L’oncogène v-erbA transforme les progéniteurs érythrocytaires primaires aviaires (T2EC) en bloquantleur engagement d’un programme d’auto-renouvellement vers un programme de différenciation. Unecomparaison trancriptomique de T2EC exprimant soit v-erbA, soit une forme non transformante de verbAa été réalisée par SAGE et RT-qPCR. Seuls quelques uns, mais pas tous les messagers codant lesprotéines ribosomiques sont réprimés. Ces résultats suggèrent que v-erbA pourrait moduler lacomposition des ribosomes et/ou moduler les fonctions extra-ribosomiques de protéines ribosomiquesspécifiques. Ainsi, nous avons décidé d’analyser le taux des protéines ribosomiques associées auxribosomes par 2D-DIGE à partir des ribosomes purifiés. L’analyse statistique effectuée sur 4expériences indépendantes avec des marquages inversées a montré de manière significative que letaux de RPL11 est inférieur dans les T2EC exprimant v-erbA comparé à ceux exprimant la forme nontransformante de v-erbA. Ces données indiquent l’existence de ribosomes dépourvus de RPL11 dansles T2EC sous l’effet de v-erbA. Les résultats des expériences d’immunoprécipitation ont conforté cettehypothèse. L’ensemble des résultats obtenus suggèrent l’implication des protéines ribosomiques, etspécialement celle de RPL11, dans les processus de transformation induite par l’oncogène v-erbA, à lafois au niveau de la traduction, et probablement par sa fonction extra-ribosomique. L’analyse de lafonction biologique de RPL11 a montré qu’une sur-expression de RPL11 dans les T2EC retarderait laprolifération cellulaire. / The v-erbA oncogene transforms chicken erythroid progenitors by blocking their differentiation andpreventing them to exit a state of self-renewal. The transcriptome of primary avian erythroidprogenitors cells (T2EC) expressing either v-erbA or a non-transforming form of v-erbA werecompared by SAGE. Only some, but not all, mRNAs encoding ribosomal proteins were shown to beaffected. These results suggest that v-erbA could modulate the composition of ribosomes and/ormodulate the extraribosomal functions of specific ribosomal proteins. We therefore decided to analyzethe level of ribosomal proteins associated to ribosomes by 2D-DIGE performed on purified ribosomes.A statistical analysis performed on 4 independent flip-flop experiments demonstrated that the level ofRPL11 is significantly lower in T2EC expressing v-erbA as compared to the non-transforming form ofv-erbA. These data suggest the presence of ribosomes without RPL11 in T2EC expressing v-ErbA.Results obtained from immunoprecipitation experiments were strengthened this hypothesis. The set ofthese data evoke the involvement of ribosomal proteins, and specially RPL11, in the v-erbAtransformation process both at the translational level and possibly in its extra-ribosomal function.Overexpression of RPL11 in T2EC showed a decrease of cell proliferation.

Proteomics based on liquid chromatography coupled to mass spectrometry has developed rapidly in the last decade and become a powerful tool for protein mixtures analysis. LC-MS based proteomics involves four steps, sample preparation, liquid chromatography, mass spectrometry and bioinformatics. Improvements in each step have extended its applications to new biological research areas. This dissertation mainly focuses on method developments in both qualitative and quantitative proteomics. The first part of this dissertation focuses on qualitative analysis of T. gondii Parasitophorous Vacuole Membrane (PVM) proteins, which is very important for T. gondii’s survival. The hypothesis of this study is that proteomic approaches coupled with immunoprecipitation using polyclonal antisera as affinity reagents can successfully characterize the proteome of the T. gondii PVM. The “Three-layer Sandwich Gel Electrophoresis” (TSGE) protocol, was developed to contend with efficient salt removal and protein concentration from challenging samples. Furthermore, the TSGE coupled to 2D-LC-MS/MS was proven to be effective with the proteomic analysis of complex protein mixtures like T. gondii whole cell lysate, allowing for high-throughput protein analysis from complex samples. By using the TSGE-2D-LC-MS/MS methodology, we successfully identified 61 proteins from the PVM samples and constructed the PVM proteome. The second part of this dissertation describes a novel method for selecting an appropriate isocyanate reagent for potential quantitative proteomics application. Our hypothesis is alteration of isocyanate structure will change fragmentation pattern and ESI property of isocyanate modified peptides. The CID property of N-terminal modified peptides by phenyl isocyanate (PIC), phenethyl isocyanate (PEIC) and pyridine-3- isocyanate (PyIC) was systematically studied using LC-ESI-MS/MS. We observed that adjustment of isocyanate structure changed both ESI and fragmentation characteristic of modified peptides. We rationalized the decrease of protonation of PIC and PEIC modified peptides results from the neutral property of the both reagents. The electron withdrawing feature of PyIC leads to significant reduction of fragments during CID. Therefore, we designed a new isocyanate reagent, 3-(isocyanatomethyl) pyridine (PyMIC). The results revealed that PyMIC modified peptides had more suitable ESI properties and generated more sequence-useful fragments compared to PIC, PyIC and even unmodified peptides. PyMIC is a more appropriate labeling reagent for quantitative proteomics applications.

