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Verticillium wilt is a serious disease in a wide range of economic crops worldwide. Verticillium wilt of potato is caused, primarily, by the fungus Verticillium dahliae. Disease management requires understanding of V. dahliae pathogenesis and interactions with potato, which was the main objective of this study. A differential potato-V. dahliae pathosystem was established where pathogenicity of four V. dahliae isolates with different levels of aggressiveness was evaluated on two potato cultivars, Kennebec (susceptible) and Ranger Russet (moderately resistant). External and internal symptoms and growth measurements revealed that isolates Vd1396-9 and Vs06-14 are highly and weakly aggressive, respectively. These two isolates were selected for transcriptomics and proteomics investigations to identify pathogenicity-related factors. Transciptomics analysis was conducted in both isolates after elicitation by root extracts from either Kennebec or Ranger Russet using a combinational approach involving subtractive hybridization and cDNA-AFLP. A total of 573 differentially expressed transcripts were detected in one or the other isolate. Among them, 185 transcripts of interest were recovered, re-amplified, sequenced and searched against NCBI and the Broad Institute V. dahliae genome databases for identification. The two contrasting-aggressiveness isolates were used for a comparative proteomics investigation. The first proteomic map of V. dahliae was established. The proteomics analysis was carried out using 2-Dimentional electrophoresis and mass spectrometry. Twenty five proteins were differentially expressed and identified in one or the other isolate. Many of the identified genes/proteins showed potential involvement in pathogenesis of V. dahliae or other fungi. Genes of stress response regulator A (oxidative stress tolerance factor), isochorismatase hydrolase (potential plant defense suppressor) and tetrahydroxynaphthalene reductase (involved in melanin and microsclerotia formation) were isolated from both isolates and cloned. Sequence analysis of these genes showed many differences that may explain their differential expression in the two isolates. Given that some of the identified genes/proteins are potentially involved in overcoming and suppressing plant defense, phenolics were profiled in Kennebec-inoculated with Vd1396-9 or Vs06-14 isolate. Chlorogenic, caffeic, ferulic acids, cis-jasmone and rutin accumulation showed variations after inoculation. The results obtained from this study will help understanding the V. dahliae-potato interactions and develop efficient strategies to control Verticillium wilt disease.

Fusarium graminearum causes Fusarium Head Blight, (one of) the most destructive cereal diseases in Canada. Yield loss, quality degradation and mycotoxin production make Fusarium a multifaceted threat. Regulated production of reactive oxygen species by Nox enzymes is indispensable for fungal pathogenesis. F. graminearum Nox mutant ∆noxAB produced equivalent mycotoxin but caused reduced virulence than wild-type. We hypothesized that Nox mediated redox signaling may participate in F. graminearum pathogenicity. Two-DE and gel-free biotin affinity chromatography, followed by LC-MS/MS analysis were employed for a comparative redox-proteomics analysis between wild-type and ∆noxAB to identify proteins oxidized by Nox activity. Total 35 proteins, 10 by 2-DE and 29 by gel-free system, were identified. 34% proteins participated in fungal metabolism, 20% in electron transfer reactions and 9% were anti-oxidant proteins. The findings suggested that Nox mediated thiol-disulfide exchange in proteins provide a switch for redox-dependent regulation of metabolic and developmental processes during induction of FHB.

In this thesis, the detection of global proteomics alteration and changes in neuropeptide distribution caused by neuropathic pain in rat spinal cord tissue was the main focus. Neuropathic pain (NP) is a major clinical syndrome caused by disease or dysfunction of the nervous system and often mediated by neuronal networks in the spinal cord. The estimated prevalence of NP is 6-8% in general population. Only in the United States, the indirect cost associated with chronic pain has been estimated to 100 billion dollars each year and NP substantially contributes to this cost. So far, the underlying mechanisms of NP are not well understood. Proteomics techniques are commonly used in biology system studies, due to its high throughput, capability of unbiased analysis and sensitivity. It builds up a bridge to link genes, peptides, proteins, and the disease. Two proteomic/peptidomic approaches were developed, evaluated and discussed in this thesis. Both of them were further applied in the studies of neuropathic pain. First approach is a quantitative proteomic approach using liquid chromatography combined with Fourier transform mass spectrometry (LC-FTMS), which is developed for quantitative analysis of proteins originated from small central nervous system (CNS) samples. This approach was successfully applied in the study of the rat spinal cord tissue proteome in a neuropathic pain model. Another approach is using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the visualization of the distribution of neuropeptides in rat spinal cord, which in the future will be applied in investigating the ongoing signal transmission under neuropathic pain conditions. Results provided by these two methods are of high importance for the general understanding of the underlying pathophysiological mechanisms and potential identification of new targets for novel treatment of neuropathic pain.

