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    • The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

      Centre for Biotechnology (Brock University, 2005-05-28)
      Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.
    • The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

      Martin, Stephanie J.; Centre for Biotechnology (Brock University, 2008-05-28)
      The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii
    • An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

      Abdalla, Moemen.; Centre for Biotechnology (Brock University, 2009-01-28)
      Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.
    • Enantiodivergent chemoenzymatic synthesis of codeine

      Leisch, Hannes G.; Centre for Biotechnology (Brock University, 2009-05-28)
      The present thesis describes our latest results in the chemistry of morphine alkaloids. An enantiodivergent synthesis of codeine utilizing a cis-cyclohexadiene diol derived from microbial whole cell oxidation of ~-bromoethylbenzene,as starting material is discussed. The total synthesis of (+)-codeine in 14 steps featuring a Mitsunobu inversion and two intramolecular Heck cyclizations is presented. Investigation of a regioselective nucleophilic opening of a homochiral vinyl oxirane, which led to a total synthesis of the natural isomer of codeine, is detailed. Furthermore, described herein are novel methodologies designed for the transformation of naturally occurring opiates into medicinally relevant derivatives. Two studies on the conversion of thebaine into the commercially available analgesic hydrocodone, two novel ·transition metal catalyzed N-demethylation procedures for opioids, and the development of a catalytic protocol for N-demethylation and Nacylation of morphine and tropane alkaloids are presented. In addition, reactions of a menthol-based version of the Burgess reagent with epoxides are discussed. The synthetic utility of this novel chiral derivative of the Burgess reagent was demonstrated by an enantiodivergent formal total synthesis of balanol. ii
    • Two enantiodivergent syntheses of balanol and the chemoenzymatic synthesis of oseltamivir

      Sulliva, Braddord Thomas; Centre for Biotechnology (Brock University, 2010-10-25)
      The present thesis outlines our latest findings on the reactivity of the Burgess reagent with oxiranes. Structural, mechanistic, and computational studies are presented. Included is the development of a (-)-menthyl version of the Burgess reagent and its application to the synthesis of enantiomerically pure ~-amino alcohols. This methodology has been exploited in the formal enantiodivergent synthesis of the (+)- and (-)-isomers of balanol. Also described is a second generation approach to both balanol enantiomers; each commencmg with the chemoenzymatic dihydroxylation of bromobenzene. This study also describes the steric and functional limitations of the toluene dioxygenase-mediated oxidation of benzoate esters. The metabolite derived from ethyl benzoate was employed in a formal synthesis of oseltamivir. Finally, several synthetic approaches to oseltamivir and its analogs are presented, each proceeding through a different vinyl aziridine derived from bromobenzene and ethyl benzoate.
    • Unique and unifying themes in the mechanisms regulating the expression of the arabidopsis thaliana PR-1 and Solanum tuberosum PR-10a inducible defense genes

      Boyle, Patrick; Centre for Biotechnology (Brock University, 2010-10-25)
      Arabidopsis is a model plant used to study disease resistance; Solanum tuberosum or potato is a crop species. Both plants possess inducible defense mechanisms that are deployed upon recognition of pathogen invasion. Transcriptional reprogramming is crucial to the activation of defense responses. The Pathogenesis-Related (PR) genes are activated in these defense programs. Expression of Arabidopsis PR-l and potato PR-10a serve as markers for the deployment of defense responses in these plants. PR-l expression indicates induction of systemic acquired resistance (SAR). Activation of SAR requires accumulation of salicylic acid (SA), in addition to the interaction of the non-expressor of pathogenesis-related genes I (NPRI), with the TGA transcription factors. The PR-10a is activated in response to pathogen invasion, wounding and elicitor treatment. PR-10a induction requires recruitment of the Whirly I (Whyl) activator to the promoter. This locus is also negatively regulated by the silencer element binding factor (SEBF). We established that both the PR-l and PR-10a are occupied by repressors under non-inducing conditions. TGA2 was found to be a constitutive resident and repressor of PR-l, which mediates repression by forming an oligomeric complex on the promoter. The DNA-binding activity of this oligomer required the TGA2 N-terminus (NT). Under resting conditions we determined that the PR-10a is bound by a repressosome containing SEBF and curiously the activator Pto interacting protein 4 (Pti4). In the context of this repressosome, SEBF is responsible for PR-10a binding, yet rWe also showed that PR-l and PR-10a are activated by different means. In PR-l activation the NPRI NT domain alleviates TGA2-mediated repression by interacting with the TGA2 NT. TGA2 remains at the PR-l but adopts a dimeric conformation and forms an enhanceosome with NPRl. In contrast, the PR-10a is activated by evicting the repressosome and recruiting Why! to the promoter. These results advance our understanding of the mechanisms regulating PR-l and PR-10a expression under resting and inducing conditions. This study also revealed that the means of regulation for related genes can differ greatly between model and crop s
    • Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

