Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Nie, Shuming; Committee Member: Parkos, Charles; Committee Member: Petros, John; Committee Member: Voit, Eberhard; Committee Member: Zhu, Cheng. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (M.S.)--Brigham Young University. Dept. of Statistics, 2007. / Includes bibliographical references (p. 46-49).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Orientador: Jorge Fernando Brandão Pereira / Coorientador: Valéria de Carvalho dos Santos Ebinuma / Banca: João Vitor Dutra Molino / Banca: Marcel Otavio Cerri / Resumo: A proteína verde fluorescente (GFP, do inglês Green Fluorescent Protein) é um biomarcador utilizado na produção de proteínas de fusão, amplamente empregado in vivo e in vitro. Este trabalho avaliou a produção da GFP expressa em Escherichia coli BL21 (DE3) [pLysS; pET28(a)] em mesa incubadora rotativa e biorreator, e sua posterior extração utilizando Sistemas Micelares de Duas Fases Aquosas (SMDFA). Na etapa de produção em mesa incubadora rotativa, estudou-se a influência da taxa de agitação, tempo de indução e concentração do composto indutor, isopropil-β-1-D-tiogalactopiranosídeo (IPTG), sobre a produção da GFP, e apenas a variação da taxa de agitação e o tempo de indução alteraram a produção de GFP. A melhor produção de GFP (314,59 mg/L) foi obtida a 30°C após 22 h de cultivo a uma agitação de 150 rpm, induzindo com 0,5 mM de IPTG realizada 10 h após o início do cultivo (meio da fase exponencial de crescimento). Posteriormente, avaliou-se menores concentrações de IPTG (0,25; 0,125 mM) na produção de GFP e observou-se que os níveis de produção foram mantidos. Estes estudos iniciais, foram a base para as condições empregadas em biorreator tanque agitado, que operou no modo batelada a uma agitação de 200 rpm, aeração de 2 vvm (volume de ar/ volume de meio) e indução com 0,125 mM de IPTG após 6 h de cultivo. Nas condições citadas, obteve-se apenas uma produção de 128,13 mg/L de GFP, desse modo, apesar da potencialidade da ampliação de escala na produção desta biomolécula, estud... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Green Fluorescent Protein (GFP) is a in vivo and in vitro biomarker widely used in the production of fusion proteins. This work evaluated the production of GFP expressed in Escherichia coli BL21 (DE3) [pLysS; pET28 (a)] in shaker and bioreactor, and its subsequent extraction using Aqueous Micellar Two-Phase Systems (AMTPS). The influence of the agitation rate, induction time and concentration of the inductor, isopropyl-β-1-D-thiogalactopyranoside (IPTG), on GFP production, was studied using a shaker. The results shown that only the agitation rate and the induction time have affect the GFP production. The best GFP production (314.59 mg/L) was obtained at 30°C after 22 h of culture at 150 rpm, induced with 0.5 mM IPTG after 10 h after of the start of cultivation (at half of exponential growth). Subsequently, lower IPTG concentrations (0.25, 0.125 mM) were evaluated for the GFP production, being maintained the production levels. From these conditions, a cultivation using agitated tank bioreactor was then carried out. The bioreactor operated in batch mode at 200 rpm with a 2 vvm aeration (air volume / volume of medium), being the GFP production induced with 0.125 mM IPTG after 6 h of microorganism' growth. Under that conditions, a production of 128.13 mg/L of GFP was obtained. Thus, despite the potential of scale-up in the production of this biomolecule, further studies are still required. After the GFP production studies, the bacterial cells were disrupted using continuous freezing / thawing cycles. GFP recovered from the cell lysate was then used to perform stability studies at different pHs (3 - 12) and temperatures (25°, 37° and 50°C). The stability studies shown that GFP was stable between pHs 7 to 11 and at temperatures of 25°C and 37°C, but not stable at a temperature of 50°C. Afterwards, liquid-liquid extraction... (Complete abstract click electronic access below) / Mestre

The author of this dissertation deals with patients with metastatic colorectal cancer to the liver. Compares the results with serum CEA, MMPs, TIMP patients with benign liver tumors and patients without cancer.

