• (A) Optimization of solid body phase synthesis of RNA using the Cpep chemistry. (B) Synthesis of BODIPY labeled oligonucleotides

      Tram, Kha; Centre for Biotechnology (Brock University, 2011-05-17)
      (A) Solid phase synthesis of oligonucleotides are well documented and are extensively studied as the demands continue to rise with the development of antisense, anti-gene, RNA interference, and aptamers. Although synthesis of RNA sequences faces many challenges, most notably the choice of the 2' -hydroxy protecting group, modified 2' -O-Cpep protected ribonucleotides were synthesized as alternitive building blocks. Altering phosphitylation procedures to incorporate 3' -N,N-diethyl phosphoramidites enhanced the overall reactivity, thus, increased the coupling efficiency without loss of integrety. Furthermore, technical optimizations of solid phase synthesis cycles were carried out to allow for successful synthesis of a homo UIO sequences with a stepwise coupling efficiency reaching 99% and a final yield of 91 %. (B) Over the past few decades, dipyrrometheneboron difluoride (BODIPY) has gained recognition as one of the most versatile fluorophores. Currently, BODIPY labeling of oligonucleotides are carried out post-synthetically and to date, there lacks a method that allows for direct incorporation of BODIPY into oligonucleotides during solid phase synthesis. Therefore, synthesis of BODIPY derived phosphoramidites will provide an alternative method in obtaining fluorescently labelled oligonucleotides. A method for the synthesis and incorporation of the BODIPY analogues into oligonucleotides by phosphoramidite chemistry-based solid phase DNA synthesis is reported here. Using this approach, BODIPY-labeled TlO homopolymer and ISIS 5132 were successfully synthesized.
    • Alternative splicing of DNA polymerase beta in vertebrates

      Bondy-Chorney, Emma; Department of Biological Sciences (Brock University, 2012-11-27)
      Alternative splicing (AS) is the predominant mechanism responsible for increasing eukaryotic transcriptome and proteome complexity. In this phenomenon, numerous mRNA transcripts are produced from a single pre-mRNA sequence. AS is reported to occur in 95% of human multi-exon genes; one specific gene that undergoes AS is DNA polymerase beta (POLB). POLB is the main DNA repair gene which performs short patch base excision repair (BER). In primate untransformed primary fibroblast cell lines, it was determined that the splice variant (SV) frequency of POLB correlates positively with species lifespan. To date, AS patterns of POLB have only been examined in mammals primarily through the use of cell lines. However, little attention has been devoted to investigating if such a relationship exists in non-mammals and whether cell lines reflect what is observed in vertebrate tissues. This idea was explored through cloning and characterization of 1,214 POLB transcripts from four non-mammalian species (Gallus gallus domesticus, Larus glaucescens, Xenopus laevis, and Pogona vitticeps) and two mammalian species (Sylvilagus floridanus and Homo sapiens) in two tissue types, liver and brain. POLB SV frequency occurred at low frequencies, < 3.2%, in non-mammalian tissues relative to mammalian (>20%). The highest POLB SV frequency was found in H. sapiens liver and brain tissues, occurring at 65.4% and 91.7%, respectively. Tissue specific AS of POLB was observed in L. glaucescens, P. vitticeps, and H. sapiens, but not G. gallus domesticus, X. laevis and S. floridanus.The AS patterns of a second gene, transient receptor potential cation channel subfamily V member 1 (TRPV1), were compared to those of POLB in liver and brain tissues of G. gallus domesticus, X. laevis and H. sapiens. This comparison was performed to investigate if any changes (either increase or decrease) observed in the AS of POLB were gene specific or if they were tissue specific, in which case similar changes in AS would be seen in POLB and TRPV1. Analysis did not reveal an increase or decrease in both the AS of POLB and TRPV1 in either the liver or brain tissues of G. gallus domesticus and H. sapiens. This result suggested that the AS patterns of POLB were not influenced by tissue specific rates of AS. Interestingly, an increase in the AS of both genes was only observed in X. laevis brain tissue. This result suggests that AS in general may be increased in the X. laevis brain as compared to liver tissue. No positive correlation between POLB SV frequency and species lifespan was found in non-mammalian tissues. The AS patterns of POLB in human primary untransformed fibroblast cell lines were representative of those seen in human liver tissue but not in brain tissue. Altogether, the AS patterns of POLB from vertebrate tissues and primate cell lines revealed a positive correlation between POLB SV frequency and lifespan in mammals, but not in non-mammals. It appears that this positive correlation does not exist in vertebrate species as a whole.
    • The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

