Motivation: The different coping styles used to respond to psychosocial stress have been linked to the development of cardiovascular disease (CVD). However, the manner in which the cardiovascular system is influenced differs between the coping styles. Of the different coping styles, defensive active coping (AC) has been shown to be the most detrimental to cardiovascular health. This is worsened by augmented α-adrenergic cardiac responses found in Africans. Furthermore, many studies have found that the prevalence of hypertension and other CVDs is much higher in urban Africans when compared to their Caucasian counterparts. This can be attributed to certain lifestyle changes implemented by Africans in the transition that occurs with urbanization, where they are forced to cope with an urban-dwelling lifestyle. One of these lifestyle factors, which also poses as a cardiovascular risk factor, is increased usage and in some cases abuse of alcohol. Certain discrepancies exist between ethnicities with regard to the metabolism of alcohol, which influences the effect of alcohol on the individual. Alcohol usage as a possible manner of coping has been supported in many instances, but the interdependent effects of alcohol usage and AC as cardiovascular risk factors has only been found in African men. Further investigation is needed to determine if coping and alcohol abuse act in tandem only in African men, or also in other ethnic or sex groups. What also needs to be discussed is whether the inconsistencies between ethnicities regarding alcohol metabolism, plays a part in the development of CVD in a bi-ethnic gender cohort. Objectives: The main aims of this study were to determine 1) receiver operated characteristic (ROC) ethnic specific cut points of alcohol usage in the prediction of ambulatory hypertension, and 2) to assess if these cut points in defensive active groups revealed increased cardiometabolic risk in a bi-ethnic sex cohort, and if so, whether the increased risk will be associated with a specific race or sex group? Methodology: This sub-study forms part of the SABPA (Sympathetic activity and Ambulatory Blood Pressure in Africans) study, conducted from 2008 to 2009. After exclusion criteria were applied, our bi-ethnic sex cohort consisted of 390 individuals. These participants were all from the Kenneth Kaunda Education District of the North-West province in South Africa, and they all signed informed consent prior to participation. The SABPA study was approved by the Ethics Review Board of the North-West University, with additional ethical approval for this sub-study. All procedures in this study complied with the guidelines of the Declaration of Helsinki. Each participant completed a psychosocial battery supervised by registered clinical psychologists, and information regarding their medication use and medical history was obtained. They also completed the Coping Style Indicator questionnaire which was developed by Amirkhan, to identify the coping style habitually used. Ambulatory blood pressure and ECG measurements were recorded for a 24h period with the Cardiotens CE120®. Anthropometric measurements were performed by ISAK (International Society for the Advancement of Kinanthropometry) level 2 accredited anthropometrists using calibrated instruments. Out of this, the body surface area were calculated. The physical activity of each participant was determined by use of the Actical® omnidirectional accelerometer. Resting blood samples were collected by a registered nurse. The following blood serum levels were determined: gamma-glutamyl transferase (γ-GT) as a marker for alcohol usage, C-reactive protein, cholesterol, high density lipoprotein, triglycerides, cotinine, reactive oxygen species and glycated haemoglobin levels. All statistical analyses were done using Statistica version 12.0. Descriptive statistics were conducted to state the baseline characteristics of the entire group, while Chi-square (X2) tests were used to determine prevalence for medications and pathology. ROC analyses were computed to establish a cut point for γ-GT predicting ambulatory hypertension in each ethnicity as well as in the entire group. Independent t-tests identified confounders, after which two-way analysis of covariance (ANCOVA) tests were computed to test a 2 x 2 main effects interaction (race x γ-GT cut points) for all cardiometabolic risk markers and to compare the different ethnic groups. ANCOVAs were then performed in the ethnic groups with high γ-GT as well as in above mean AC for the graphs that followed. Lastly, odds ratios (OR‟s) with 95% confidence intervals (CI‟s) were calculated in several models to highlight the odds of high alcohol intake to predict ambulatory hypertension in the ethnic-sex groups as well as in AC ethnic-sex groups. Significant values were noted as p ≤ 0.05. Results: The Africans revealed higher cardiometabolic risk markers, above mean defensive active coping, seeking social support with less avoidance coping scores. ROC analyses revealed that ambulatory hypertension commences at a much higher level of γ-GT in the Africans [55.7U/l (AUC=0.69; 95% CI: 0.61; 0.76)] with sensitivity /specificity of 47%/83% compared to the Caucasians [19.5U/l (AUC=0.747; 95% CI: 0.68; 0.82)] with sensitivity/specificity of 70%/73%. The Caucasians thus reveal an increased sensitivity for alcohol ingestion at a much lower γ-GT cut point compared to the Africans. When comparing ethnic specific ROC cut point groups, we found that certain levels of cardiometabolic risk factors such as C-reactive protein, systolic blood pressure, waist circumference and silent ischemic events, were significantly higher in the African group, especially in above mean AC groups. Out of the Africans with high γ-GT levels, 73% used the AC style, suggesting hypervigilant AC coping and increased CVD risk in Africans. Clinical significance was determined by OR‟s, which demonstrated that high γ-GT levels in AC African men predicted ambulatory hypertension with an OR of 7.37 (95% CI: 6.71 – 8.05). Higher alcohol intake predicted ambulatory hypertension in AC Caucasians with an OR of 2.77 (95% CI: 2.31 – 3.23) in men and 6.42 (95% CI: 5.85 – 7.0) in women respectively. Conclusion: γ-GT cut-points in defensive active groups revealed increased cardiometabolic risk markers in a bi-ethnic sex cohort. A possible hypermetabolic state in African men may initially protect them against CVD morbidity but if chronically challenged with no forthcoming social support, CVD risk is imminent. / MSc (Physiology), North-West University, Potchefstroom Campus, 2015