Thesis (Masters Diploma (Technology)--Cape Technikon, Cape Town, 1986 / Child psychiatrists have become increasingly aware of the existence. of affective disorders in prepubertal and pubertal patients. This has led to the investigation of possible biological factors contributing to the disorders. Due to the lack of availability of human brain material, different parameters have been investigated in the periphery in order to obtain information regarding the aetiology of major depressive disorder. The neurotransmitters, NA, 5-HT and DA have been implicated in depression. Levels of the metabolites of these transmitters have been measured in plasma, urine and CSF of adult depressed patients. Two other peripheral "tools" used in the study of major depressive disorder are blood platelets and lymphocytes. The former contain cr 2 -adrenoceptors and imipramine binding sites (indicative of 5-HT uptake into the platelet) and the latter S-adrenoceptors. Platelets have been widely used as a model for indirectly evaluating changes in central cr2-adrenoceptor and imipramine binding whereas lymphocytes have been used to measure changes in S-adrenoceptor binding and activity in adults with major depressive disorder.

Thesis (DTech (Environmental Health))--Cape Peninsula University of Technology, 2017. / Metal pollution in aquatic environments is considered a major environmental concern because of variation in several abiotic factors that impose severe restrictions on organisms living in these areas. Ceratophyllum demersum L. (family Ceratophyllaceae), a hornwort or coontail, free floating rootless macrophyte has been suggested a suitable model for investigating metal stress and was used in the current study. This study assessed the use of selected biological responses, namely antioxidant responses and changes in chlorophyll concentration in Ceratophyllum demersum L., as biomarkers of metal exposure, and also investigated the field application of these responses in the Diep River. The ultimate aim was also to determine the usefulness of C. demersum as model of metal contamination and as phytoremediator after a pollution event. An investigation of metal bioaccumulation in this macrophyte exposed to different concentrations of a combination of metals over a five-week exposure period in a greenhouse, was undertaken, as well as a field study in the Diep River, Milnerton, Cape Town and a pond (reference site) at the Cape Peninsula University of Technology, Cape Town, to validate experimental results. In the laboratory study the water was contaminated once off at the beginning of the study, to simulate a pollution event. The metal concentrations in the water and plants were measured in the four treatments and the control every week over a five-week exposure period. The samples were acid-digested and analysed with an Inductively-Coupled Plasma-Mass Spectrophotometer (ICP-MS). The results showed that concentrations of the metals in the water varied in all treatments over time with no specific patterns amongst the treatment groups. This macrophyte proved highly effective in the bioaccumulation of these metals at all four exposure concentrations. The metals bioaccumulated rapidly in the plants after the water was spiked. The main focus of the study was to investigate the possible use of biochemical responses in C. demersum as possible biomarkers for metal exposure. A range of antioxidant/oxidative stress parameters were measured in the plant exposed to a combination of metals (Al, Cu, Fe, Zn) in four different treatments over the five week exposure period. Total antioxidant capacity (TAC) was measured using Total Polyphenols (TP), Ferric Reducing Antioxidant Power (FRAP) and Oxygen Radical Absorbance Capacity assay (ORAC), enzyme activity was determined using Catalase (CAT), Superoxide Dismutase (SOD), Ascorbate Acid (AsA) and Total Glutathione (GSHt) and lipid peroxidation was measured by using Thiobarbituric Acid Reactive Substances (TBARS) and Conjugated Dienes (CDs). The cocktail of the four metals induced significant changes in the antioxidant defence system of C. demersum, including the antioxidant enzyme activities. The different metal exposures disturbed the cellular redox status in the plant. The current study has demonstrated that this macrophyte shows tolerance to metal-induced oxidative stress and that it can survive under relatively high concentrations of these metals by adapting its antioxidant defence strategies. Chlorophyll was extracted in 80% chilled acetone in the dark and the absorbance values were determined using a spectrophotometer. Chlorophyll a (chl a), chlorophyll b (chl b) and total chlorophyll (chl t) contents were measured under different exposure concentrations of metals in the macrophyte. The results of this study indicated that chlorophyll contents were variable over the exposure period and no significant differences in chlorophyll concentrations were found between weeks. A field study in the Diep River and the pond located at the CPUT campus (reference site) was conducted to validate experimental results. Plants in a polluted section of the