Maize is the staple food for a majority of people in Southern Africa, but plant diseases are responsible for at least 10% of crop production losses. Bacterial leaf streak (BLS) of maize was first reported in South Africa in 1949 and has not been reported elsewhere. Very little is known about the pathogen involved and therefore it is deemed necessary to compile a characteristic profile for the pathogen to prevent the possibility of major crop losses as a result of this disease. This study aimed to use biochemical and phenotypic methods to determine the specific characteristics of the causal agent of BLS. Diseased plant material showing symptoms of BLS were collected during the maize production seasons of 2012 and 2013 within South Africa’s maize production regions namely the North West, Free State, Gauteng and Northern Cape provinces. To prevent contamination, maize leaves were surface sterilised thoroughly before bacterial isolation commenced. Sections of the infected maize leaves were placed on GYC agar plates on which yellow, mucoid bacterial colonies after incubation for 24 to 48 hrs. The isolated bacteria were purified and the molecular identification of the bacteria was conducted in a related study. Although literature indicates that Xanthomonas campestris pv. zeae is the causal agent of BLS, pure cultures obtained from maize leaves showing characteristic symptoms of BLS were identified as species of Xanthomonas, Pantoea, and Enterobacter. To elucidate the pathogenicity of the isolated strains, pathogenicity tests based on Koch’s postulates were performed. Results from the pathogenicity tests confirmed that only the isolate Xanthomonas species was capable of inducing the characteristic BLS symptoms when healthy maize plants were inoculated with the suspected pathogens. It is important to inoculate the maize seedlings at the correct age (four-leaf stage) and the spray method is recommended. Re-isolation was repeated from the same plant material used during the initial isolation process but the isolation method was amended. The optimised isolation method involved the use of a dilution range and spread plate method. Colonies from this isolation technique grew as bright yellow colonies that were identified as Xanthomonas spp. This outcome indicates the importance of surface sterilisation, pulverisation and subsequent dilution of plant materials for isolation of bacterial pathogens from diseases plants. These isolates were used to create protein profiles with SDS-PAGE electrophoresis and carbon utilisation patterns with the Biolog® GN2 system. Protein profiling banding patterns was assessed based on presence/absence criteria. Highly similar protein profiles were observed among the X. campestris pv. zeae isolates but groupings of different protein profiles were determined when minor differences in the protein profiles was taken into account. Xanthomonas campestris pv. zeae was successfully distinguished from the X. axonopodis pv. vasculorum reference strain through unique SDS banding patterns. Banding patterns obtained from cultures grown in a liquid medium (tryptic soy broth) were of a higher quality than the banding patterns obtained from bacteria harvested from solid media (CYG agar plates). Carbon source utilisation data was used to evaluate the average well colour development obtained from each isolate. Statistically significant differences were found among some of the isolates, with some isolates being metabolically more active than other isolates. Substrate utilisation patterns produced by the isolates corresponded to previously published studies on various Xanthomonas species. The cell count of the samples used during carbon utilisation patterns must be standardised in order to obtain reliable results. During this study, the application of Koch’s postulates and two inoculation techniques confirmed that Xanthomonas campestris pv. zeae is the pathogen responsible for bacterial leaf streak of maize. Members of the Pantoea and Enterobacter genera were found on the leaf surface of maize plants infected with BLS but inoculations of healthy maize plants with these bacteria did not result in bacterial leaf streak symptoms on the maize plants. These bacteria were not pathogenic and were considered endophytes. The identified pathogen was characterised through protein and metabolic profiling. The protein profiles of the pathogen obtained through analysis of the major bands of the SDS-PAGE gels were highly similar and distinguishable from the Xanthomonas reference culture. Groupings within the X. campestris pv. zeae group was found when major and minor bands were considered, this may however be altered when the intensities of the bands are used during analysis. Carbon utilisation patterns were assessed using Biolog® GN2 plates. A metabolic fingerprint was created for the pathogen of BLS, it was possible to distinguish between X. campestris pv. zeae and other Xanthomonas strains based on the fingerprint. This fingerprint could be used to identify the pathogen. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