      Zhang, Wen Xiao; Centre for Biotechnology (Brock University, 2010-10-26)
      Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.
    • Regulation of Systemic Acquired Resistance through the Interaction of Arabidopsis thaliana Transcription factors TGAI and TGA2 with NPRI

      Rochon, Amanda; Centre for Biotechnology (2012-07-31)
      Arabidopsis thaliana is an established model plant system for studying plantpathogen interactions. The knowledge garnered from examining the mechanism of induced disease resistance in this model system can be applied to eliminate the cost and danger associated with current means of crop protection. A specific defense pathway, known as systemic acquired resistance (SAR), involves whole plant protection from a wide variety of bacterial, viral and fungal pathogens and remains induced weeks to months after being triggered. The ability of Arabidopsis to mount SAR depends on the accumulation of salicylic acid (SA), the NPRI (non-expressor of pathogenesis related gene 1) protein and the expression of a subset of pathogenesis related (PR) genes. NPRI exerts its effect in this pathway through interaction with a closely related class of bZIP transcription factors known as TGA factors, which are named for their recognition of the cognate DNA motif TGACG. We have discovered that one of these transcription factors, TGA2, behaves as a repressor in unchallenged Arabidopsis and acts to repress NPRI-dependent activation of PRJ. TGA1, which bears moderate sequence similarity to TGA2, acts as a transcriptional activator in unchallenged Arabidopsis, however the significance of this activity is J unclear. Once SAR has been induced, TGAI and TGA2 interact with NPRI to form complexes that are capable of activating transcription. Curiously, although TGAI is capable of transactivating, the ability of the TGAI-NPRI complex to activate transcription results from a novel transactivation domain in NPRI. This transactivation domain, which depends on the oxidation of cysteines 521 and 529, is also responsible for the transactivation ability of the TGA2-NPRI complex. Although the exact mechanism preventing TGA2-NPRI interaction in unchallenged Arabidopsis is unclear, the regulation of TGAI-NPRI interaction is based on the redox status of cysteines 260 and 266 in TGAl. We determined that a glutaredoxin, which is an enzyme capable of regulating a protein's redox status, interacts with the reduced form of TGAI and this interaction results .in the glutathionylation of TGAI and a loss of interaction with NPRl. Taken together, these results expand our understanding of how TGA transcription factors and NPRI behave to regulate events and gene expression during SAR. Furthermore, the regulation of the behavior of both TGAI and NPRI by their redox status and the involvement of a glutaredoxin in modulating TGAI-NPRI interaction suggests the redox regulation of proteins is a general mechanism implemented in SAR.
    • Icewine fermentation by Saccharomyces cervisiae : fundamental stress responses and comparative fermentation dynamics

      Pigeau, Gary M.; Centre for Biotechnology (2012-12-13)
      Icewine is a unique dessert wine fermented from the juice of grapes that have frozen naturally on the vine. Fermentations of 40 Brix (>400g/L soluble solids) juice typically used in Canada to produce Icewine are osmotically stressful, frequently sluggish, may take months to reach desired ethanol levels and are accompanied by high levels of glycerol and volatile acidity. Yeast placed under osmotic stress produce glycerol to restore cellular turgor and it has been suggested that acetate is produced to maintain redox balance. The primary objective of this project was to examine, at the molecular level, the cause of and metabolic pathway utilized by Saccharomyces cerevisiae wine yeast in the production of glycerol and acetic acid, the major contributor to volatile acidity. Northern analyses of glycerol-3-phosphate dehydrogenases (GPDl and GPD2) and aldehyde dehydrogenases (ALD2,ALD3,ALD4,ALD5 andALD6) were performed to ascertain their contributions to glycerol and acetate production respectively. It was determined that only GP Dl and ALD3 had transcriptional patterns which correlate to the elevated levels of glycerol and acetate found in Icewine. Acetaldehyde, a precursor to acetate, was found to stimulate only the ALD3 isoform, but increased expression of ALD3 alone does not cause increased acetate production. This indicates that upregulation of ALD3 is not sufficient to increase acetic acid in wine and requires additional responses found in cells lmder hyperosmotic stress. The secondary objective of this project was to provide the first comprehensive, descriptive analysis of the fermentation of Canadian Icewine. The effect of increasing juice soluble solids from 40 to 42, 44 and 46 Brix on wine yeast's ability to grow and ferment, with a focus on acetic acid production, titratable acidity changes and the maximum amount of sugar consumed by the yeast, was investigated. It appears from this study that yeast divert metabolic resources away from growth and towards combating osmotic stress since yeast growth is negatively correlated and glycerol and acetic acid production are positively correlated with Icewine juice concentration. Based on this work, it appears that the upper limit of Riesling Icewine juice concentration that can be fermented to produce an Icewine with 10% v/v ethanol is 42 Brix. These results are of importance to the commercial production of Icewines. The compositional profile of 346 Icewine juices collected in the Niagara Peninsula of southern Ontario is also reported. Malic acid was found to be a main contributor to juice titratable acidity, while the increase in titratable acidity of the finished wines can be accounted for by the acetic acid produced during fermentation. The large proportion of Icewine titratable acidity represented by acetic acid may require winemakers to further adjust the acidity of the wine to achieve the sugar to acid balance required in this wine style. The utilization of nitrogen sources during fermentation was also investigated to determine icewine juice induced osmotic stress affected nitrogen uptake.
    • ORGANOSILICON BIOTECHNOLOGY: A BIO-INSPIRED APPROACH TO THE HYDROLYSIS OF ALKOXYSILANES and THE LIPASE-CATALYZED SYNTHESIS OF SILOXANE-CONTAINING POLYESTERS AND POLYAMIDES