Diep River were shown to bioaccumulate metals to high concentrations. Bioaccumulation of metals in C. demersum might have induced oxidative stress, and other environmental factors such as temperature- and chemical stress might have caused chlorophyll degradation. The chlorophyll concentrations in the plants of the pond (reference site) might also have been affected by temperature and chemical stress of the water. Significantly higher AsA, CAT, ORAC, SOD and TBARS concentrations in the Diep River plants might be an indication that the plants in the river might be well adapted to the constant exposure to metals and that the plants might have developed a tolerance mechanism to cope with oxidative stress compared to those of the pond. The results show that metals are bioaccumulated quickly by C. demersum after the water is contaminated with metals, i.e. after the "pollution event". However, over time, metals are continuously exchanged between the plants and the water, accounting for the fluctuations in metal concentrations observed over time. This study has shown that C. demersum has phytoremediation potential because it was able to remove high concentrations of metals from the contaminated water. Therefore, C. demersum, can be applied as a model for metal contamination and a phytoremediator after a pollution event. The potential to antioxidant responses and chlorophyll content as biomarkers of metal exposure in C. demersum have been demonstrated.

M.Sc. (Zoology) / There has been an increasing concern worldwide regarding the possible adverse effects of pharmaceutical supplements present in our aquatic ecosystems and whether or not they modify the physiological functioning in humans and wildlife. Trenbolone acetate (TBA) and zeranol (Z) for example, are two commonly used synthetic anabolic growth promoting hormones in cattle production. TBA is metabolized into trenbolone-β and excreted as both trenbolone-α and -β. In liquid manure trenbolone-β has a half-life of over 270 days and Z, 120 days. Therefore if released into the surrounding environment there is the possibility for long-term severe ecological impacts i.e. fish reproduction and general health. The aim of this study was to determine the physiological effects of several growth promoting hormones used as growth promoting hormones in cattle production on the Mozambique Tilapia – Oreochromis mossambicus. The growth promoting hormones assessed in this study were; Trenbolone acetate, Methyltestosterone, Diethylstilbestrol and Zeranol. The aim was accomplished by making use of histology (gills, liver and gonads) and three biomarker assays; Glutathione-S-transferase (GST), Uridine-Diphosphate Glucuronosyltransferase (UDPGT) and Cellular Energy Allocation (CEA). Stock solutions of Trenbolone acetate (14 μg/l and 15 μg/l), Methyltestosterone (7 μg/l and 7.5 μg/l), Zeranol (2.8 μg/l and 3 μg/l) and Diethylstilbestrol (0.28 μg/l and 0.29 μg/l) were prepared. Fish were exposed under controlled conditions for a period of 24-hours, 4-, 15- and 30-days respectively using a flow-through system. The aquarium water was changed (45 L removed and replaced with 45 L of prepared growth hormone containing bore-hole water) every 48 – 72 hours to remove all waste material thus ensuring the aquariums were clean. Upon performing the necropsies, gills, liver and gonads were removed and examined using standard histological techniques. Muscle tissue was used to determine the CEA, liver and kidney tissue was used for both GST and UDPGT assays. The results obtained from the histology revealed that the gills and liver were not severely affected by exposure to the growth promoting hormones although possible exposure related alterations were evident. The gonads results indicated that exposure to the growth promoting hormones severely affected the morphology and functioning of the organs to the point where reproduction is questionable. The results obtained from the hepatosomatic index (HSI) and gonadosomatic index (GSI) revealed no significant differences (p<0.05) although a trend of increasing HSI and decreasing GSI was evident in the male fish exposed to the androgens. With regards to the biomarker assays there were minor decreases in CEA in the exposed fish but no significant differences (p<0.05) could be established. The GST assay revealed that Zeranol prompted a significant increase (p<0.05) in GST activity in the kidney after 4- and 15-days of exposure while the liver displayed no change in GST activity. The UDPGT assay revealed minor fluctuation in UDPGT activity in both the kidney and liver throughout the study, however, no significant differences (p<0.05) could be established. To conclude, exposure to these growth promoting hormones at the selected concentrations and exposure periods severely compromised the fish’s reproductive capabilities thus challenging the fish’s fitness. Further studies examining the energy metabolism and xenobiotic detoxification pathways of the Mozambique tilapia and other indigenous fish species are recommended to better comprehend the effects that these growth promoting hormones may possess.

