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.

Mass spectrometry is the primary technology of proteomics. For the analysis of complex proteomes, protein identities and quantities are inferred from their peptides that are generated by cleaving all proteins with the endopeptidase trypsin. But there is one major disadvantage that is due to biophysical differences, different peptides cause different intensities. Miscellaneous approaches have been developed to circumvent this problem based on the chemical or metabolic introduction of heavy stable isotopes. This enables to monitor protein abundance differences of two or more samples on the same tryptic peptides that differ in mass only. Absolute quantification can be achieved similar by spiking-in synthetic isotopical labeled counterparts of a sample’s tryptic peptides. However, labeling technics suffer from high prices, introduced biases, need for extensive manual control, laborious implementation and implementation restrictions. Therefore, a multiplicity of label-free approaches have been developed that profit from instrumental improvements targeting reliability of identifications and reproducibility of quantitative values. No extensive systematic comparison of label-free quantitative parameters has been published so far presumably because of the laborious implementation. An analysis of primary label-free parameters and associated normalization methods is presented here that compares dynamic and linear ranges and accuracies in the estimation of protein amounts. This facilitated the establishment of label-free procedures addressing three fundamental questions in proteomics: what is a sample’s composition, are proteins that share a specific property enriched and what are the differences between two (or more) samples. A new mathematic model is presented that defines and elucidates enrichment. The procedures were applied first to analyze and compare stem cell plasma membrane proteomes. This is an ambitious model for proteomics because of only small amounts of arduous to analyze, partial hydrophobic proteins in a complex proteomic and chemical background. It is of scientific relevance, as membrane proteins are the cell’s communication interface that enable cell type specific processes and hence can be used to define, isolate and quantify those. The success of cell surface proteome enrichment, the quantitative composition of the proteome and the proteomic difference between stem cells isolated from the dental pulp and cultivated in different media is shown. Secondly, the procedures were applied to the analysis of transient protein networks that assemble onto proteo-liposomes in a newly designed recruitment assay that fully recapitulates membrane sorting as seen in vivo. All transmembrane proteins need to be trafficked to other organelles’ membranes by vesicular trafficking. Sorting signals within the cytosolic regions of the protein cargos trigger the formation of trafficking complexes around those. The transient membrane complexes additionally recognize organelle or organelle-domain specific membrane lipids, such as phosphatidylinositol phosphates. Different trafficking ways are characterized by different trafficking complexes. The elucidation of trafficking complexes that form around a transmembrane protein of interest discloses its trafficking routes and involved signaling processes. The synthetic proteo-liposomes were prepared from chemically defined lipids and heterologous expressed cytosolic domains of type I or type II membrane receptors. The proteomic analyses of such samples are challenging because of huge proteomic backgrounds of proteins binding to the liposomes irrespective of the receptor and relatively small amounts and numbers of receptor-specific binders. Though the basic idea is to elucidate sorting machineries and study membrane trafficking processes, such experiments are untargeted and miscellaneous discoveries were achieved. We elucidated that the apical determinant crumbs 2 is a cargo of the retromer complex. This revealed a fameless level of control for the establishment of cell polarity. We found retromer along with the adapter complexes AP 4 and AP 5 trafficking the beta amyloid precursor protein APP. This confirmed recent publications and yielded new insights. Moreover, many more proteins and complexes appeared to associate with the cytosolic part of APP (AICD) in a membrane context-dependent or -independent manner. Among those, some were so far unknown to interact with AICD, like mTORC1 and the PIKFyve complex.