Cytosolic glutathione transferases (GSTs) are a large family of enzymes that play an important role in detoxification of xenobiotics. They catalyse the conjugation of the glutathione tripeptide (GSH) to a wide range of toxic electrophilic acceptors. The overall 3D folds and architectures of the catalytic sites of many GSTs are conserved. They are composed of a well conserved glutathione binding site (G-site) and a promiscuous hydrophobic binding site (H-site). The 3D structure and ligand specificity has allowed the sub-classification of the multiple isoforms within the soluble GST superfamily. GSTs are involved in the drug detoxification and so are the target of medicinal chemistry programmes but it has proven difficult to generate isoform-specific inhibitors due to their inherent promiscuity. In this project, Venughopal Bhat (University of Edinburgh, laboratory of Dr. Mike Greaney) and I have explored a new platform to probe enzyme specificity. Protein-directed dynamic combinatorial chemistry (DCC) allows the assembly and amplification of a ligand within the confines of a binding site. DCC was used as a tool to explore the promiscuous H-site of four eukaryotic GSTs. I purified recombinant forms of SjGST, hGST P1-1, mGST M1-1 and mGST A4-4 from E. coli and assayed them with the universal, synthetic GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Venughopal Bhat prepared a ten-member, thermodynamically-controlled, dynamic combinatorial library (DCL) of acyl hydrazones from a 1-chloro-2-nitrobenzene aldehyde and ten acylhydrazides. This DCL was incubated with each of the four GST isozymes (spanning diverse classes) and distinct amplification effects were observed for SjGST and hGST P1-1. I subsequently carried out several biophysical experiments in an attempt to rank each of the ligands. These experiements, coupled with molecular modelling, provided insight into the basis of the observed selectivity. Bacterial GSTs are thought to play a role in primary metabolism and display a different GSH-conjugation mechanism compared to the eukaryotic GSTs. A recombinant form of the beta-class GST from the pathogenic bacterium Burkholderia cenocepacia was isolated, purified and biochemically characterised. The same ten-member acylhydrazone DCL was interfaced with the bacterial GST which was shown to amplify a hydrophobic library member that shared structural features with the known substrate 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (HOPDA). With the collaboration of Venughopal Bhat, I attempted to explore the putative active site of a GST-like protein with an unknown function using the same DCL. Although no amplification was observed, a new aldehyde template was suggested for future DCC experiments on this protein. GSTs are widely employed in biotechnology as protein fusion tags to enhance target protein solubility coupled with a facile enzyme assay. Manish Gupta and Juan Mareque-Rivas (University of Edinburgh) used the N-terminal, hexahistidine-tagged SjGST to demonstrate that quantum dots (QDs) coated with nitrilotriacetic acid (NTA) bound to Ni2+ ions can be used to reversibly and selectively bind, purify, and fluorescently label a His6-tagged GST in one step with retention of enzymatic activity. For this prupose, I purified and characterized both the untagged and hexahistidinetagged – SjGST prior to their experiments.

