procollagen gene and since a number of studies have characterised the mouse CCAAT binding protein, this study focused on purification and identification of the CME binding protein(s). Purification was performed by conventional biochemical techniques using heparin-agarose and sequence-specific DNA affinity chromatography, as well as separation on SDS-polyacrylamide gels. Two cycles of DNA affinity chromatography yielded two polypeptides with apparent molecular weights of 50 and 67 kDa. Automated N-terminal sequencing of the polypeptides indicated that they were blocked and therefore no sequence could be obtained. In addition, these polypeptides failed to raise an immune response in mice and rabbits. Subsequently, polypeptides were digested with trypsin in situ in polyacrylamide gels and the eluted peptides were analysed by MAWITOF-mass spectrometry. The mass:charge ratios (mlz ratios) obtained were used to search the database using a mass tolerance of 1.5 Da and only one hit was obtained. The match obtained was that of a mouse zinc finger protein of which not much is known, except that it might be a transcription factor. This result supports previous observations of Collins et al (J Cell Biochem 1998, 70: 455-467) that complex formation requires the presence of zinc. The primary structure of the CME binding protein remains to be determined. Transformation of fibroblasts is normally accompanied by changes in the expression of extracellular proteins, including type I procoUagen. Although CT-I fibroblasts, show very little change in a2(I) procollagen gene expression, the c-f os gene is drastically down-regulated. This study sought to establish if there is any relationship between the unusually high levels of the a2(I) procollagen gene in this transformed cell line and failure of the cells to stimulate c-fos expression in response to serum. CT-1 :fibroblasts that overexpressed wild type Fos were established and changes in the expression of the a,2(1) procollagen gene were measured. Overexpression of Fos down-regulated the a,2(1) procollagen gene, which was not due to increased turnover of the a1(I) procollagen mRNA. Analysis of promoter activity showed that the promoter and first intron, which has been reported to contain negative regulatory elements, did not harbour any Fas-responsive elements. The -343 bp and the -2300 bp promoter constructs were transactivated in cells overexpressing Fos. Thus, although overexpression of Fos resulted in a significant decrease in the levels of the a2(1) procollagen WA, it does not involve the region between -2300 bp and +1800 bp of the a2(1) procollagen gene. Furthermore, there was no change in the stability of the message, indicating that constitutive expression of Fos did not activate a factor that could play a role in altered turnover of the a2(I) procollagen mRNA. It is therefore possible that constitutively high levels of Fos may trigger the expression of a number of other genes, which have a negative impact on the expression of the a2(I) procollagen gene.

Since its discovery in the early 1940s the element plutonium has been seen by mankind as both an opportunity and a threat. As a radioactive nuclide plutonium presents health hazards in its handling and if mankind is to make the most of this element's potential benefits it is essential that these hazards be understood. Both overestimation and underestimation of these hazards are damaging to its proper utilisation. Many studies have been carried out to determine the effects of plutonium exposure and a broad picture of the biological behaviour of plutonium has been built up. Radiological protection standards are based on such broad understanding and a "Central Dogma" has arisen viz, plutonium is bound avidly in liver and bone; clearance half-lives from these organs differ (by a factor of 2.5) but are very long - a minimum of 50 years for bone; this is why plutonium urinary excretion levels are very low. Despite all the research work that has been carried out there are many important areas of plutonium behaviour which are not well understood or in which the central ideas adopted for radiological protection purposes are questionable. One such questionable area is extended half-life in the body. Two rather different areas relate to the molecular binding interactions which plutonium enters into in body tissues and transfer mechanisms from blood into cellular organelles. Very little is known about these processes and the speciation that plutonium demonstrates within the body. This thesis explores understanding of plutonium behaviour by application of pharmacokinetic theory to observed human behaviour, both following occupational exposure and experimental injection. Occupational exposure data demonstrated behaviour consistent with pharmacokinetic expectations over periods of 25 years or more. Long-term half-lives were 10 to 30 years rather than 50 to 100 years or more. There was no evidence of differing half-lives between liver and bone. Very low renal clearance was seen in intravenous injection studies suggesting either very extensive plutonium binding to the protein transferrin in blood or pointing to reabsorption in the kidney tubule after glomerular filtration. This latter possibility might lead to a "Plutonium blood pressure" which effectively forces activity into tissues irrespective of the strength of binding forces. Experimental work indicated species differences in transferrin binding which may have relevance for extrapolation from animals to humans.