Mass spectrometry coupled with liquid chromatography and gel electrophoresis enables separation and detection of components in a complex mixture. During the last two decades, its applications were dramatically extended and remarkable progress has been made in many fields, in particular, environmental and biological analyses. This dissertation focuses on identification and characterization of biologically active compounds and comparative analysis of protein expression changes. The first two projects (Chapters 2 and 3) focus on the application of LC/MS approach to profile the bioactivated intermediates of 4, 4'-methylenedianiline (DAPM) from rat vascular smooth muscle cells (VSMCs) and bile. In our study, several DAPM metabolites were detected and characterized in detail by liquid chromatography-electrospray tandem mass spectrometry. The structural assignments of these metabolites from VSMCs and rat bile significantly improve our understanding of DAPM biotransformations and toxicity. The third project described in Chapter 4 focuses on using electrospray tandem mass spectrometry (ES-MS/MS) and theoretical calculation (GAUSSIAN 03 program) to investigate the unusual methyl radical loss and consecutive fragment ions that dominate the low-energy collision induced dissociation (CID) mass spectra of prodiginine compounds. Structures of the fragment ions are proposed and explanations are given to rationalize the observed competition between the formation of even-electron ions and radical ions. Our study shows that the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. In Chapter 5, hypoxia-induced changes of zebrafish skeletal muscle were studied using two-dimensional difference in-gel electrophoresis (2D-DIGE) in vivo after 48 h in hypoxia vs. normoxia. The results showed that proteins involved in mitochondrial oxidative metabolism are down-regulated, whereas glycolytic enzymes are up-regulated to compensate for the loss of ATP synthesis in aerobic metabolism. The up-regulation of two spots identified as hemoglobin variants was also observed. These protein expression changes are consistent with a hypoxic response that enhances anaerobic metabolism or O2 transport to tissues.

ABSTRACT Few antibiotics are left that are effective against methicillin-resistant Staphylococcus aureus (MRSA) and even strains resistant to these agents have been isolated. Previous studies have identified five distinct MRSA clonotypes, which are present globally. No comprehensive national study has previously been undertaken to investigate the MRSA types in South Africa, and this study was aimed at elucidating the genotypic population structure of South African MRSA isolates. SmaI digested genomic DNA, separated by pulsed-field gel electrophoresis, was used to characterise 349 S. aureus isolates, obtained from various state and private diagnostic laboratories. PFGE results were complemented with those of spa typing and staphylococcal cassette chromosome mec (SCCmec) typing results. Two-hundred-and-five different PFGE patterns were identified, which were grouped into twenty-four clusters. Three were major lineages, containing more than 20% of the isolates with a similarity cut-off of 70%. Only thirty-seven spa types were identified (fourteen novel spa types), which clustered into six spa-Clonal Complexes after BURP analysis. SCCmec types I-IV were identified, including variants of each type. Data suggest that the Archaic clone (RSA05), oldest of the epidemic clones, represents one of the major clones in South Africa. Strains that were part of this complex (n=98 (28.2%); t064; SCCmec type I-pls) clustered together with strain E2125/ATCC BAA-38 (t051; SCCmec type I). Another major complex, RSA16 (n=90 (25.7%); t012; SCCmec type II/IIB) possessed a single-locus variant (SLV) spa type and the same or a SLV SCCmec types as EMRSA-16 (t018; SCCmec type II). The third major complex, RSA03 (n=74 (21.2%); t037; SCCmec type III/IIIE), had similar spa and SCCmec types to control strainANS46 (t037; SCCmec type III). One MRSA and twelve MSSA isolates were also identified as carrying genes for the toxin Panton-Valentine leukocidin, which was confirmed by DNA nucleotide sequencing.