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

As glutationas transferases compõem uma superfamíla de proteínas que atuam na fase II do sistema de desintoxicação das células. Participam principalmente através do processo de conjugação da glutationa com moléculas hidrofóbicas e eletrofílicas, como por exemplo os herbicidas. No entanto, outras funções foram descritas como a tolerância ao estresse oxidativo, inseticidas, antibióticos microbianos, transporte de produtos secundários tóxicos, sinalização da célula durante as respostas ao estresse e fenômenos de resistência envolvendo agentes de quimioterapia contra o câncer. Nesta tese procurou-se estabelecer uma relação entre a seqüência, estrutura, função e afinidade das GSTs. A estrutura, de modo geral, determina a função da enzima, mas por si só, não dita sua especificidade. Esta última informação é fundamental para o desenvolvimento de novos agroquímicos ou para o desenho racional de novas proteínas. A relação entre a seqüência, estrutura, função e afinidade mostra que o paradigma estrutura-função deveria ser ampliado para incluir a seqüência de aminoácidos e a afinidade da enzima. Apesar da grande diversidade de substratos e seqüências encontradas nas GSTs há pelo menos um caso de convergência funcional em duas classes distintas desta superfamília. Uma encontrada apenas no reino Animalia (classe Pi) e outra exclusiva do reino Plantae (classe Phi). Ferramentas da bioinformática estrutural, como docking molecular e minimização de energia foram utilizadas para analisar as interações entre a enzima e o substrato. Estas ajudam a explicar como duas proteínas com aproximadamente 22% de identidade de seqüência apresentam afinidades semelhantes. Finalmente, foram propostos mutantes da GST de Saccharum officinarum utilizando a informação estrutural da enzima, visando uma alteração na afinidade da mesma. / Glutathione Transferases comprehend a superfamily of proteins that plays the phase II of the detoxification system of the cells. Their major catalysis is the conjugation of glutathione with hydrophobic and eletrophilic molecules, for example herbicides. However, other functions were described like oxidative stress, insecticides, microbial antibiotics, transport of secondary products, cells signalization during response to stress and resistance of chemotherapy drugs against cancer. This work aimed to establish a relation between sequence, structure, function and affinity of GSTs. The structure, in general, determines the function, but alone, can not determine the enzyme specificity. This last information is essential to the development of new agrochemicals or for the rational design of proteins. The relation between sequence, structure, function and affinity shows that the paradigm of structure-function should be enlarged in order to include the information of amino acid sequences and the enzyme affinities. Despite the wide variety of substrates and sequences found in the GSTs, there is at least one case of functional convergence between two distinct classes in this superfamily. One is found in the Animalia kingdom (class Pi) and the other is exclusively found in Plantae (class Phi). The structural bioinformatic tools, such as molecular docking and energy minimization were used to analyze the interactions between the enzyme and the substrate. These help to understand how two enzymes with approximately 22% of sequence identity can show the same affinities. Finally, GST mutants of Saccharum officinarum were proposed, using the enzyme structural information in order to modify the enzyme affinities.

<p>Current techniques for nucleic acid analysis often involve extensive sample preparation that requires skilled personnel and multiple purification steps. In this dissertation, we introduce an on-chip, isothermal, post-hybridization labeling and signal amplification technique that can directly interrogate unmodified DNA and RNA samples on a microarray format, eliminating the need for microarray sample pre-processing. </p><p>We name this technique Surface Initiated Enzymatic Polymerization (SIEP), where we exploit the ability of a template independent DNA polymerase called Terminal Deoxynucleotidyl Transferase (TdT) to catalyze the formation of long single-stranded DNA (ssDNA) chain from the 3'-end of a short DNA primer, which is tethered on the surface, and TdT's ability to incorporate unnatural reporter nucleotides, such as fluorescent nucleotides. We hypothesize that polymerization of a long ssDNA chain while incorporating multiple fluorescent nucleotides on target DNA or RNA hybridized to probe printed on a surface will provide a simple and powerful, isothermal method for on-chip labeling and signal amplification. </p><p>We developed the SIEP methodology by first characterizing TdT biochemical reaction to polymerize long homopolymer ssDNA (> 1000 bases) starting from the 3'-OH of ten bases oligonucleotides. We found that the preferred monomers (deoxynucleotide, dNTP) are dATP and dTTP, and that the length of the ssDNA extension is determined by the ratio of input monomer (dNTP) to initiator (short oligonucleotides). We also investigated TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the initiation efficiency, the degree of fluorophore incorporation, the length and the polydispersity of the polymerized DNA strand. These experiments allowed us to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using SIEP as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a up to ~45 fold increase in signal amplification compared to the incorporation of a single fluorophore.</p><p>SIEP was then employed to detect hybridization of DNA (25 bases), short miRNA (21 bases) and long mRNA (1400 bases) by the post-hybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of hybridized target strands. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection (LOD) and a 2-log linear dynamic range while the detection of short miRNA and fragmented mRNA targets resulted in ~2 pM and ~10 pM LOD, respectively with a 3-log linear dynamic range.</p><p>We further developed SIEP for colorimetric detection by exploiting the presence of negatively charged phosphate backbone on the surface as target DNA or RNA hybridizes on the immobilized probe. The net negative charge on the surface is further increased by TdT catalyzed polymerization of long ssDNA. We then used positively charged gold nanoparticles as reporters, which can be further amplified through electroless metallization, creating DNA spots that are visible by eye. We observed an increase of 100 fold in LOD due to SIEP amplification.</p><p>Overall, we demonstrated the use of SIEP methodology to label unmodified target DNA and RNA on chip, which can be detected through fluorescence signal or colorimetric signal of metallized DNA spots. This methodology is straightforward and versatile, is compatible with current microarray technology, and can be implemented using commercially available reagents.</p> / Dissertation