Despite our growing knowledge of virus biology they continue to present a problem to global public health. This problem arises from their high mutation rates that allow them to evade antiviral therapies that we have developed to date. An alternative solution for developing antiviral therapies could be to target host cell factors that are hijacked by the virus. The basis of this hypothesis is that if we can stop the virus from using host cell machinery or from evading host immune mechanisms we could treat the infection more efficiently. With the major research focus being on viral proteins and how we can prevent their functions, there is a lot of work to be done in finding host factors that could be the key to treating an infection. The three themes presented in this thesis broadly focus on this goal. The first theme looks at miRNAs, their interacting partners, and their dysregulation during HCV infection. A microRNA is identified from a small molecule screen of miRNAs that are dysregulated during HCV infection and its role in liver immunometabolism is examined to determine its antiviral potential and identify host factors that could be of interest to target with antiviral therapeutics. The second theme examines the potential of activity-based protein profiling techniques for complementing existing antiviral therapies. An azauracil probe is characterized to examine its ability to interact with viral polymerases and its suitability as a building block for antiviral research or therapies. The final theme uses activity-based protein profiling techniques to study a novel carbamate-hydrazone chemotype and establish its suitability as a chemical probe. The hydrazone probe’s reactivity with the mammalian proteome was determined and its interacting partners were identified using chemoproteomic techniques with an overall goal of examining its suitability for antiviral research. Overall, this thesis uses chemical and molecular biology techniques to present three differing perspectives on how to approach the discovery of host factors and develop novel antiviral therapies.

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer. The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided. Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD. In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. / <p>QC 20170302</p>

La tuberculose (TB) reste la maladie infectieuse la plus meurtrière au monde. L’absence de développement de médicaments accompagnée de l'émergence de la résistance aux molécules existantes se traduit principalement par l'échec du traitement et la mort. M. tb infecte l'homme par l'inhalation. Le patient peut rester en bonne santé sans aucun symptôme, on parle alors de tuberculose latente. Dans ce cas, la bactérie résider dans le macrophage. La principale caractéristique de M. tb est de rester cacher à l'intérieur de ces cellules, puis se réactiver lorsque le système immunitaire s’affaibli.Mon travail a été consacrée à l'étude de nouveaux analogues de Cyclophostin / Cyclipostins (CyCs) et Oxadiazolone. Nombreuses molécules présentaient des activités antibactériennes lorsque la bactérie était cultivée dans le milieu liquide ou à l'intérieur du macrophage sans aucun effet toxique sur les macrophages.En utilisant le meilleur inhibiteur de CyC17, nous avons identifié 23 protéines cibles potentielles, étant impliquées dans le métabolisme des lipides de M. tb et / ou dans la biosynthèse des lipides de la paroi cellulaire. Parmi ces cibles, Ag85C et TesA, ont été caractérisées biochimiquement et structurallemnent.De plus, nous avons établi que les CyC sont des inhibiteurs puissants et sélectifs de la croissance des mycobactéries. De manière intéressante, ces composés étaient également actifs contre de nombreuses souches cliniques de M. abscessus, un pathogène opportuniste également appelés « cauchemars pour les antibiotiques », ouvrant ainsi la voie à la recherche de solutions alternatives pour lutter contre les souches mycobactériennes résistantes. / Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. The stagnation in drug development accompanied with the emergence of drug resistance mainly results in treatment failure and death. M. tb infect human through inhalation. Patient can remains healthy without any symptom in which case it is known as latent TB. In latent TB, the bacteria is known to be reside in the macrophage - a type of white blood cell of the immune system, that engulfs and digests infected microorganisms. The famous characteristic of M. tb is the ability to stay inside and avoid to be killed by these cells, then reactivate when the immune system is weak.My work was devoted to the study of two series of new compounds, namely analogs of Cyclophostin/Cyclipostins (CyCs) and Oxadiazolone (OXs). Many molecules exhibited antibacterial activities when the bacteria was grown in the liquid medium or inside the macrophage without any toxic effect to the macrophages.Using the best CyC17 inhibitor, we identified 23 potential candidate target proteins, being involved in M. tb lipid metabolism and/or in cell wall lipid biosynthesis. Among these targets, two important protein, i.e. Ag85C and TesA, were biochemically and structurally characterized.Moreover, we clearly established that the CyCs are powerful and selective growth inhibitors of mycobacteria, with no effect on Gram-negative or Gram-positive bacteria. Interestingly, these compounds were also active against numerous M. abscessus clinical strains, an opportunistic pathogens also called “nightmare for antibiotic”, thus opening the way to find alternative solutions to fight against resistant mycobacterial strains.