The magnesium ion (Mg2 ) is involved in a variety of physiological and biochemical processes including the activation of over 300 enzymes and the control of transmembrane movement of cations. In most mammalian cells intracellular Mg2+ is kept well below electrochemical equilibrium. In cardiac muscle cells recent studies suggest that the intracellular and extracellular concentrations of Mg2+ are around I and 0.5 mIs4, respectively. The equilibrium potential for Mg2 is approximately - 10 mV. In resting muscle the membrane potential is far more negative than this and M g2+ extrusion must take place aganist an electrochemical gradient. Factors which may be important in preserving intracellular Mg2+ include; changes in membrane potential which occur during the action potential, intracellular buffering, intracellular compartmental redistribution, the permeability characteristics of the plasma membrane and transport stystems in the plasma membrane. The aims of the study are to investigate the effects of extracellular Mg2+ on contractility and coronary flow and the effects of hormonal and extracellular cationic control of Mg2+ homeostasis in the rat heart. Subsequently, the effects of dietary magnesium on the magnesium, calcium, sodium and potassium content of the heart (and other tissues) in young rats is also investigated. Perfusion of the isolated heart with elevated extracellular Mg2+ (1.2 - 7.2 mM) caused a profound reduction in the force of contraction and an associated increase in the coronary flow rate. The selective 13-adrenoceptor agonist isoprenaline (10 M), evoked a large Mg2+ efflux and an associated increase in the force and rate of contraction in the isolated perfused heart. Quantitativley, similar increases in Mg2+ efflux were seen during stimulation of the isolated heart with the adenylate cyclase activator, forskolin (10 M). Stimulation of superfused electrically paced ventricle segments with either isoprenaline, noradrenaline or adrenaline (10-6 M) also evoked a large net Mg2+ efflux. The isoprenaline-evoked Mg2+ efflux was significantly reduced during treatment of the heart with either the 0- antagonist, propranolol (I o-5 M), or the Ca2 -channel blocker, verapamil (1 o-5 M) Stimulation of mag-ftira-2 AM loaded cardiac myocytes in suspension with isoprenaline did not result in any change in intracellular M g2+. Elevations of extracellular Na+ concentration (as NaCl or sodium isethionate), elevated chloride (as choline chloride) and sucrose (at concentrations osmotically equivalent to elevated Na+) all evoked large Mg2+ eftiuxes in the isolated perfused heart. The elevated Na (as NaCl) -evoked Mg 2 efflux was partially, though not significantly, inhibited by the Na channel blocker, amiloride (10 M) and propranolol (lOs M). During stimulation of the heart with elevated Na+ there was no increase in lactate dehydrogenase activity indicating that the release of Mg2+ was not due to cell damage. Treatment of the heart with verapamil (I o-6 M), which dramatically reduced the force of contraction, had little effect on the Mg2+ efflux evoked by elevated Na+ (as sodium sulphate). Acute perfusion of the isolated heart with nominally Mg2+ free physiological solution over 15 min caused a significant reduction in the magnesium content of the heart, thereafter, magnesium was well conserved. Magnesium deficient diets fed to young rats over a period of approximately one month caused reduction in food consumption, retardation of growth, reductions in some organ weights and various cationic disturbances in the heart and other tissues. In the heart there were no significant changes to sodium or potassium but there was a significant increase in calcium and reduction in magnesium. Collectively, these results suggest that magnesium is well conserved in the heart and that the catecholamines and changes in extracellular osmolarity may be important physiological regulators of magnesium homeostasis in the heart.

Hypertension is the commonest cardiovascular disorder and poses a major health challenge to populations of countries in socio-economic transition as well as those of developed countries. In the United Arab Emirates (UAE), hypertension has emerged as a leading cause of death. However, there is little first hand information with regard to the status of specific known risk factors for hypertension, nor of the extent to which the rapid changes in life style, especially dietary habits and lack of exercise, have contributed to the growing number of hypertensives in this population. The aim of this study was to document the circulating levels of various biochemical markers of hypertension and chronic renal failure (CRF), a disease which is normally associated with hypertension. The study population consisted of hypertensive and CRF patients and healthy Emirati subjects, who are mainly Gull Arabs of Bedouin descent. It has been hypothesized that the population lacks a number of confounding risk factors for cardiovascular diseases such as alcohol consumption, chronic stress and cigarettesmoking. Plasma levels of immunoreactive endothelin-1 (ET-l), homocysteine (Hcy), growth hormone (GH), insulin-like growth factor-I (IGF-l), leptin, and insulin were measured by standard enzyme-linked immunosorbent assay (ELISA) methods. Plasma glucose, blood urea nitrogen (BUN), creatinine, lipids and lipoproteins were measured by standard colorimetric assays. With regard to hypertension, a major finding of this study is related to the levels of ET-1 in Etniratis with untreated essential hypertension. These were significantly (p < 0.01) higher in hypertensives (mean 10.1 ± 1.0 pmol ') than in normotensives (mean 2.2± 0.5 pmol F'). For all subjects ET-1 levels correlated closely with systolic blood pressure (SBP), but less significantly with diastolic blood pressure (DBP) and body weight. In a more extensive study, hypertensives were found to have higher body mass index (BMI) and triacylglycerols (TG), non-esterified fatty acids (NEFA) and ET-1 and lower high-density lipoprotein- (HDL)-cholesterol. Further, both SBP and DBP weresignificantly correlated with age, BMI. NEFA, ET-1 and log insulin and TG and negatively with HDL-cho!esterol. The independence of associations between blood pressure and ET-1 or HDL-cholesterol and ET-1 and HDL-cholesterol or NEFA were confirmed by partial regression, but not that of correlations between NEFA and blood pressure or HDL-cholesterol. Further study of the racial differences in the levels of biochemical markers among treated hypertensive subjects of four ethnic groups, has revealed that whilst ET-1 was decreased to normal levels. Hcy was significantly increased in hypertensives than age-, gender- and ethnic-matched healthy normotensives. In all hypertensive subjects, total cholesterol (TC), HDL-cholesterol, and low-density lipoprotein- (LDL)—cholesterol were significantly decreased and TG was significantly increased while NEFA was unchanged. Emirati subjects, either hypertensives or normotensives, had the highest BMI with tendency towards obesity compared to the non- Gulf Arab, African, and Asian groups respectively. In patients with renal disease, results have shown that IGF-1 is notably decreased in CRF patients and negatively correlated with both systolic and DBP indicative of a possible role for IGF-1 as a marker of renal disease progression among hypertensive patients. Moreover, significantly elevated levels of leptin were observed, particularly in female patients, and leptin was shown to correlate significantly (p < 0.0]) with insulin, total and LDL-cholesterol and TG. Leptin was negatively correlated with GH concentrations, but was not correlated with IGF-1 levels. Further, the results have also shown that in CRF, reduced activity of lecithin: cholesterol acyltransferase (LCAT), has been documented and this has been shown to be associated with low levels of HDLcholesterol and other lipid changes including membrane lipid composition. in untreated hypertensives, low I-IDL-cholesterol have also been documented, but apparently without impaired LCAT activity. When combined all CRF subjects, regardless of dialysis, total and LDL-cholesterol were unchanged, TO and free cholesterol (FC) were raised and HDL-cholesterol levels and the percentage of esterified cholesterol (EC) were significantly decreased compared to controls. Plasma NEFA levels for untreated patients were similar to controls, but were decreased in peritoneal dialysis patients and markedly increased both before and, even more so, after dialysis in haemodialysis patients. In conclusion, the results of this study have shown that plasma concentrations of ET-1 in Emiratis are significantly (p c 0.01) higher in untreated hypertensives than in normotensives. The increased lelevls of FT-I were associated with raised NEFA and decreased HDL-cholesterol concentrations. It is suggested that ET-1 either alone or in conjunction with dislipidaemia, could be a risk factor for cardiovascular disease in this population. in treated hypertensive subjects however, Hcy levels rather than FT-I were increased in all ethnic groups compared to age- gender and ethnic-matched controls. In addition, the pattern of correlations and hormone and lipid changes found in this study suggest that raised insulin levels may be an important determinant of leptin levels in CRF, but that OH, IGF-1 and lipid status are not.

Proteins are often able to distinguish between closely related ligands, thus achieving specificity. A major goal in biophysical chemistry is to understand the molecular basis for protein-ligand recognition. This level of understanding is necessary for developing methods to accurately predict protein-ligand binding energetics from structural data. The goal of this thesis was to identify features of protein-ligand interactions that may not be adequately accounted for in structure energetics calculations in order to improve our ability to predict binding energetics for these interactions. Specifically, the features of protein-nucleotide binding were studied using the small, guanine-specific ribonuclease, RNase Sa binding to two closely related nucleotide inhibitors, guanosine-3'-monophosphate (3'GMP) and inosine-3'-monophosphate (3'IMP) as a model system. Comparing the binding of these two inhibitors using isothermal titration calorimetry (ITC), x-ray crystallography, NMR and molecular dynamics (MD) simulations has revealed important determinants of guanine base recognition by proteins, specifically the role of the exocyclic amino group (N2) of the guanine base. Importantly, due to the high conservation of guanine binding sites in proteins, the observations for RNase Sa can potentially be extended to other systems. In addition, RNase Sa has provided a well-defined system for the investigation of changes in heat capacity and changes in backbone dynamics upon ligand binding. All of the data presented here support the idea that fluctuations in protein structure can contribute significantly to protein-nucleotide binding energetics even for an apparently rigid-body interaction. These fluctuations make a significant contribution to the enthalpy, entropy, and heat capacity changes associated with the RNase Sa-nucleotide interaction. This implies that fast time-scale motions must be accounted for to optimize structure-based calculations for protein-nucleotide binding. The use of molecular dynamics simulations is presented as a promising way to account for these motions for surface area based calculations.