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Increasing evidence has shown that abnormal metabolic phenotypes in body fluids reflect the pathogenesis and pathophysiology of Parkinson's disease (PD). However, the relationship between metabolic phenotypes and PD is not fully understood. Mass spectrometry (MS) based metabolomics is a powerful technique, which was frequently used for the sensitive and reproducible detection of hundreds to thousands of metabolites in biofluid samples.. Here we developed and performed MS-based metabolomics studies involving hundreds of human urine samples with data acquired from multiple analytical batches for surveying potential biomarkers of PD. A new software statTarget was developed and introduced. Protocols for liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were developed, including sample preparation, data acquisition, quality controls, quality assurance and data analysis. Urinary metabolites from a total of 401 clinical urine samples collected from 106 idiopathic PD patients and 104 normal control subjects were profiled by using LC-MS. Quality control (QC) strategy has been performed in MS-based metabolomics for high reproducibility and accuracy of MS data. GC-MS with methyl chloroformate (MCF) derivatization was used for profiling highly polar metabolites in patients with early-, middle- and advanced-stage PD. Our study revealed the significant correlation between clinical phenotypes and urinary metabolite profiles. Comprehensive metabolomics was successfully developed with the goal of identifying urinary metabolite markers that can be used for evaluating the development of PD. A group of 18 metabolites have shown not only a high discriminating ability for the early-stage PD patients but also accurately distinguished the middle- and advanced- stages patients from control subjects. For the evaluation of PD, 18 metabolites showed good potential as metabolite markers with related metabolic pathway variations observed in branched chain amino acid metabolism, glycine derivation, steroid hormone biosynthesis, tryptophan metabolism, and phenylalanine metabolism.. We have further performed targeted analysis of potential biomarkers by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and GC-MS. The UPLC-MS/MS method was developed and optimized for detecting the concentration variation of metabolites in tryptophan metabolism for alpha-synuclein over-expressed flies (Parkinson's disease model). The altered tryptophan metabolism was proved as one of the common metabolite signatures between PD patients and alpha-synuclein over-expressed fly model of PD, and thus may be used for developing potential markers of the disease and evaluating the efficacy of novel therapeutic agents. An asymmetric labeling strategy and positive chemical ionization gas chromatography-tandem mass spectrometry (PCI-GC-MS-MS) approach was developed for the determination of non-amino organic acids and amino acids, as well as short chain fatty acids. Carboxylic and amino groups could be selectively labelled by propyl and ethyl groups, respectively. The specific neutral losses of C3H8O (60 Da), C3H5O2 (74 Da) and C4H8O2 (88 Da) were useful in the selective identification for qualitative analysis of organic acids and amino acid derivatives. The developed PCI-GC-MS/MS method showed good reproducibility and linear range.. In summary, metabolomics study has its inherent advantage in the characterization of biomarkers for the development of PD and may bring new scientific knowledge as well as impact on the progression of PD and other related neurodegenerative diseases.

Increased blood plasma concentrations of the aminothiol homocysteine (Hcy) are associated with a variety of disease states including those which cause impaired renal function, many forms of cardiovascular disease, and neurodegenerative diseases such as Alzheimer's. Therefore, Hcy has the potential to be a significant diagnostic biomarker. Routine monitoring of Hcy plasma concentration is encumbered by the time and resources required to quantify Hcy using currently accepted instrumental analysis methods. As part of the continuing effort to develop a quick, reliable, inexpensive, and user-friendly test to quantify Hcy at the point of care, we have designed a series of novel colorimetric and fluorescent chemical probes based on bridged viologen structures. The absorbance at 540 nm for the para-bridged bis-nitrile viologen probe (pCN) was found to be proportional to the concentration of Hcy analyte, with LOD = 2.17 μM and LOQ = 6.10 μM where unhealthy Hcy plasma concentrations are > 15 μM. The mechanism of reactivity between pCN and Hcy encompasses a dynamic set of reactions which involve pimerization of radical probe species and thioether adduct formation of pCN with Hcy. Preliminary results with fluorometric analogs of the bridged viologen probes are also presented.