No description available.

published_or_final_version / abstract / toc / Pathology / Master / Master of Philosophy

This study shows that resistance to killing by reactive oxygen intermediates (ROI) increases during migration and development in Schistosoma mansoni. Resistance is associated with the protective role of antioxidants as shown by the increased levels of superoxide dismutase and of the glutathione system enzymes. Hydroperoxide-dependent glutathione peroxidase activity was not detectable in newly transformed schistosomula, however the activity was present in the liver stages. The antischistosomal drug oltipraz (OPZ) decreased in an irreversible manner the activity of S. mansoni glutathione S-transferase (GST), an important protective enzyme, both in vivo and in vitro. The inhibition of GST activity was not isoenzyme restricted and was non-competitive with respect to the two substrates essential for GST activity. On the other hand, OPZ treatment increased the levels of mouse (S. mansoni host) liver GST activity in an isoenzyme specific manner, with the $ mu$ class subunit induction accounting for most of the increase. However, mammalian GST activity was inhibited by OPZ in vitro. However, the inhibition of mammalian GST activity was reversible upon addition of dithiol reducing compounds. OPZ inhibited the binding of ($ sp{14}$C) N-ethylmaleimide (specifically alkylates SH groups), suggesting that OPZ interacts with SH-groups of GST to inhibit its enzymatic activity. Another SH-dependent enzyme, hexokinase, from yeast and S. mansoni was reversibly inhibited by OPZ. The oxy-analogue of OPZ, in which the thione sulphur is replaced with oxygen, did not inhibit the enzymatic activity of GST and hexokinase. Many of the biochemical effects of OPZ on S. mansoni and its mammalian hosts may be related to its ability to bind to SH groups and inactivation of the functions of many essential proteins.

Glutathione transferases (GSTs) are good candidates for investigations of enzyme evolution, due to their broad substrate specificities and structural homology. The primary role of GSTs is to act as phase II detoxifying enzymes protecting the cell from toxic compounds of both endo- and exogenous origins. The detoxification is conducted via conjugation with glutathione (GSH), which facilitates their removal from the body. The work presented in this thesis has supported a theory for enzyme evolution when the multiple pathway to novel functions can been seen to involve a “generalist” state from which “specialist” states with a new activities can evolve. The generalist has broader specificity and lower activity than the specialist. The term quasi-species is used for a group or cluster of enzyme variants with similar functional properties, and this entity has been suggested as the fittest group for further evolution. This is based on studies of the evolution of new GST variants in two generation. Three diverging clusters or quasi-species, with diverging substrate selectivity, were identified from a GST M1/M2 library, by using directed evolution (family DNA shuffling), multiple substrate screening and multivariate statistics as tools. One of the clusters was M1-like and the other was M2-like, both functionally and structurally. The third quasi-species diverged orthogonally from the parent-like distributions. Its functional character can be referred to as a “generalist” as it had lower activities with most of the substrates assayed except for epoxy-3-(4-nitrophenoxy)-propane (EPNP) and p-nitrophenyl acetate (pNPA). Another round of family DNA shuffling was made with selected variants from the “generalist” quasi-species. From the second generation three quasi-species emerged with diverging functions and sequences. The major cluster contained enzyme variants that represented a direct propagation of the generalists. Diverging from the generalists was a cluster with high specificity with isothiocyanates (ITCs). Increased ITC specificity and decreased epoxide specificity was observed among the novel variants (specialists). The change in functional properties was attributed to a Tyr116His substitution in the active site. These results demonstrate the usefulness of multivariate analysis in the quest for novel enzyme quasi-species in a multi-substrate space, and how minimal changes in the active site can generate distinctive functional properties. An application of our method could be identification of enzyme quasi-species that have lost their sensitivity with alternative inhibitors.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.