A esquizofrenia é uma doença crônica, grave e incapacitante que afeta cerca 24 milhões de pessoas em âmbito mundial. É caracterizada por uma desorganização no pensamento que prejudica a funcionalidade do indivíduo. Existem intervenções que são efetivas e contribuem para a diminuição da prevalência do transtorno, pois ajudam o portador a levar uma vida produtiva e integrada à sociedade, porém devem ser ministradas nos estágios iniciais da doença. No entanto, existe uma grande dificuldade em se diagnosticar a esquizofrenia precocemente devido a sua complexidade e às sutilezas dos seus sintomas apresentados antes do surgimento da psicose. O cérebro não é acessível a exames invasivos in vivo e por esse motivo a exploração de fluidos periféricos é de grande importância. As plaquetas e neurônios serotonérgicos possuem características bioquímicas e morfológicas em comum que possibilitam a comparação entre a estrutura e a função de ambos e, por causa dessa similaridade, muitos trabalhos utilizam plaquetas como modelo para o estudo de doenças neuropsiquiátricas, inclusive a esquizofrenia. A detecção precoce da esquizofrenia é um objeto de investigação atual e relevante não somente para revolucionar os meios atuais de diagnóstico, mas também para desenvolver novos tratamentos aplicados aos estágios iniciais da doença, diferenciar os subgrupos de doentes e monitorar as intervenções preventivas. A proposta do presente trabalho é fazer o estudo da expressão de proteínas em plaquetas de pacientes esquizofrênicos e controles com o objetivo de identificar proteínas candidatas a biomarcadores utilizando técnicas proteômicas quantitativas e confiáveis, como 2D-DIGE e a espectrometria de massas. / Schizophrenia is a disabling, serious, and chronic illness, which affects about 24 million people worldwide. It is characterized by a severe disorganization of the thoughts that harms the social life of patients becoming them dependent of the family and/or government. There are effective treatments that contribute to decrease the prevalence of the disorder because they improve the life and social conditions of the patients, but they are only advantageous if the intervention is made in the early stages of the disease. It is difficult to obtain early diagnosis due to the complexity of the disease and its insidious symptoms before the beginning of the psychosis. The brain is not easily accessed in vivo and, because of this, it is very important to study the peripheral tissues like blood, which makes the use of the platelets very interesting. Furthermore, platelets and serotonergic neurons share biochemical and morphological characteristics that allows the comparison between structure and function of both. From these similarities many authors has used platelets as a neuron model to study many neurodegenerative diseases including schizophrenia. The early detection of schizophrenia is a current and suitable goal, not only to improve the early diagnosis but also to develop new treatments, differentiate the subtypes, and monitor the preventive interventions. The purpose of this project is to do a comparative screening of expressed proteins in platelets from schizophrenics and controls with the objective of finding differently expressed proteins that could be candidates to biomarkers using 2D-DIGE and mass spectrometry.

<p>The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed.</p><p>The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. <i>N,N’</i>-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, <i>i.e.,</i> a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power.</p><p>A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.</p>

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed. The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power. A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

