This research investigated the mechanism of action in bacteria of 3,4-methylenedioxy-β-nitropropene (BDM-I), a very broad spectrum antimicrobial lead compound in development as an anti-infective drug. The thesis proposes that BDM-I inhibits bacterial protein tyrosine phosphatases, a novel mechanism of action for an antimicrobial agent and a new target in microorganisms. This very open investigation was directed by considerable biological information on the effects of BDM-I in microorganisms and animals which provided insights into possible and improbable cellular targets. The biological effects of BDM-I were investigated using biochemical and cell-based assays, transmission electron microscopy and whole genome DNA microarray analysis. The specific experiments and order of execution were largely dependent on information gained as the project progressed. BDM-I was shown not to target the metabolic pathways of the major classes of antibacterial drugs, which supports a novel mechanism of action. Investigation of several species-specific effects suggested that cell signalling pathways were a possible target. Based on the structure of BDM-I and review of the scientific literature on cell signalling in bacteria, the hypothesis that BDM-I acted by inhibition of protein tyrosine phosphatases (PTP) was supported by demonstrating inhibition of human and bacterial PTP's in an enzyme assay. This mechanism was consistent with other demonstrated effects: inhibition of the intracellular pathogen, Chlamydia trachomatis; inhibition of swarming in Proteus spp. and inhibition of pigment production in Serratia marcescens; and with kill kinetics in bacteria and yeast. A pilot global genome analysis of BDM-I treated Bacillus subtilis did not detect differential expression of PTP genes but has provided many avenues for further investigation. This research further supports the development of BDM-I as a broad spectrum anti-infective drug.

G-substrate is a 23 kDa protein named as a specific substrate of cGMP-dependent protein kinase and found predominantly in cerebellar Purkinje cells. As a component of the NO/cGMP/PKG pathway, G-substrate is potentially involved in several important cellular processes and has so far been associated with a number of disease conditions: a single point mutation in G-substrate has been linked to hypercholesterolaemia, while the potent inhibition of PP2A by phosphorylated Gsubstrate possibly influences Tau protein hyperphosphorylation and contributes to Alzheimer's disease pathology. Conversely, overexpression of G-substrate protein in dopaminergic neurons has been found to protect neurons from Parkinson's disease toxins, making G-substrate a possible target of interventions for mitigating the debilitating effects of Parkinson's disease on patients. A shorter splice variant, which only retains one of the phosphorylatable threonine motifs, has recently been described for G-substrate and given the importance of phosphorylation to its action as a phosphatase inhibitor, this study focuses on determining whether both variants of the protein exhibit similar levels of phosphatase inhibition and interact with the same/similar proteins in vivo. We were also interested in determining whether the 51 amino acid section absent from short G-substrate resulted in any significant differences in protein structure, which potentially has implications on functions in vivo. My results indicate the association of G-substrate with a wide range of proteins involved in processes including cell cycle regulation, endocytosis and signalling and the two variants do not always interact with the same proteins. Among these interactors is the PARK 7/ DJ-1 protease, which like G-substrate has been shown to be neuroprotective. I have found that G-substrate is proteolysed by DJ-1 in its active form and interactions between these two proteins is affected by the anti-vertigo drug Tanganil. Phosphatase inhibition studies suggest that the G-substrate variants affect phosphatase activity to different extents under similar conditions, while NMR and circular dichroism structural studies suggest that in solution, the full length Gsubstrate variant is slightly more compactly folded. Understanding the details of G-substrate action in the cell will lead to a better understanding of its roles including the protection of dopaminergic neurons from Parkinson's disease toxins and shed more light on the intricacies of the NO/cGMP/PKG signalling pathway as a whole, thus providing important information that might help improve strategies for dealing with conditions involving this pathway and help develop interventions for diseases such as Alzheimer's and Parkinson's.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

Certain types of cyanobacteria have the potential to produce toxins including microcystin, a hepatotoxin. Toxic cyanobacterial blooms are becoming increasingly common worldwide. They are a concern in the Great Lakes and surrounding waters. In this study, Lake Ontario’s Bay of Quinte, Lake Erie’s Maumee Bay, and three reservoirs along the Grand River were studied. Environmental variables, cyanobacterial biomass inferred from the Fluoroprobe, and microcystin concentrations were measured. In 2005 the three reservoirs, Belwood Lake, Conestogo Lake, and Guelph Lake were sampled every two weeks from July to September. Belwood Lake was also sampled in October when a cyanobacterial bloom occurred. In 2006 the Bay of Quinte was sampled twice, in July and September, and Maumee Bay was sampled twice, in June and August. Physical variables measured included water transparency and temperature. All species of nitrogen (N) and phosphorus (P) were measured, along with extracted chlorophyll a and particulate carbon (C), N, and P. The distribution of chlorophyll and major algal groups throughout the water column was profiled in situ using a spectral fluorometer (Fluoroprobe).Variable fluorescence of phytoplankton was assessed using Pulse Amplitude Modulated (PAM) fluorometry to measure photosynthetic parameters. Phytoplankton counts were performed on selected samples from the Bay of Quinte and Maumee Bay. Total and dissolved microcystin were measured using the protein phosphatase inhibition assay (PPIA). PPIA was chosen over alternative detection methods because it is a functional assay that measures the level of microcystin in a sample via the amount of protein phosphatase inhibition that it exerts. This yields ecologically relevant data as protein phosphatase inhibition is the main mode of microcystin toxicity. The PPIA formulation used in our lab was based on variations in the literature that use unconcentrated water samples directly in the assay. The assay was optimized to employ both a higher and lower standard curve through the use of two enzyme concentrations. The lower enzyme concentration allowed the method detection limit to be decreased to 0.05 µg/L to accommodate our low-microcystin samples. In the Bay of Quinte, microcystin levels were higher in July 2006 (total mean=2.25 μg/L ) than in September 2006 (total mean=0.58 μg/L). In July a cyanobacterial bloom consisting of 97% Microcystis spp. was present. In September 83% of the cyanobacterial biomass was composed of Anabaena spiroides and only 8% was Microcystis spp. In the Bay of Quinte elevated microcystin concentrations were associated with higher soluble reactive P levels, lower seston C:P molar ratios, and lower total N. In Maumee Bay microcystin levels were higher in August 2006 (total mean= 4.45 μg/L) than they were in June 2006 (<0.05 μg/L). In August a cyanobacterial bloom consisting of 22% Microcystis spp. and 48% Aphanizomenon flos-aquae was observed. Higher microcystin concentrations in Maumee Bay were associated with decreased total N: total P molar ratios, increased total P, and decreased water transparency as measured by Secchi depth. Belwood Lake had the highest microcystin levels of the three reservoirs but only once exceeded the recommended World Health Organization concentration of 1.0 μg/L. Belwood Lake’s largest cyanobacterial bloom in October 2005 was accompanied by relatively low microcystin levels (<0.2 μg/L). Conestogo and Guelph lakes always had microcystin levels below 0.2 μg/L and 0.6 μg/L, respectively. In the Grand River reservoirs, increased microcystin concentrations were associated with higher chlorophyll a, higher light attenuation coefficients, lower total N, lower total N: total P molar ratios, higher C:P molar ratios, lower nitrate, higher cyanobacterial biomass, and higher total P. When data from the Bay of Quinte, Maumee Bay, and Grand River reservoirs were pooled, total microcystin had the most significant positive correlation with total P. Total microcystin and water temperature also had a significant positive correlation.

The main objective of this study was the application and comparison of different assays in assessing toxicity of cyanobacterial samples, and also characterizing toxicity of the field samples. Therefore, toxicity of purified microcystin-LR (MC-LR) and cyanobacterial samples collected from the Hartbeespoort (HBP) Dam (winter and summer seasons of 2005/2006) and Kruger National Park (KNP) were investigated and compared using the ELISA, mouse bioassay, catfish primary hepatocytes (in vitro assay) and protein phosphatase inhibition (PPi) assays. During sampling in the summer season at the HBP Dam, the dam surface was covered with a thick-green layer of cyanobacterial scum and a foul smell coming from the water surface was always present. Only blue-green streaks of cyanobacteria covered the dam surface during the winter season. All HBP Dam samples (winter and summer samples) and KNP samples (Nhlanhanzwani Dam, Mpanama Dam and Sunset Dam) were dominated by Microcystis aeruginosa with the exception of Makhohlola Dam samples which were found to have no cyanobacteria. The World Health Organization (WHO) has proposed a guideline value for human use of 1.0 µg/L (0.001 mg/L) for MC-LR, the most common microcystin (MC) variant, in drinking water (WHO 1998), whereas 2 000 Microcystis cells/mL have been recommended as the limit of cyanobacteria in drinking water for animals (DWAF 1996). Cyanotoxin concentrations exceeding the prescribed guideline value were detected in all HBP Dam samples (ELISA results ranging between 3.67 to 86.08 mg/L; PPi results ranging between 2.99 to 54.90 mg/L) and KNP samples (ELISA results ranging between 0.1 to 49.41 mg/L; PPi results ranging between 0.006 to 10.95 mg/L) using both the ELISA and PPi assays. In the current study, a dose of about 175 µg/kg of purified MC-LR was demonstrated to be lethal in male CD-1 SPF mice. The HBP Dam summer samples and Nhlanganzwani Dam samples were the only cyanobacterial samples that resulted in death (acute toxicity) of mice. In order to be able to investigate further the in vivo effects of cyanotoxins, transmission electron microscopy (TEM) was used to complement results obtained from the in vivo assay. Ultrastructural changes of varying degree were observed in livers of mice exposed to both the HBP Dam winter and summer samples. Early stages of hepatocyte to hepatocyte disassociation, slight vesiculation of endoplasmic reticulum (ER) and swollen mitochondria were the most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam winter samples. The most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam summer samples were massive hepatic haemorrhage indicated by the appearance of erythrocytes between