      Frampton, Mark B.; Centre for Biotechnology (Brock University, 2013-02-22)
      The first part of this thesis studied the capacity of amino acids and enzymes to catalyze the hydrolysis and condensation of tetraethoxysilane and phenyltrimethoxysilane. Selected amino acids were shown to accelerate the hydrolysis and condensation of tetraethoxysilane under ambient temperature, pressure and at neutral pH (pH 7±0.02). The nature of the side chain of the amino acid was important in promoting hydrolysis and condensation. Several proteases were shown to have a capacity to hydrolyze tri- and tet-ra- alkoxysilanes under the same mild reaction conditions. The second part of this thesis employed an immobilized Candida antarctica lipase B (Novozym-435, N435) to produce siloxane-containing polyesters, polyamides, and polyester amides under solvent-free conditions. Enzymatic activity was shown to be temperature dependent, increasing until enzyme denaturation became the dominant pro-cess, which typically occurred between 120-130ᵒC. The residual activity of N435 was, on average, greater than 90%, when used in the synthesis of disiloxane-containing polyesters, regardless of the polymerization temperature except at the very highest temperatures, 140-150ᵒC. A study of the thermal tolerance of N435 determined that, over ten reaction cycles, there was a decrease in the initial rate of polymerization with each consecutive use of the catalyst. No change in the degree of monomer conversion after a 24 hour reaction cycle was found.
    • Plant rhizosphere specificity and variability in the insect and plant adhesins, Madl and Mad2, within the genus Metarhizium suggest plant adaptation as an evolutionary force

      Wyrebek, Michael; Centre for Biotechnology (2013-04-19)
      Metarhizium is a soil-inhabiting fungus currently used as a biological control agent against various insect species, and research efforts are typically focused on its ability to kill insects. In section 1, we tested the hypothesis that species of Metarhizium are not randomly distributed in soils but show plant rhizosphere-specific associations. Results indicated an association of three Metarhizium species (Metarhizium robertsii, M. brunneum and M. guizhouense) with the rhizosphere of certain types of plant species. M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense, which was supported by in vitro experiments with grass root exudate. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types. In section 2, we explored the variation in the insect adhesin, Madl, and the plant adhesin, Mad2, in fourteen isolates of Metarhizium representing seven different species. Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions. Phylogenetic analysis of 5' EF-Ia, which is used for species identification, as well as Madl and Mad2 sequences demonstrated that the Mad2 phylogeny is more congruent with 5' EF-1a than Madl. This suggests Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation. While other abiotic and biotic factors cannot be excluded in contributing to divergence, it appears that plant associations have been the driving factor causing divergence among Metarhizium species.
    • STUDYING ABERRANT METHYLATION AND miRNA PROFILING FOR THE DETECTION OF LUNG CANCER BIOMARKERS