      Sasan, Ramanpreet Kaur; Centre for Biotechnology (2013-04-15)
      The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.
    • Analysis of the pattern and trend of human genomic variations in the form of single nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs)

      chundi, vinay kumar; Department of Biological Sciences
      Single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) are the most common genetic variations in the human genome. They have been shown to associate with phenotype variation including genetic disease. Based on data in a recent version of the NCBI dbSNP database (Build 150), there are 305,651,992 SNPs and 19,177,943 INDELs, and together as all small sequence variants, they represent approximately 11% of the human reference genome sequences. In this study, we aimed first to examine the characteristics of SNPs and INDELs based on their location and variation type. We then identified the ancestral alleles for these variants and examined the patterns of variation from the ancestral state. Our results show that the occurrence of small variants averages at 104 SNPs/kb and 6.5 INDELs/kb for a total of ~11% of the genome. Chromosome 16 and 21 represent the least and most conserved autosomes, respectively, while the sex chromosomes are shown to have a much lower density of SNPs and INDELs being more than 30% lower in the X chromosome and more than 85% lower in the Y chromosome. By gene context, SNPs are biased towards genic regions and INDELs are biased towards intergenic regions, and further, INDELs are biased towards protein-coding genes and intron regions within the genic regions and SNPs are biased towards non-coding genes in the genic regions. Within the coding regions, SNPs and INDELs are biased towards missense and frameshift variations, respectively. Some of the biases were due to biased sources of the variation data targeting at genic regions, while the bias towards intron regions is due to selection pressure. Further, genes with the highest level of variation showed enrichment in functions related to environmental sensing and immune responses, while those with least variation associate with critical processes such as mRNA splicing and processing. Through a comparative genomics approach, we determined the ancestral state for most of these variants and our results indicate that ~0.79% of the genome has been subject to SNP and INDEL variation since the last common human ancestor. Our study represents the first comprehensive data analysis of human variation in SNPs and INDELs and the determination of their ancestral state, providing useful resources for human genetics study and new insights into human evolution.
    • Biofilm Formation and Quorum Sensing in Pseudomonas fluorescens Pf0-1

      Bordeleau, Emily; Centre for Biotechnology
      A bacterial biofilm is a community of microorganisms adhering to a surface, exhibiting biochemical and phenotypic differences from their planktonic counterparts. The transition from a free-floating to sessile cell type has been shown to be, in part, mediated by high intracellular levels of the nucleotide second messenger c-di-GMP. It is suggested that one of the environmental cues for biofilm formation, recognized by members of the c-di-GMP network, is local cell density. In areas of high cell density, cells can communicate through a system called quorum sensing. In gram negative bacteria, acyl-homoserine lactone (AHL) molecules are excreted into the surrounding medium and recognized by cells in close proximity. It is hypothesized that upon recognizing AHLs through c-di-GMP signaling, gene expression is altered leading to a sessile lifestyle. Thus, the long-term goal of this research is to provide evidence for the link between c-di-GMP and quorum sensing-mediated mechanisms in biofilm formation in Pseudomonas fluorescens Pf0-1. The first objective towards this goal was to identify the AHLs utilized by P. fluorescens Pf0-1 in quorum sensing mechanisms. Through gas-chromatography mass-spectrometry (GCMS), two AHLs were identified in the supernatant of P. fluorescens Pf0-1; N-butyryl-HSL and Ndecanoyl- HSL. Subsequent work will focus on the identification of AHLs with longer acyl-chain and varying levels of acyl chain oxidation. The second objective towards this goal was to utilize the 96-well static microtiter plate biofilm assay as a platform for studying the relationships between c-di-GMP and AHL-mediated mechanisms in biofilm formation. As a protocol for 96-well static biofilm assays that was previously successful was no longer reproducible, different microtiter plate surfaces were surveyed for their ability to support P. fluorescens Pf0-1 biofilm and to investigate potential factors that could interfere with development on the abiotic surface. Throughout the troubleshooting process, biofilm assay experiments carried out in microtiter plates with the same type of surface chemistry, but from different manufacturers and batches, resulted in variable quantities of biofilm. This observation then inspired the production of a surface that would create more favorable interactions with bacterial cells and offer increased points of attachment to further promote biofilm formation. In this new platform, microtiter plates are pre-treated by abrasive forces such as sandblasting and drilling before biofilm assays, which gives robust biofilm formation that will allow for future investigation into connections between c-di-GMP and AHL-controlled mechanisms of biofilm formation in P. fluorescens Pf0-1.
    • Bioinformatic and morphological characterization of Catharanthus roseus mutants