Biomarker validation remains one of the most important constraints to development of new clinical diagnostic assays. To address this challenge, an immuno-mass spectrometric assay known as SISCAPA has been developed for quantitation of protein biomarkers in human blood. The SISCAPA assay overcomes the sensitivity barrier facing most mass spectrometric approaches by utilizing high affinity antibodies for enrichment of specific surrogate peptide analytes from complex mixtures such as trypsin-digested human plasma. However, several technological barriers remain before the SISCAPA technology gains widespread use for biomarker validation. Improvements are required in areas such as selection of high affinity anti-peptide antibodies, peptide detection sensitivity and increasing sample throughput to allow biomarker validation on large sample sets. The work presented in this dissertation describes the development of new methods for antibody selection and for high-throughput application of SISCAPA technology to biomarker measurement in human plasma. Specifically, two technological developments are described: 1) an assay called MiSCREEN was developed, which allows high-throughput screening of anti-peptide antibodies, enabling selection of high affinity reagents for de novo SISCAPA assays and 2) a liquid chromatography (LC)-free SISCAPA assay was developed that enables quantitation of surrogate peptides using both MALDI-TOF and RapidFire/MS platforms. Taken together, these technological advances provide a meaningful solution to the biomarker validation dilemma and allow a unified system for biomarker qualification, verification, validation and development of clinical assays for diagnosis and monitoring of a variety of diseases. To demonstrate the utility of the unified SISCAPA system for biomarker measurement, an assay was developed for protein C inhibitor (PCI) as a marker for prediction of biochemical recurrence in prostate cancer patients. The PCI-specific analyte was shown to predict biochemical recurrence of prostate cancer after radiation/hormone treatment. Early stage detection of recurrence was achieved, when compared to the ‘gold standard’ marker for prostate cancer, prostate specific antigen (PSA). Two-dimensional gel electrophoresis studies on PCI, revealed unique protein spots in a serum sample from a biochemically recurrent patient. Studying such alterations at the protein level may enable understanding of the molecular mechanisms by which PCI is involved in prostate cancer progression. / Graduate

Research was undertaken to discover and study trypanosome proteins that may play important roles in host-parasite or vector-parasite interactions. The methods used mass spectrometry based proteomics ideally suited for analysis of low abundance molecules. First, isobaric tags were used to monitor changes in proteins expression throughout the life cycle of Trypanosoma congolense, an economically important livestock pathogen. This was the first large scale survey of protein expression in trypanosomes. In addition to generating protein expression data for approximately 2000 different parasite proteins, 6 previously undescribed T. congolense proteins were discovered. Several of the proteins with interesting expression trends were selected for molecular characterization and monoclonal antibody derivation. Second, immunoenrichment and mass spectrometry were used to identify the cognate antigen recognized by a T. congolense-specific monoclonal antibody. The antigen, a flagellar calcium binding protein, was expressed as a recombinant protein and used to test its utility as a potential serodiagnostic antigen for diagnosis of T. congolense infections. Third, a “deep-mining” protein discovery mass spectrometric method was used to identify trypanosome proteins present in the plasma of late-stage African sleeping sickness patients. A total of 254 trypanosome proteins were unequivocally identified by tandem mass spectrometry. These findings are unprecedented since never before have such a large number of pathogen proteins been discovered in human blood using a non-biased approach (i.e. without using a targeted assay). The proteins discovered provide insights into host-parasite interactions and are strong candidates as targets for new diagnostic assays. / Graduate

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

Glaucoma is a leading cause of irreversible blindness worldwide. Primary Open Angle Glaucoma is the most common form of the disease and can be characterized by the slow and irreversible apoptotic death of retinal ganglion cells, a unique optic nerve neuropathy resulting in loss of vision. Increased intra-ocular pressure is known to be a leading risk-factor for glaucoma, and lowering IOP is currently the only evidence based method for the clinical management of the disease. However the exact mechanism by which an elevated IOP leads to the death of the retinal ganglion cells is still poorly understood. By using previous finite element models of glaucoma to quantify the biomechanical environment within the optic nerve head we have built human primary cell culture models in an attempt to replicate aspects of early glaucomatous optic neuropathy. In these models we mimic the in vivo biomechanical environment in the lamina cribrosa by growing human optic nerve head astrocytes and lamina cribrosa cells on compliant substrates and subjecting the cells to deformation. Specifically, a global protein scan using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on all the experiments to identify potential biomarkers for glaucoma. A secondary analysis using enzyme-linked immunosorbent assay (ELISA) identified extracellular proteins of interest. Over 520 proteins were identified in response to biomechnical strain from both cell types. Many of these proteins centred on TGF-, p53 and TNF, which have previously been shown to play a role in the pathogenesis of glaucoma. Proteins found in astrocytes were astrocytic phosphoprotein (PEA15), UDP-glucose dehydrogenase (UGDH), and annexin A4 (ANXA4). LC proteins were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66) and Eukaryotic translation initiation factor 5A (eIF-5A). These proteomic results will enable a series of functional studies looking into the role select markers play in ONH glial cell activation, a process still not well understood. Candidates for this work will be prioritized based on novelty and relevance to mechanisms of cellular stress and death. We hypothesize that study of these molecular pathways will provide insight into this process, as well as improve our understanding of how glial activation contributes to the development of glaucomatous optic neuropathy.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.