      ZAMAKHSHARI, HADEEL; Centre for Biotechnology (2013-04-19)
      Lung cancer is a major chronic disease responsible for the highest mortality rate, among other types of cancer, and represents 29% of all deaths in Canada. The clinical diagnosis of lung carcinoma still requires a standard diagnostic approach, as there are no symptoms in its early stage. Therefore, it is usually diagnosed at a later stage, when the survival rate is low. With the recent advancement in molecular biology and biotechnology, a molecular biomarker approach for the diagnosis of early lung cancer seems to be a potential option. In this study, we aimed to investigate and standardize a promising Lung ,Cancer Biomarker by studying the aberrant methylation of two tumour suppressor genes, namely RASSFIA and RAR-B, and the miRNA profiling of four . commonly deregulated miRNA (miR-199a-3p, miR-182, miR-lOO and miR-221). Four lung cancer cell lines were used (two SCLC and two NSCLC), with comparisons being made with normal lung cell lines. Our results, we found that none of these genes were methylated. We then evaluated TP53, and found the promoter of this gene to be methylated in the cancer cell lines, as compared to the normal cell lines, indicating gene inactivation. We carried out miRNA profiling of the cancer cell lines and reported that 80 miRNAs are deregulated in lung cancer cell lines as compared to the normal cell lines. Our study was the first of its kind to indicate that hsa-mir-4301, hsa-mir-4707-5p and hsa-mir-4497 (newly discovered miRNAs) are deregulated in lung cancer cell lines. We also investigated miR-199a-3p, mir-lOO and miR-182, and found that miR-199a -3p and mir-l00 were down-regulated in cancer lines, whereas miR-182 was up-regulated in the cancer cell lines. In the final part of the study we observed that mir-221 could be a putative biomarker to distinguish between the two types of lung cancer because it was down-regulated in SCLC, and up-regulated in the NSCLC cell lines. In conclusion, we found four miRNA molecular biomarkers that possibly could be used in the early diagnosis of the lung cancer. More studies are still required with larger numbers of samples to effectively establish these as molecular biomarkers for the diagnosis of lung cancer
    • Prostate Cancer and the Search for Novel Biomarkers

      Haj-Ahmad, Taha Alexander; Centre for Biotechnology (Brock University, 2013-05-06)
      The primary objective of this research project was to identify prostate cancer (PCa) -specific biomarkers from urine. This was done using a multi-faceted approach that targeted (1) the genome (DNA); (2) the transcriptome (mRNA and miRNA); and (3) the proteome. Toward this end, urine samples were collected from ten healthy individuals, eight men with PCa and twelve men with enlarged, non-cancerous prostates or with Benign Prostatic Hyperplasia (BPH). Urine samples were also collected from the same patients (PCa and BPH) as part of a two-year follow-up. Initially urinary nucleic acids and proteins were assessed both qualitatively and quantitatively for characteristics either unique or common among the groups. Subsequently macromolecules were pooled within each group and assessed for either protein composition via LC-MS/MS or microRNA (miRNA) expression by microarray. A number of potential candidates including miRNAs were identified as being deregulated in either pooled PCa or BPH with respect to the healthy control group. Candidate biomarkers were then assessed among individual samples to validate their utility in diagnosing PCa and/or differentiating PCa from BPH. A number of potential targets including deregulation of miRNAs 1825 and 484, and mRNAs for Fibronectin and Tumor Protein 53 Inducible Nuclear Protein 2 (TP53INP2) appeared to be indicative of PCa. Furthermore, deregulation of miR-498 appeared to be indicative of BPH. The sensitivities and specificities associated with using deregulation in many of these targets to subsequently predict PCa or BPH were also determined. This research project has identified a number of potential targets, detectable in urine, which merit further investigation towards the accurate identification of PCa and its discrimination from BPH. The significance of this work is amplified by the non-invasive nature of the sample source from which these candidates were derived, urine. Many cancer biomarker discovery studies have tended to focus primarily on blood (plasma or serum) and/or tissue samples. This is one of the first PCa biomarker studies to focus exclusively on urine as a sample source.
    • Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

      Salim, Vonny; Centre for Biotechnology (Brock University, 2013-08-23)
      The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.
    • The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

      Levac, Dylan Edward Ryan; Department of Biological Sciences (Brock University, 2013-09-19)
      The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.
    • The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

      Bosak, Jan; Centre for Biotechnology (Brock University, 2014-08-05)
      During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.
    • Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

      Slavickova, Nina; Centre for Biotechnology (Brock University, 2014-08-05)
      Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.
    • Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

      Mahboob, Abdullah; Centre for Biotechnology (Brock University, 2014-10-01)
      ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems.
    • Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

      Behie, Scott William; Centre for Biotechnology
      While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.
    • UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

      Abbas, Basma; Centre for Biotechnology
      Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.