      Jones, Graham; Centre for Biotechnology
      Catharanthus roseus, a member of the Apocynaceae family, has been studied extensively for its valuable chemotherapeutic monoterpenoid indole alkaloids (MIAs). Ethyl methanesuphonate (EMS) mutagenesis is a screening tool that has been used to look for altered MIA profiles in hope of discovering mutations of crucial MIA biosynthetic genes. Without a high-throughput mutation detection screen for C. roseus sequencing data, a range of techniques must be used to discover the EMS-induced changes within the plant. Bioinformatic and morphological analysis revealed the likely alterations leading to unique MIA profiles in two C. roseus EMS mutants: the high-ajmalicine accumulating line M2-0754 and the low-MIA accumulating line M2-1582. Expression of geissoschizine synthase (GS) was downregulated almost seven-fold in the leaves of M2-0754, leading to the accumulation of an alternate pathway MIA from the labile intermediate. The low-MIA profile and increased auxin sensitivity of M2-1582 is likely due to the expression of a dysfunctional auxin influx transport protein homologue.
    • A Catharanthus roseus mutant with trace levels of secologanin and monoterpenoid indole alkaloids does not express BIS1/BIS2 transcription factors and fails to activate iridoid biosynthesis.

      Kidd, Trevor; Centre for Biotechnology
      The Madagascar periwinkle (Catharanthus roseus) is the sole source of the monoterpenoid indole alkaloids (MIAs) that result in several essential anti-cancer chemotherapies as well as being an important source for other MIA derived pharmaceutical agents. Most of the alkaloid and pre-alkaloid iridoid pathway has been elucidated, but some critical areas remain uncharacterized. The early iridoid pathway is localized to internal phloem associated parenchyma (IPAP) cells, with the latter part of the pathway localized to the epidermal cells indicating intercellular transport within the leaf tissue does occur. However, possible transport or translocation of MIAs or pre-MIAs between organs within Catharanthus roseus has not been studied. Previously, 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants had been screened with a simple TLC (thin layer chromatography) to identify mutants with altered MIA profiles yielded one plant with trace MIA production. This trace MIA status was confirmed using UPLC-MS (Ultra Performance Liquid Chromatography and Mass Spectrometry) and the plant had a thick, stocky, short root system, small leaves, a rigid stem, premature senescence and was susceptible to infection resulting in its rescue to in-vitro. Quantitative real-time PCR analysis of iridoid gene expression showed significant downregulation of several iridoid pathway genes as well as the downregulation of transcription factors BIS1 and BIS2. Feeding the trace MIA mutant roots the iridoid secologanin resulted in alkaloid production in the leaves. While grafting trace MIA mutant shoots onto MIA producing WT roots also resulted in alkaloid production in the mutant leaves. This study establishes that MIAs or pre-MIA iridoids, such as secologanin, may be translocated between the plant organs and this may be useful for the identification of novel transporters, enzymes, and transcription factors.
    • Chemical, biochemical, and molecular characterization of a low vindoline Catharanthus roseus mutant.

      Edge, Alison; Centre for Biotechnology
      The Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drug vinblastine, which is formed via the coupling of monoterpenoid indole alkaloids (MIAs) catharanthine and vindoline. A mutant line of C. roseus (M2-1865) with an altered MIA profile was identified in a screen of 4000 M2 lines generated by ethylmethanesulfonate (EMS) chemical mutagenesis. While this line did not accumulate vinblastine due to reduced levels of vindoline within the leaves, significant levels of 2,3-epoxide derivatives of tabersonine accumulated on the leaf surface. Detailed nucleotide, amino acid, and enzyme activity analyses of tabersonine 3-reductase in the M2-1865 line showed that a single amino acid substitution (H189Y) diminished the biochemical activity of T3R by 95%. Genetic crosses showed the phenotype to be recessive, exhibiting standard Mendelian single-gene inheritance. The usefulness of EMS mutagenesis in elucidating MIA biosynthesis is highlighted by the results of this study.
    • The Development of a Time-Resolved, Vesicle-Based, Fluorescence Assay for the Activity of the Lipid Kinase Pik1