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.

The biological integrity of aquatic ecosystems has become threatened by the effects of high nutrient loads, inorganic and organic chemicals. The effect of these xenobiotics needs to be investigated by the application of biotests in whole effluent toxicity testing and biomarkers in active biomonitoring. Whole effluent toxicity (WET) testing determines the toxicity/effect of whole effluent on aquatic organisms. Sub-lethal effects can be determined by analysing the levels of subcellular/physiological indicators/enzymes in aquatic organisms exposed to in situ conditions. The procedure used for in situ assessments was active biomonitoring (ABM). The aim of this study was to assess the instream health of a known contaminated system (Rietvlei Wetland System, Gauteng, South Africa) using WET and ABM methodologies. Three sites in the Rietvlei Wetland System were selected and sampling was undertaken during high flow (April 2003) and low flow (August 2003) periods. The ABM sampling protocol involved the deployment of aquatic molluscs (Melanoides tuberculata) and fish (Oreochromis mossambicus) for a period of four weeks at the sites. Following the four week exposure period the organisms were transported back to the laboratory and the following biomarkers were assessed: ethoxyresorufin o-deethylase (EROD), acetylcholine esterase (AChE) and metallothione (MT). Water samples were also collected for WET testing during the low flow period, since this was the only period where mortality was observed in the ABM organisms. Standard WET protocols were used to assess the toxicity of the water from the three sites. These protocols were: 96 h Poecilia reticulata lethality test, 48 h Daphnia pulex lethality test and 72 h Selenastrum capricorutum growth inhibition test. In addition the same biomarker analyses that were done on the ABM organisms were carried out on WET exposed D. pulex and P. reticulata. The WET testing and ABM indicated highest toxicity at Sites 1 and 3. Algal growth inhibition test showed highest stimulation of algal growth at Site 2 and inhibition at Site 3. Sites 1 and 2 showed signs of contamination by organophosphates and carbamates due to elevated AChE and reduced EROD. However there were no significant differences in AChE activity between fish tested in the 96h toxicity test and those used in ABM. Very low AChE activity was recorded in D. pulex. Snails also had lower AChE activity when compared to the exposed fish species. Metallothionein content was higher in field-exposed fish than those used in WET, however both assessment protocols indicated that Site 3 was affected the greatest by metals. Snails exhibited higher MT content than fish and D. pulex showed no MT activity. There were no significant differences in MT content between the sites. Acetylcholine esterase appears to be a relevant means of investigating biological effects of many neurotoxic contaminants on aquatic habitats and trophic levels. Metallothionein content is a good indicator of toxicity due to heavy metals for active biomonitoring as well as the WET test. Ethoxyresorufin-O-deethylase is a more difficult biomarker to work with since it shows no differences in activity between control and exposed organisms. The EROD assay does not detect very low levels of EROD induction. Acetylecholinesterase and MT are the recommended biomarker assays for the detection of sublethal responses in the WET laboratory toxicity test. The AChE activities and MT exhibit comparable results in both ABM and WET assessment protocols. In future studies WET testing needs to be complimented with a suite biomarker analyses to determine the type of pollutants in a system and sufficiently describe the pollution status of a system. / Dr. V. Wepener