The most abundant biological thiols, homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) have been the subject of intense research due to their association with a wide range of diseases. They play a key role in maintaining the redox status of biological systems. Selective detection methods for these thiols are challenging due to their similar structures and properties. Current commercially available detection methods use separations, fragile and expensive enzymatic or immunogenic materials and complex instrumentation. This has led to a global effort towards developing simple and inexpensive optical probes and indicators selective for specific biological thiols. Highly selective chemical probes and simple methods for detection and potential quantification of Hcy and GSH in their natural biological media have been developed. These indicators and methods are relatively simple and inexpensive for potential application at point of care. The selective detection of Hcy using novel asymmetric viologen chemical probes at room temperature is described as well as the use of commercially available materials under photochemical conditions. These probes respond linearly proportional to increasing Hcy concentrations, potentially enabling the monitoring of Hcy levels in human plasma. Additionally, new methods for the selective determination of GSH in human plasma, as well as its quantification in whole blood deposited on filter paper (dried blood spots), is also presented herein.

The Effect of Sulindac was studied on an animal model of ischemic stroke. Sulindac, a non steroid anti inflammatory drug (NSAID) could protect cell death due to hypoxia/reoxygenation. This drug was given 2 days before and 24 hrs after ischemia until animals were sacrificed on 3rd or 11th day. Infarct size was measured for these animals. Sulindac induced Hsp 27 in ischemic penumbra and core on Day 3 & 11 with uncoated nylon suture which shows its cell-survival and anti-apoptotic activity. Also, it increased expression of cell survival markers such as Akt, Bcl2 & Grp 78 in ischemic penumbra and core. With silicon suture it reduced expression of Hsp 27 in ischemic penumbra and core, alleviating cell stress and having pro-survival and anti-stress effects. In conclusion sulindac may have excellent potential as neuro protective agent against oxidative stress in cerebral ischemia. / by JIgar Modi. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Two major troponin I (TnI) genes, fetal TnI (ssTnI) and adult TnI (cTnI), are expressed in the mammalian heart under the control of a developmentally regulated program. In this study, the up-stream domain (~1,800 bp) of mouse fetal TnI gene has been cloned and characterized. There is a high homology of this region among mouse, rat and human. Transfection assays indicated that conserved GA-rich sequences, CREB and a CCAAT box within the first 300 bp upstream of the transcription start site were critical for the gene expression. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays revealed binding proteins to CREB site in nuclear extracts from myocardial cells. Thyroid hormone (T3) caused a significant inhibitory effect on ssTnI expression in myocardial cells. Cardiac troponin I (cTnI) mutations have been linked to the development of restrictive cardiomyopathy (RCM) in human patients. We modeled one mutation in human cTnI Cv terminus, arginine1 92 histidine (R192H) by cardiac specific expression of the mutated protein (cTnI193His in mouse sequence) in transgenic mice. The main functional alteration detected in cTnI193His mice by ultrasound cardiac imaging examinations was impaired cardiac relaxation manifested by a decreased left ventricular end diastolic dimension (LVEDD) and an increased end diastolic dimension in both atria. Echocardiography revealed a series of changes on the transgenic mice including a reversed E-to-A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. At the age of 12 months, cardiac output in cTnI193His mice was significantly declined, and some transgenic mice showed congestive heart failure. The negative impact of cTnI193His on ventricular contraction and relaxation was further demonstrated in isolated mouse working heart preparations. / Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO.The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation. The results demonstrate a critical role of the COOH-terminal domain of cTnI in the diastolic function of cardiac muscle. This mouse model provides us with a tool to further investigate the pathophysiology and the development of RCM. / by Jianfeng Du. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