      Meehan, Kailey; Centre for Biotechnology
      Pik1 is a yeast phosphatidylinositol-4 kinase that is critical for vesicular traffic to the plasma membrane and endosomes from the trans-Golgi (1, 2). Pik1 is a soluble enzyme, and the small myristoylated, Ca2+ binding, EF hand protein, Frequenin (Frq1) facilitates its membrane localization (3). It has been suggested that Pik1 finds its substrate, phosphatidylinositol (PI), within phospholipid bilayers with assistance from the PI/PC transfer protein Sec14 (4). It is proposed that Sec14 stimulates the activity of Pik1 by partially removing PI from the bilayer and presenting it to the kinase as a more suitable substrate (4). In order to test this hypothesis, the Bellbrook Labs Transcreener ADP2 FI Assay kit, a fluorescence-based enzyme assay kit that reports the production of ADP, was adapted to create a time-resolved, vesicle-based assay. The assay was developed and validated with the catalytic subunit of Protein Kinase A (PKA) and the commercial human PI kinase PIK3C3. An expression system for Pik1 was created in yeast. To preserve the stability of the 125 kDa enzyme it was co-expressed with both Frq1 and the heat shock protein, Cdc37, producing the Pik1-Frq1 complex with a removable 10xhistidine tag on Pik1. The growth of the yeast transformants was investigated using different culture conditions, media, and methods of inducing protein expression. The expression of the Pik1-Frq1 complex was confirmed on a Western blot that showed a band corresponding to the molecular weight of the complex. Next steps will involve optimizing the purification of the Pik1-Frq1 complex on a metal affinity resin, with the ultimate goal being to test the Sec14 presentation mechanism by measuring the kinase activity of Pik1 from purified fractions in the presence and absence of Sec14.
    • Development of a Transcriptome-Based Genome Assembly Tool and Whole Genome Sequencing for Autism Spectrum Disorders

      Baldwin, Robert; Centre for Biotechnology
      This thesis consisted of two independent projects. The first involved developing a software tool that uses transcriptome data to improve genome assemblies. The second involved processing and analyzing whole genome sequencing (WGS) from the ASPIRE autism spectrum disorder (ASD) cohort. The first project produced the bioinformatics software called RDNA. This free tool was written in Perl and should be valuable for users interested in genome assembly. Comparative assessment between RDNA and the leading transcript based scaffolding software showed that RDNA can significantly improve genome assemblies while making relatively few scaffolding connection errors. RDNA also makes possible the assembly of scaffolding connections, including gap filling, using BLAST. The second project was undertaken with collaborators and involved processing and analyzing whole genome sequencing (WGS) data from the ASPIRE ASD cohort. The ASPIRE ASD cohort consisted of several hundred probands from both simplex and multiplex families. Sequencing occurred for 120 of these individuals who were selected based upon membership in two phenotype clusters (C1 and C2). These individuals had a relatively high rate of intellectual disability (ID) compared to heavily studied ASD cohorts such as the Simons Simplex Collection (SSC), indicating a significant involvement of de novo sequence variants. Analysis of rare single nucleotide variants (SNVs) and insertion/deletions (indels) identified large risk factors for severe neurodevelopmental disorders (NDDs), two of which were previously observed de novo among individuals with severe, undiagnosed NDDs. On this basis, ABCA1 was found to be a novel candidate risk gene. Gene Ontology (GO) analysis of rare loss of function and missense SNVs indicted the importance of lipid metabolic processes and synaptic signalling. Overall, the genetic variation examined by this study pertained to a modest number of cases, consistent with previous findings that ASD is a genetically heterogeneous disorder with a complex genetic architecture.
    • DNA-Directed DNA Polymerases Evolve From Reverse Transcriptase