hepatocytes and the extensive vesiculation of ER. This is the first time that the African sharptooth catfish primary hepatocyte model has been used to assess the hepatotoxicity of purified MC-LR and cyanotoxin-containing water samples. In this study, the toxicity of cyanobacterial samples and purified MC-LR to cause hepatotoxicity in mice was confirmed in vitro using the catfish primary cell line. A comparison among the cyanobacterial samples using EC50 showed the following hepatotoxicity trend in the catfish primary cell line: HBP Dam summer samples > Nhlanganzwani Dam samples > HBP Dam winter samples > Mpanama Dam samples > Sunset Dam samples > Makhohlola Dam samples. The HBP Dam samples were the most hepatotoxic and Makhohlola Dam samples were the least hepatotoxic. The EC50 for purified MC-LR using the catfish primary hepatocytes was about 91 nM. A statistical comparison of the assays used in this study (i.e. ELISA, PPi, mouse test and cytotoxicity [catfish primary hepatocyte] assays) was performed based on the Kappa coefficient (K). An almost perfect agreement (K > 0.80) was observed between the mouse test and cytotoxicity assay; mouse test and ELISA; cytotoxicity assay and ELISA; and ELISA and PPi assay. In conclusion, field samples collected during the summer season were found to have very high levels of toxins and a higher degree of toxicity when compared to the winter samples. The cytotoxicity assay using African sharptooth catfish (Clarias gariepinus) primary hepatocytes has been shown for the first time to produce results similar to those observed when using the mouse bioassay in assessing cyanobacterial toxicity. Therefore, this primary cell line may be used as a potential alternative to the mouse assay in toxicity testing of cyanotoxins. Three KNP dams (Nhlanganzwani Dam, Mpanama Dam and Sunset Dam) investigated in this study were found to contain Microcystis aeruginosa. All four KNP dams (Nhlanganzwani Dam, Mpanama Dam, Makhohlola Dam and Sunset Dam) had cyanotoxin levels above the prescribed guideline value, which is of concern and warrants further investigations to the effects on wildlife in the park. Future studies will include use of High Performance Liquid Chromatography (HPLC) to investigate the toxin profile of the field samples in order to fully describe the different classes/or types of toxins present in the samples. More validation studies that could give a more comprehensive understanding about the sensitivity of the catfish primary cell line for microcystins will also be undertaken. / Dissertation (MSc (Paraclinical Studies))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T09:33:31Z (GMT). No. of bitstreams: 1 Costa_BrunaZucolotoda_M.pdf: 3730198 bytes, checksum: aee2e21aa8a8998b409bff4636299519 (MD5) Previous issue date: 2011 / Resumo: A versatilidade enzimática foi abordada nesta dissertação desde a triagem de microorganismos para a seleção de biocatalisadores adequados, e estudos de interações intermoleculares presentes em sistemas complexos de promiscuidade enzimática, até avaliação da inibição de enzimas relacionadas a ocorrência de diversas enfermidades. As triagens enzimáticas de micro-organismos isolados de água de drenagem de mina de prospecção de cobre permitiram a identificação de hidrolases e monoxigenases, sendo que as cepas de bactérias que apresentaram as melhores atividades oxidativas mostraram-se promissoras para oxidação de diversas classes de sulfetos, em ensaios de biocatálise convencional. A atuação promíscua de lipases foi avaliada frente a oxidação de cetonas cíclicas em meio orgânico, e as interações intermoleculares deste sistema foram estudadas por RMN, onde foi observado características de um sistema organizado em micelas reversas. A partir destas evidências, foi proposto que a condição necessária para a reação enzimática é o confinamento da enzima num ambiente aquoso restrito limitado por uma barreira molecular de ácido graxo. Finalmente a inibição da atividade enzimática de proteína tirosina fosfatases foi avaliada com implementação de uma metodologia fluorimétrica rápida e eficiente. Logo, este trabalho apresentou uma visão ampla no campo da enzimologia com o emprego de biocatalisadores, sejam eles enzimas isoladas ou células íntegras, em estudos relacionados a aspectos químicos, bioquímicos e terapêuticos das enzimas / Abstract: In this thesis the enzymatic versatility was evaluated in microorganisms selecting specific biocatalysts, in studies of intermolecular interactions of complex systems focusing enzyme promiscuity, and last in enzyme inhibition related to the occurrence of various diseases. The enzymatic screening of microorganisms isolated from copper mine water drainage led to the identification of hydrolases and monooxygenases, and bacterial strains with relevant oxidative activities were further investigated in larger scale oxidation of several sulfides. The promiscuous activity of lipases was evaluated in organic medium catalyzing the oxidation of cyclic ketones. The supramolecular interactions involved in these reactions were studied by NMR, revealing the presence of reverse micelles in a highly organized system. Therefore confinement of the enzyme in a restricted aqueous environment limited by a narrow molecular barrier of fatty acids in an organic medium is the necessary condition for this enzymatic reaction to occur. Finally, inhibition of enzymatic activity of protein tyrosine phosphatases was assessed with the implementation of a quick and efficient fluorimetric method. The data presented herewith provide a broad vision of enzyme application, as isolated enzymes or whole cells, in studies addressing chemical, biochemical and therapeutic issues / Mestrado / Quimica Organica / Mestre em Química