Transgenic mice were generated to express a restrictive cardiomyopathy (RCM) human cardiac troponin I (cTnI) R192H mutation in the heart. My study's objective was to assess cardiac function during the development of diastolic dysfunction and to gain insight into the pathophysiological impact of the RCM cTnI mutation. Cardiac function was monitored in cTnI193His mice and wild-type littermates for a period of 12 months. It progressed gradually from abnormal relaxation to diastolic dysfunction characterized with micro- echocardiography by a reversed E/A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. The negative impact of cTnI193His on cardiac function was further demonstrated in isolated mouse working heart preparations. Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO. The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation characterized morphologically by enlarged atria and restricted ventricle and functionally by diastolic dysfunction. / by Nariman Gobara. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2012. / Oxidative stress, in recent times appears to be a major underlying risk factor in the occurrence of various diseases such as cardiovascular disease (CVD) and ischemic heart disease (IHD). During oxidative stress, there is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence mechanisms in favour of ROS. This results in severe cellular damages in the heart, vascular membranes and other organs. Potential benefits of dietary supplements as one of the major quenching elements against oxidative stress have been highlighted. Thus, a growing interest has been stimulated in finding natural alternatives for the treatment and! or prevention of oxidative stress-mediated diseases. Red palm oil (RPO), refined from the tropical plant Elaeis guineensis was used in this study since it has captivated much attention in the health sector lately. The effects of RPO-supplementation on oxidative stress biomarkers as well as homocysteine, a cardiovascular disease risk factor in an oxidative stress-induced rat model were investigated in this in vivo study. All experiments were conducted for a period of six weeks. Male Wistar rats (120-150g) were randomly divided into six groups (n=5) where all the rats received a standard diet. Two groups (groups C, D) were supplemented with 0.175g RPO (7g RPO/kg chow) for four weeks whereas groups (groups E, F) were given 0.175g RPO (7g RPO/kg chow) supplementation for six weeks. Rats in control groups (groups A, B) were not given any RPO-supplementation. Groups B, 0, F were induced with oxidative stress by injection of 0.5ml (20IlM/100g of body weight) organic tertiary-butyl hydroperoxide. All parameters were determined using appropriate methods in plasma, serum and erythrocytes. Data were expressed as mean ± SEM. No significant differences were obtained between groups for total antioxidant capacity and glutathione peroxidase activity. Red palm oil supplementation significantly increased superoxide dismutase activity after 6 weeks consumption, total glutathione levels after 4 weeks consumption and homocysteine levels after four and six weeks consumption in rats not subjected to oxidative stress. Under oxidative stress conditions, malondialdehyde (MOA) level, a marker of oxidative stress related damage, significantly increased in rats receiving a standard diet. However, when RPO diet was supplemented for 4 and 6 weeks, MOA levels significantly decreased towards the value of normal controls. In conclusion, our findings suggest that RPO-supplementation could ameliorate antioxidant status in the body through its potential ability to increase some antioxidant enzymes activity. Similarly, it is suggested that RPO-supplementation could protect the rat against oxidative stress induced damage in diseased state.

Homocysteine is a natural occurring aminothiol. It is an intermediate product in the metabolism of methionine. Methionine is an essential amino acid required for protein synthesis. Metabolic irregularities disrupt homocysteine levels in plasma. Elevated homocysteine levels are directly linked to folate and cobalamin (vitamin B12) deficiencies, and are an independent risk factor for cardiovascular diseases. High homocysteine levels have also been associated with Alzheimer's, osteoporosis, renal failure, cancer, birth defects and pregnancy complications. The association of elevated homocysteine levels with cardiovascular disease and other diseases has generated great interest in the detection of homocysteine. An optical method for the detection of homocysteine has been developed using fluorescein mono- and dialdehydes. Selectivity for homocysteine was achieved based on the characteristic differences between 5- and 6-membered ring heterocyclic amines formed upon the reaction with fluorescein mono- and dialdehydes. 6-membered ring homocysteine-derived thiazinane-4-carboxylic acids were found to be more basic than 5-membered cysteine-derived thiazolidine-4-carboxylic acids. Fluorescence enhancement in response to homocysteine was thus attained by tuning pH and excitation wavelengths. Furthermore, the design and synthesis of a more sensitive fluorophore, fluorescein tri aldehyde has been accomplished based on the aforementioned findings to enable the detection of homocysteine at physiological levels. Calculations of Mulliken charges revealed that the formation of thiazinanes results in modulation of the electron density on the fluorophore leading to higher fluorescence.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biomedical Technology, Durban University of Technology, 2012. / Antiretroviral therapy (ART) was introduced because it has shown to reverse the Acquired Immunodeficiency syndrome (AIDS), by reducing the HIV replication, allowing the regeneration of the patient’s immune system. ART is given to patients for the rest of their lives as part of HIV clinical care, but the use of ART has shown evidence of metabolic side effects which range from manageable to life threatening complications. Aims and objectives of the study The aim of the study was to investigate whether patients on ART developed metabolic side effects such as pancreatitis, dyslipidaemia and hepatotoxicity. These metabolic side effects were determined by laboratory testing of blood levels of specific biomarkers at stipulated intervals. Any significant change in the blood levels of these specific biomarkers was identified. Methodology : The study included 92 patients who were already selected for the ART programme which is in accordance to the South African National Antiretroviral Therapy Guidelines of 2003 Laboratory blood analysis was conducted. The repeated measures analysis of variance (ANOVA) was used to compare changes in biomarkers over time. The severity of each side effect was assessed by grading each biomarker laboratory result through the use of an established toxicity grading table. Results : It was found that the biomarker blood levels were not significantly altered within 12 months of ART, however, there was a gradual increase of most biomarker values, indicating that abnormalities may be detected after a longer period of treatment. Conclusion : Within 12 months of treatment, life-threatening toxicities were not detected. It may be speculated that if ART is monitored correctly, life-threatening toxicities may be avoided in many patients.