      Zhang, Lei; Centre for Biotechnology (2013-05-07)
      Scientists have been debating for decades the origin of life on earth. A number of hypotheses were proposed as to what emerged first RNA or DNA; with most scientists are in favour of the "RNA World" hypothesis. Assuming RNA emerged first, it fellow that the RNA polymerases would've appeared before DNA polymerases. Using recombinant DNA technology and bioinformatics we undertook this study to explore the relationship between RNA polymerases, reverse transcriptase and DNA polymerases. The working hypothesis is that DNA polymerases evolved from reverse transcriptase and the latter evolved from RNA polymerases. If this hypothesis is correct then one would expect to find various ancient DNA polymerases with varying level of reverse transcriptase activity. In the first phase of this research project multiple sequence alignments were made on the protein sequence of 32 prokaryotic DNA-directed DNA polymerases originating from 11 prokaryotic families against 3 viral reverse transcriptase. The data from such alignments was not very conclusive. DNA polymerases with higher level of reverse transcriptase activity were non-confined to ancient organisms, as one would've expected. The second phase of this project was focused on conditions that may alter the DNA polymerase activity. Various reaction conditions, such as temperature, using various ions (Ni2+, Mn2+, Mg2+) were tested. Interestingly, it was found that the DNA polymerase from the Thermos aquatics family can be made to copy RNA into DNA (i.e. reverse transcriptase activity). Thus it was shown that under appropriate conditions (ions and reactions temperatures) reverse transcriptase activity can be induced in DNA polymerase. In the third phase of this study recombinant DNA technology was used to generate a chimeric DNA polymerase; in attempts to identify the region(s) of the polymerase responsible for RNA-directed DNA polymerase activity. The two DNA polymerases employed were the Thermus aquatic us and Thermus thermophiles. As in the second phase various reaction conditions were investigated. Data indicated that the newly engineered chimeric DNA polymerase can be induced to copy RNA into DNA. Thus the intrinsic reverse transcriptase activity found in ancient DNA polymerases was localized into a domain and can be induced via appropriate reaction conditions.
    • Elucidation of the components involved in the antioxidant activity of honey

      Miotto, Danielle; Centre for Biotechnology (Brock University, 2011-10-14)
      Canadian honeys were analyzed for sugar concentration, honey colour, total phenolic content, the level of brown pigments, and antioxidant activity in order to elucidate the main components involved in the antioxidant activity of honey. By employing size-exclusion chromatography in combination with activity-guided fractionation, it was demonstrated that the antioxidant components are of high molecular weight (HMW), brown in colour and absorb at both 280nm and 450nm. The presence of brown HMW antioxidant components prompted an investigation on the influence of heattreatment on the Maillard reaction and the formation of melanoid ins. Heat-treatment of honey resulted in an increase in the level of phenolics in the melanoidin fractions which correlated with an increase in antioxidant activity. The preliminary results of this study suggest for the first time that honey melanoidins underlie the antioxidant activity of unheated and heat-treated honey, and that phenolic constituents are involved in the melanoidin structure and are likely incorporated by covalent or non-covalent interaction.
    • Honey proteins and their interaction with polyphenols

      Alvarez, Liset Maldonado; Centre for Biotechnology (Brock University, 2011-10-14)
      This project aimed to determine the protein prof i les and concent rat ion in honeys, ef fect of storage condi t ions on the protein content and the interact ion between proteins and polyphenols. Thi r teen honeys f rom di f ferent botanical or igins were analyzed for thei r protein prof i les using SDS-PAGE, protein concent rat ion and phenol ic content , using the Pierce Protein Assay and Fol in-Ciocal teau methods, respectively. Protein-polyphenol interact ions were analyzed by a combinat ion of the ext ract ion of honeys wi th solvents of di f ferent polar i t ies fol lowed by LCjMS analysis of the obtained f ract ions. Results demonst rated a di f ferent protein content in the tested honeys, wi th buckwheat honey possessing the highest protein concent rat ion. We have shown that the reduct ion of proteins dur ing honey storage was caused, partially, by the protein complexat ion wi th phenolics. The LCjMS analysis of the peak elut ing at retent ion t ime of 10 to 14 min demonst rated that these phenolics included f lavonoids such as Pinobanksin, Pinobanksin acetate, Apigenin, Kaemferol and Myricetin and also cinnamic acid.
    • Human Endogenous Retrovirus (HERV) Insertional Polymorphisms