<p>Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.</p>

Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.

This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction. Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding arylalkylamide by the enzyme arylalkylamine N-acyltransferase (AANAT). In insects, this enzyme is responsible for controlling melanism, the inactivation of biogenic amines, the sclerotization of the insect cuticle, photoperiodism, and the penultimate intermediate in the production of melatonin. We studied a pair of AANAT enzymes from B. mori: Bm-AANAT and Bm-AANAT3. The former was found to catalyze the direct formation of long-chain acylarylalkylamines (only the second enzyme discovered to do such chemistry), while the latter exhibited potent inactivation of several amines through acetylation. We conducted a full kinetic characterization of Bm-AANAT3, including double-reciprocal plots, pH-rate profiling, dead-end inhibition, and the construction of mutants to elucidate catalytically-relevant amino acids. Our hope is that new insights and discoveries on these enzymatic pathways in model organisms will yield novel molecular targets for human health and disease. We then developed an innovative, microwave-assisted synthesis of a binding-based probe capable of enriching proteins that bind adenine ribose derivatives (AdoRs). We employed this probe in activity-based protein profiling studies to qualitatively assess the AdoR-binding proteome in three bacterial cell lines from the genus Bacillus. This proof of concept experiment demonstrated a unique subset of proteins distinctive to each species, and confirmed the efficacy of the probe tagging and subsequent enrichment. This technology can be used in clinical applications for the detection and identification of cancerous biomarkers. Finally, we successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these TPP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other TPP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Herkömmliche Zytostatika greifen vornehmlich in den Zellzyklus ein und somit werden Zellen mit hoher Proliferationsrate geschädigt. Allerdings fallen hierunter nicht ausschließlich Krebszellen, sondern auch gesunde, schnell proliferierende Gewebearten. Auf Grund dessen verursacht eine klassische Chemotherapie schwerwiegende Nebenwirkungen. Neuere Therapieansätze greifen daher geno- sowie phänotypischer Unterschiede zwischen malignen und gesunden Zellpopulationen auf und können selektiv den zytotoxischen Wirkstoff in die Tumorpopulation einbringen. Dazu werden sogenannte Prodrug-Konzepte verfolgt, bei denen ein möglichst „untoxisches” Prodrug gezielt im entarteten Gewebe enzymatisch zum zytotoxischen Wirkstoff (Drug) aktiviert wird. In diesem Rahmen werden Substrate für die Antibody-Directed Enzyme Prodrug Therapy (ADEPT) hergestellt. Bei diesem Konzept wird eine hohe Tumorspezifität durch Konjugate aus Enzymen und Antikörpern erlangt, indem das Immunglobulin selektiv an tumorassoziierte Antigene bindet und durch das konjugierte Enzym die Drugfreisetzung ermöglicht wird. Die natürlichen zytotoxischen Antibiotika (+)-CC 1065 und (+)-Duocarmycin SA dienen hierbei als Leitstrukturen für die Synthese entsprechender Prodrugs. Im Rahmen der vorliegenden Arbeit wurden insgesamt zwei neue Duocarmycin-analoge Prodrugs sowie neun neue seco-Drugs synthetisiert, wobei vier Vertreter eine terminale Alkinfunktion aufweisen. Für die Darstellung der Prodrugs wurden auf die Galaktose als Glykosideinheit zurückgegriffen. Zudem wurde ein neuartiges dimeres seco Drug hergestellt, das aus zwei pharmakophoren Einheiten sowie einem verbrückenden Linker mit Alkineinheit besteht. Die jeweiligen Substanzen wurden auf ihre In-vitro-Zytotoxizitäten sowie die Eignung für eine Anwendung im ADEPT-Ansatz evaluiert. Neun der neuen Duocarmycin-Analoga wurden in Form von seco- und Prodrugs wurden im Rahmen des aktivitätsbasierten Protein-Profilings untersucht. Hierbei konnte die Aldehyddehydrogenase 1 als wichtiges Angriffsziel der Duocarmycin-Familie verifiziert werden.