A major obstacle to achieving commercially viable lignocellulosic biofuels through consolidated bioprocessing (CBP) is the lack of “industry-ready” microorganisms. Ideally, a CBP-relevant organism would achieve efficient and complete hydrolysis of lignocellulose, simultaneous utilization of the diverse hydrolysis products and high yields of the desired biofuel. To date, no single microbe has been identified that can perform all of these processes at industrially significant levels. As such, thermophilic decaying woodchip compost was investigated as a source of novel lignocellulolytic, biofuel producing bacteria. From a single sample, a collection of physiologically diverse strains were isolated, which displayed differences in substrate utilization and biofuel production capabilities. Molecular characterization of these isolates, and development of a genome relatedness prediction model based on the chaperonin-60 universal target sequence, identified these isolates as strains of Thermoanaerobacter thermohydrosulfuricus. Application of this model to other Thermoanaerobacter spp. further identified that these isolates belong to a divergent and lesser characterized lineage within the genus. Based on this, the CBP-potential of a single isolate, T. thermohydrosulfuricus WC1, was selected for further investigation through metabolic, genomic and proteomic analyses. Its ability to grow on polymeric xylan, potentially catalyzed by an endoxylanase found in only a few Thermoanaerobacter strains, distinguishes T. thermohydrosulfuricus WC1 from many other strains within the genus. The simultaneous consumption of two important lignocellulose constituent saccharides, cellobiose and xylose was also observed and represents a desirable phenotype in CBP-relevant organisms. However, at elevated sugar concentrations, T. thermohydrosulfuricus WC1 produces principally lactate, rather than the desired biofuel ethanol, as the major end-product. Proteomic analysis identified that all likely end-product forming proteins were expressed at high levels suggesting that the end-product distribution patterns in T. thermohydrosulfuricus WC1 are likely controlled via metabolite-based regulation or are constrained by metabolic bottlenecks. The xylanolytic and simultaneous substrate utilization capabilities of T. thermohydrosulfuricus WC1 identify it as a strain of interest for CBP. However, for its development into an “industry-ready” strain as a co-culture with a cellulolytic microorganism, improved biofuel producing capabilities are needed. The practical implications of CBP-relevant phenotypes in T. thermohydrosulfuricus WC1 in relation to other Thermoanaerobacter spp. will be discussed.

The origin of fluorous interaction was explored and experimentally examined based on both HPLC and CEC data in this project. It was found that the selective fluorous interaction is a kind of reduced instantaneous or induced dipole interaction compared to the hydrophobic interaction. A series of FPPM preparation parameters were optimized. The optimized FPPM column can resolve the components in a manner that was otherwise not possible with its non-fluorous (hydrocarbon) counterpart. Following, the CEC separation of fluorous analytes on FPPM stationary phase based upon fluorous-fluorous interaction was realized for the first time. It was also found that, quantitatively, hydrophobic stationary phases have better methylene selectivity (〖 α〗_(-CH_2-)), while fluorous stationary phases have better perfluoromethylene selectivity (〖 α〗_(-CF_2-)). Thermodynamically, ∆G_(-CF_2- → -CF_2-)^° : ∆G_(-CH_2- → -CH_2-)^° (Gibbs free energy change of transferring a –CF2– unit to pure fluorous stationary phase versus Gibbs free energy change of transferring a –CH2– unit to pure hydrophobic stationary phase) is approximately equal to 8:1. A new concept, hypothetical water percentage (HWP) based on the comparison of 〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-) was proposed for the first time to quantitatively evaluate the hydrophobicity/fluorophilicity of a stationary phase. A stationary phase can be classified as fluorous stationary phase when the HWP is less than 0 (more negative indicates more fluorous), or as a hydrophobic stationary phase when the HWP is larger than 100. For the range between 0 and 100, the stationary phase can be treated as either fluorous or hydrophobic due to the similar values of〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-). Fluorous tagged peptides and proteins (up to 5800 Da) were effectively separated from their non-fluorous counterparts on the FPPM stationary phase in capillary-based columns and detected both on-line with ESI-MS and off-line with MALDI-MS. Finally, the FPPM solid-phase extraction (SPE) stationary phase was transplanted from the capillary to a microchip format. This microchip exhibits the merits of both selective fluorous interaction and micro total analysis system (µTAS). / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-11-19 23:11:16.636

Proteome analysis techonologies are commonly employed for discovery-based biomarker identification studies. This thesis aims to help bridge the gap between analytical technology development and clinical application by improving and appling a proteomics workflow for biomarker discovery in congenital urinary tract obstruction (UTO). By accentuating the importance of experimental design, and evaluating the biological relevance of quantitative proteome analyses, the results of this research provide confidence in a number of identified candidate biomarkers of UTO. A sensitive method for quantification of proteome samples was developed using temperature controlled reversed-phase liquid chromatography (TPLC). The TPLC system provides high recovery (> 90 %), as well as high accuracy and precision in estimating the concentration across a number of protein sample types (CV < 10 %). The need for extensive fractionation strategies coupled with LC-MS analysis challenges the throughput of the overall experiment. Development of a dual column LC-MS interface reduced the total analysis time by a factor of 2 over conventional single column LC-MS systems. The system was applied to a quantitative proteome analysis of proximal tubule cells exposed to mechanical stretch, mimicking the conditions they experience during UTO and a urinary exosomal proteome analysis for candidate biomarker identification of this disease. A total of 1636 proteins were identified in the whole cell proteome analysis, of which 317 were found to be significantly altered in abundance. Analysis of the urinary exosomal proteome yielded 318 proteins, of which 189 were found to be altered in abundance due to obstruction. Western blot confirmation of a few select proteins provided backing to the quantitative proteome analysis, while gene ontology and KEGG pathway analysis yielded functional information. The results from the quantitative analyses of the urinary exosomes and proximal tubule cells identified candidates for both diagnosis and prognosis of UTO. In addition, activation of a novel pathway was identified, presenting a potential drug target which could be exploited to improve recovery of children following relief of UTO. This thesis therefore contributes useful technological and methodological advancements towards routine proteome analysis, as well as providing candidate biomarker identification for the leading cause of renal functional loss in children.

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.