      Golem, Scott Matthew Bradley; Centre for Biotechnology (Brock University, 2013-08-07)
      Human endogenous retroviruses (HERVs) are the result of ancient germ cell infections of human germ cells by exogenous retroviruses. HERVs belong to the long terminal repeat (LTR) group of retrotransposons that comprise ~8% of the human genome. The majority of the HERVs documented have been truncated and/or incurred lethal mutations and no longer encode functional genes; however a very small number of HERVs seem to maintain functional in making new copies by retrotranspositon as suggested by the identification of a handful of polymorphic HERV insertions in human populations. The objectives of this study were to identify novel insertion of HERVs via analysis of personal genomic data and survey the polymorphism levels of new and known HERV insertions in the human genome. Specifically, this study involves the experimental validation of polymorphic HERV insertion candidates predicted by personal genome-based computation prediction and survey the polymorphism level within the human population based on a set of 30 diverse human DNA samples. Based on computational analysis of a limited number of personal genome sequences, PCR genotyping aided in the identification of 15 dimorphic, 2 trimorphic and 5 fixed full-length HERV-K insertions not previously investigated. These results suggest that the proliferation rate of HERVKs, perhaps also other ERVs, in the human genome may be much higher than we previously appreciated and the recently inserted HERVs exhibit a high level of instability. Throughout this study we have observed the frequent presence of additional forms of genotypes for these HERV insertions, and we propose for the first time the establishment of new genotype reporting nomenclature to reflect all possible combinations of the pre-integration site, solo-LTR and full-length HERV alleles.
    • Hyperosmotic Stress and the Impact on Metabolite Formation and Redox Balance in Saccharomyces cerevisiae and Saccharomyces bayanus strains

      Heit, Caitlin; Department of Biological Sciences (Brock University, 2014-02-21)
      Wine produced using an appassimento-type process represents a new and exciting innovation for the Ontario wine industry. This process involves drying grapes that have already been picked from the vine, which increases the sugar content due to dehydration and induces a variety of changes both within and on the surface of the grapes. Increasing sugar contents in musts subject wine yeast to conditions of high osmolarity during alcoholic fermentations. Under these conditions, yeast growth can be inhibited, target alcohol levels may not be attained and metabolic by-products of the hyperosmotic stress response, including glycerol and acetic acid, may impact wine composition. The further metabolism of acetic acid to acetylCoA by yeast facilitates the synthesis of ethyl acetate, a volatile compound that can also impact wine quality if present in sufficiently high concentrations. The first objective of this project was to understand the effect of yeast strain and sugar concentration on fermentation kinetics and metabolite formation, notably acetic acid and ethyl acetate, during fermentation in appassimento-type must. Our working hypotheses were that (1) the natural isolate Saccharomyces bayanus would produce less acetic acid and ethyl acetate compared to Saccharomyces cerevisiae strain EC-1118 fermenting the high and low sugar juices; (2) the wine produced using the appassimento process would contain higher levels of acetic acid and lower levels of ethyl acetate compared to table wine; (3) and the strains would be similar in the kinetic behavior of their fermentation performances in the high sugar must. This study determined that the S. bayanus strain produced significantly less acetic acid and ethyl acetate in the appassimento wine and table wine fermentations. Differences in acetic acid and ethyl acetate production were also observed within strains fermenting the two sugar conditions. Acetic acid production was higher in table wine fermented by S. bayanus as no acetic acid was produced in appassimento-style wine, and 1.4-times higher in appassimento wine fermented by EC-1118 over that found in table wine. Ethyl acetate production was 27.6-times higher in table wine fermented by S. bayanus, and 5.2-times higher by EC-1118, compared to that in appassimento wine. Sugar utilization and ethanol production were comparable between strains as no significant differences were determined. The second objective of this project was to bring a method in-house for measuring the concentration of pyridine nucleotides, NAD+, NADP+, NADH and NADPH, in yeast cytosolic extract. Development of this method is of applicative interest for our lab group as it will enable the redox balance of the NAD+/ NADH and NADP+/ NADPH systems to be assessed during high sugar fermentations to determine their respective roles as metabolic triggers for acetic acid production. Two methods were evaluated in this study including a UV-endpoint method using a set of enzymatic assay protocols outlined in Bergmeyer (1974) and a colorimetric enzyme cycling method developed by Sigma-Aldrich® using commercial kits. The former was determined to be limited by its low sensitivity following application to yeast extract and subsequent coenzyme analyses, while the latter was shown to exhibit greater sensitivity. The results obtained from the kits indicated high linearity, accuracy and precision of the analytical method for measuring NADH and NADPH, and that it was sensitive enough to measure the low coenzyme concentrations present in yeast extract samples. NADtotal and NADPtotal concentrations were determined to be above the lower limit of quantification and within the range of the respective calibration curves, making this method suitable for our research purposes.
    • IMPACT OF CROP LEVEL AND HANG TIME ON THE COMPOSITION OF FOUR WINE GRAPE CULTIVARS FROM THE NIAGARA REGION