Methyl tert-butyl ether (MTBE) has been used as a gasoline additive to improve the combustion efficiency and to replace lead since 1978. It is the most commonly used oxygenate now due to its low cost, convenience of transfer, and ease of blending and production. MTBE has become a prevalent groundwater contaminant because it is widely used and it has been disposed inappropriately. MTBE has been demonstrated an animal carcinogen. The US Environmental Protection Agency (US EPA) has temporarily classified MTBE as a possible human carcinogen and has set its advisory level for drinking water at 20-40 &#x00B5;g/L based on taste and odor concerns. The Taiwan Environmental Protection Administration (TEPA) also classifies it as the Class IV toxic chemical substances. Currently, natural attenuation (NA) as well as natural bioremediation or enhanced bioremediation are attractive remediation options for contaminated sites due to their economic benefit and environmental friendly. In general, in situ microorganisms at the contaminated site play a very important role in site restoration. Although early studies suggested that the biodegradability of MTBE was not significant, recent laboratory and field reports reveal that MTBE can be biodegraded under aerobic and anaerobic conditions. In addition, evidences and some successful cases of MTBE attenuation have been reported that make natural attenuation a considerable remedial strategy. However, the biodegrading rate might decrease if the nutritional and physiological requirements are not met. Thus, it is important to assess the biodegradability of natural microorganisms under various site conditions to obtain optimal remedial conditions. Contributions of intrinsic biodegradation and other abiotic mechanisms to the removal and control of contaminants should also be evaluated to provide sufficient information for remedial option determination. Moreover, isolation and identification of the dominant native microorganisms will be helpful to following remediation tasks. In the first part of this study, microcosm study and microbial identification technologies (denaturing gradient gel electrophoresis, DGGE) were applied to assess the biodegradability of MTBE by indigenous microbial consortia and to identify the dominant microorganisms at a MTBE-contaminated site (Site A). In the second part of this study, thorough field investigations were performed to evaluate the occurrence of natural attenuation of MTBE at two MTBE-contaminated sites (Site A and Site B). In addition, a natural attenuation model, BIOSCREEN, was performed to assess the effectiveness of natural attenuation on MTBE containment. The main objectives of this study contained the following: (1)Evaluate MTBE biodegradability under different redox conditions by the indigenous microorganisms. (2)Determine the dominant native microorganisms in MTBE biodegradation for further application. (3)Assess the feasibility of using natural attenuation to control the MTBE plume. (4)Evaluate the contributions of intrinsic biodegradation patterns on natural attenuation processes by BIOSCREEN. Results from the microcosm study reveal that MTBE could be biodegraded by aquifer sediments without the addition of extra carbon sources under aerobic conditions. The production of tert-butyl alcohol (TBA), a degradation byproduct of MTBE, was detected. Complete removal of TBA was also observed by the end of the experiment. Results from aerobic microcosms study indicate that oxygen might be the major limiting factor of MTBE biodegradation at Site A. Thus, MTBE at this site could be removed via natural biodegradation processes with the supplement of sufficient oxygen. Microcosm study with extracted supernatant of aquifer sediments as the inocula show that the indigenous microorganisms were capable of using MTBE as the sole carbon and energy source. The calculated MTBE degradation rate was 0.597 mg/g cells/h or 0.194 nmole/mg cells/h. No MTBE removal was observed under various anaerobic conditions. Results suggest that aerobic biodegradation was the dominant degradation process and aerobic bioremediation might be a more appropriate option for the site remediation. According to the results of DGGE analysis, aerobic MTBE-biodegrading bacteria, Pseudomonas sp. and Xanthomonas sp., might exist at this site. Although results of microcosm study show that MTBE could not be degraded under anaerobic conditions, the microbial identification indicates that some novel anaerobic microbes, which could degraded MTBE, might be present at this site. In addition, anaerobic microbes caused the consumption of electron acceptors (e.g., nitrate, ferric iron) and removal of benzene, toluene, ethylbenzene, xylenes (BTEX), 1,2,4-trimethyl benzene (1,2,4-TMB), and 1,3,5-trimethyl benzene (1,3,5-TMB) (TMBs) in the anaerobic microcosms. These results also indicate that the potential of anaerobes activities was high at Site A. Based on the results from the field investigation, natural attenuation of MTBE was occurring at both sites. MTBE plume at Site B could be effectively controlled via natural attenuation processes. Nevertheless, MTBE plume at Site A has migrated to a farther downgradient area and passed the boundary of the site. Field investigation results indicate that the natural attenuation mechanisms of MTBE at both sites were occurring with the first-order attenuation rates of 0.0021 and 0.0048 1/day at Sites A and B, respectively. According to BIOSCREEN simulation, biodegradation was responsible for 78% and 59% of MTBE mass reduction at Sites A and B, respectively. The intrinsic biodegradation had significant contributions on the control of MTBE plumes. Moreover, the dilution and dispersion processes might be the major mechanisms for the attenuation of MTBE in the downgradient areas. However, results also reveal that intrinsic biological processes might still fail to contain the plume if the selected point of compliance is not appropriate. Results of this study suggest that natural attenuation might be feasible to be used as a remedial option for the remediation of MTBE-contaminated site on the premise that (1) detailed site characterization has been conducted, and (2) the occurrence and effectiveness of natural attenuation processes have been confirmed. Based on the results from the field investigation and laboratory microcosm studies, MTBE could be biodegraded by natural microbial populations at the studied sites under both aerobic and anaerobic conditions and natural attenuation would be applied as a remedial option at MTBE-contaminated sites. Results from this study would be useful in determining the favorable bioremediation conditions and designing an efficient and cost-effective bioremediation system such as monitored natural attenuation (MNA) or in situ or on-site MTBE bioremediation system for field application.

The genus Enterococcus comprises of a group of commensal organisms of the human gut which has been associated with cases of endocarditis and urinary tract infections. In the present study, 12 Enterococcus isolates were obtained from clinical specimens and characterized using genotyping techniques that have become an integral part of clinical research. There were three different genotyping methods used to identify the enterococci to species level and to determine the level of genetic diversity among the selected strains. These techniques were, randomly amplified polymorphic DNA-PCR (RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis (PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and vancomycin were also determined using a disc diffusion assay and a microtitre plate dilution assay. All twelve strains were found to be vancomycin resistant enterococci (VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on MIC values were categorized into three groups, susceptible (< 0.25 μg/ml), intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was performed using four random primers. Primers yielding the highest discriminative power were used for phylogenetic analysis. The phylogenetic analysis indicated that all 12 strains yielded clonal dissemination, therefore a low genetic diversity between them. The 16S rDNA of all strains were used to identify the enterococci at species level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and found to belong to three species of Enterococcus. These were E.faecalis, E.faecium and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE gives a higher discriminatory power as a higher dissimilarity between the strains was observed. Similar penicillin MICs for each of the strains in the three categories are grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR is a sensitive, faster, specific and cost effective technique, PFGE analysis has given a higher discriminatory power, higher reproducibility of the results and the polymorphism seen in the patterns suggest that PFGE has a potential of being an essential tool in clinical diagnostics. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.