      Moreno Luna, Luis Hugo; Centre for Biotechnology (Brock University, 2014-02-13)
      This study analyzed the use of two viticultural practices: “crop level” (half crop; HC, and full crop; FC) and “hang times”, and their impact on the composition of four grape cultivars; Pinot gris, Riesling, Cabernet Franc and Cabernet Sauvignon from the Niagara Region and wine volatile composition by GC-MS. It was hypothesized that keeping a full crop with a longer hang time would have a greater impact on wine quality than reducing the crop level. In all cultivars, a reduction of crop level induced reductions in yield, clusters per vine and crop load, with increases in Brix. Extended hang time also increased Brix related to desiccation. The climatic conditions at harvest had an impact on hang time effects. The GC-MS analysis detected the presence of 30 volatile components in the wine, with different odour activity values. Harvest time had a positive impact than crop reduction in almost all compounds.
    • The impact of grape clone, yeast strain and protein on sparkling wine quality

      Onguta, Esther; Centre for Biotechnology
      The foaming properties in sparkling wine are an indicator of quality as it is the first quality perception consumers have upon opening a bottle of sparkling wine. Proteins, which are derived from both grapes and yeast during sparkling wine production, are known to impact the foaming properties in finished sparkling wines. The objectives of this project were to (1) understand the role and relationship that proteins have on the overall foaming properties and overall quality in sparkling wine, (2) determine the role different yeast and grape clones from different varietals have on sparkling wine quality and (3) understand how bentonite affects sparkling wine quality. The protein concentration in sparkling wine produced from Mariafeld Pinot noir appeared to impact the foaming properties. The longest elapsed time for foam dissipation was observed in the control treatment where bentonite was not used to strip protein and the shortest time was observed in the treatment where bentonite was used to remove grape and yeast proteins. In Riesling sparkling wines, the largest protein concentrations were observed in non-bentonite treated juices while the lowest were observed in the bentonite treatments. The prevalence of foam observed in both bentonite treatments, where grape proteins were completely removed, indicated that proteins derived over the course of secondary fermentation were foam forming in Riesling sparkling wine. It was also observed that different yeast, varietals, clones and soil compositions may impact the protein concentrations, chemical compositions and overall quality of sparkling wine. The results of this research aim to better understand sparkling wine quality to optimize production in the Niagara Peninsula.
    • An in vivo, ex vivo, and in vitro exploration of the use of chronic hypoxia/physioxia and ROS/RNS-mediated alteration of physiological function in mitochondrial disease

      Messner, Holt; Centre for Biotechnology
      Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are by-products of cellular O2 metabolism and participate in cell signalling and normal physiological homeostasis. Although ROS/RNS are normal and important molecules in cell physiology, production in excess or in absence of sufficient cellular antioxidant capacity can lead to critical cellular damage which has shown to contribute to a plethora of disease pathologies. Experimental evidence has also supported the use of chronic limitation of ambient O2 gas as a means to reduce the amount of ROS/RNS in both animals and cell culture. The purpose of this thesis was to explore the use of a regulated O2 environment to determine if chronic hypoxia and/or physioxia may influence ROS/RNS production in animal and cell culture models of various known mitochondrial diseases. The absence of superoxide dismutase 2 (SOD2) has been proven to be extremely lethal, so I attempted to breed and house entire mouse dams in hypoxia (11% O2) to investigate if limiting O2 might reduce the amount of ROS/RNS-mediated damage and extend the life span of SOD2 knockout (KO) mouse pups. Many attempts to rescue these KO mice failed due to premature death and/or maternal cannibalism, however, the SOD2 heterozygote (SOD2+/-) – which experience halved SOD2 expression compared to wildtype controls – were viable and body mass data was examined. Although no main effect was found of genotype on body mass over time, male, but not female, mice housed in chronic hypoxia gained significantly less weight than their normoxia (20% O2) counterparts. After examining live animals, I focused on measuring the effects of regulating the O2 environment on ROS production in a variety of cellular models of different mitochondrial disease. Some measure of structural and/or functional integrity is compromised in mitochondrial disease, typically leading to exacerbated proton leak, subsequent superoxide/hydrogen peroxide (H2O2) formation, and, unsurprisingly, a significant potential for cellular damage. These features of mitochondrial disease are further compounded by the fact that a great deal of published cell culture work does not actively regulate O2 levels and thus cells often experience an environment with O2 levels hyperoxic relative to what is typically experienced in vivo. The purpose of this second study was to investigate whether growing and assaying mito-disease cell lines in a regulated, physiologically-relevant O2 environment would reduce H2O2 output to levels similar to wildtype controls. Measuring H2O2 production from various mito-disease cell lines, almost all cell lines produced more H2O2 when grown in normoxia (18% O2) culture conditions compared to physioxia (5% O2), however, only a few of the mitochondrial disease cell lines tested here produced the expected increase in cellular H2O2 efflux than their wildtype counterparts at 18% O2. As expected, almost all mitochondrial disease cell lines produced H2O2 at a similar level to their respective controls when grown in 5% O2. Furthermore, NADPH Oxidases (NOX) were explored as a potentially significant source of elevated ROS production at 18% O2. However, upon NOX inhibition, no significant measurable changes in H2O2 production were reported in any of the cell lines tested here. Finally, the last study in this thesis explored structural and functional consequences which may accompany halved SOD2 expression in adult SOD2+/- female mice over time. Sarco-endoplasmic reticulum ATP-ase (SERCA) is an enzyme responsible for calcium handling in myocytes and its function is critical for proper muscular relaxation and contraction. The soleus and extensor digitorum longus (EDL) were analyzed to determine whether muscle type (ie. slow-oxidative muscle or fast-glycolytic muscle) would influence the effects of heterozygous SOD2 deletion. Interestingly, the soleus muscle showed significant impairments in SERCA function with a reduction in SERCA’s apparent affinity for calcium, whereas there were no differences between genotypes in the EDL muscle. This corresponded well with the fact that SERCA tyrosine nitration was significantly elevated in the soleus, particularly on SERCA2a. Conversely, there were no signs of elevated SERCA tyrosine nitration on SERCA1a, the predominant SERCA isoform in the EDL. In conclusion, the results from these studies provide some insight to the roles that O2 and ROS generation have on physiological function in vivo and in vitro, though it also prompts further investigation due to mixed results in many cases.
    • Investigating Flavivirus Infection, Dissemination, and Transmission Dynamics Using Zika virus, West Nile virus, and their Mosquito Vectors

      Agbulos, Darrell; Centre for Biotechnology
      Flaviviruses (family Flaviviridae, genus Flavivirus) are a group viral pathogens responsible for causing disease and death in both humans and animals. Mosquito saliva potentiates Flavivirus infection in both in vitro and in vivo models; however, it remains unknown whether saliva from different species differentially potentiates infection. By inoculating the saliva of different mosquito species plus WNV onto Vero cells, plaque assays were used to study if saliva could differentially potentiate WNV infection. It was found that while there was no significant difference between Ae. aegypti and Ae albopictus saliva (p=0.19), more interestingly was that both saliva treatments had a significant reduction in plaques formed compared to virus alone (p= 0.01 and p=0.00). The presence of mosquito saliva appears to exert a protective effect in vitro when WNV is present. It also remains to be elucidated as to whether Canadian mosquitoes are able to spread Zika virus. By orally infecting wild caught mosquitoes with a ZIKV infected sugar meal and detecting the presence of virus 10 and 14 days post infection (d.p.i.), the vector competence of Canadian mosquitoes was evaluated. It was found that after 10 (n=50) and 14 d.p.i. (n=32), 2% and 0% of a population of Culex pipiens mosquitoes were found to be able to become infected and transmit the virus, respectively. Although Culex pipiens mosquitoes from the Niagara region may not be vectors of ZIKV, that does not negate other Canadian mosquitoes as being potential vectors.
    • Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

      Naoum, Ravi; Centre for Biotechnology (Brock University, 2015-01-21)
      Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.