Brunetti, Craig R

Ustilago maydis (DC) Corda is the causal agent of 'common smut of corn'. Completion of the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms of U. maydis infection include chlorosis and the formation of tumours on all aerial corn tissues. Within the tumours, thick-walled diploid teliospores form; these are the reproductive and dispersal agent for the fungus. U. maydis is the model to study basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is required to fully realize the value of this resource. The research presented here improved U. maydis genome annotation through the analysis of cDNA library sequences and comparative genomics. These analyses identified and characterized pathogenesis-related genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model for investigating 'host-induced' meiosis. Further, the cDNA library analyses identified non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are endogenous RNA molecules with regions complementary to a protein-coding transcript. Although NATs have been identified in a wide variety of mammals, plants, and fungi, very few have been functionally characterized. Over 200 U. maydis NATs were annotated by analyzing full-length cDNA sequences. NAT structural features were characterized. Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut fungus, U. hordei. The data supported a common role for NATs in smut teliospore development, independent of the RNA interference pathway. Analysis of the expression of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U. maydis during teliospore dormancy. This model proposed NATs facilitate the maintenance of stored mRNAs through the formation of double-stranded RNA. In testing this model, it was determined that the deletion of two separate upstream regulatory regions, one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased pathogenesis. These studies strengthened U. maydis as a model organism, and began the functional investigation of NATs in U. maydis, which identified a new class of fungal pathogenesis genes.

Author Keywords: cDNA library analysis, genome annotation, mRNA stability, natural antisense transcripts, pathogenesis, Ustilago maydis

Ustilago maydis (D.C.) Corda is a biotrophic pathogen that secretes effectors to establish and maintain a relationship with its host, Zea mays. In this pathosystem, the molecular function of effectors is well-studied, but the regulation of effector gene expression remains largely unknown. This study characterized Zfp1, a putative U. maydis Zn(II)2Cys6 transcription factor, as a modulator of effector gene expression. The amino acid sequence of Zfp1 indicated the presence of a GAL4-like zinc binuclear cluster as well as a fungal specific transcription factor domain. Nuclear localization was confirmed by tagging Zfp1 with enhanced green fluorescent protein. Deletion of zfp1 resulted in attenuated hyphal growth, reduced infection frequency, an arrest in pathogenic development, and decreased anthocyanin production. This phenotype can be attributed to the altered transcript levels of genes encoding predicted and confirmed U. maydis effectors in the zfp1 deletion strain during pathogenic growth. Complementation of zfp1 deletion strain with tin2, an effector involved in anthocyanin induction, suggested this effector is downstream of Zfp1 and its expression is influenced by this transcription factor during in planta growth. When wild-type zfp1 was ectopically inserted in the zfp1 deletion strain, pathogenesis and virulence were partially restored. This, coupled with zfp1 over-expression strains having a similar phenotype as the deletion strains, suggested Zfp1 may interact with other proteins for full function. These findings show that Zfp1, in conjunction with one or more binding partners, contributes to U. maydis pathogenesis, virulence, and anthocyanin production through the regulation of effector gene expression.

Author Keywords: effector, pathogenesis, transcription factor, Ustilago maydis, Zea mays, zinc finger

Neuroblastoma (NB) has one of the highest mortality rates in pediatric oncology due to relapsed and refractory disease. Current aggressive multi-modal treatments are inhibited by dose-limiting toxicities and are associated with late-effects and secondary malignancies, emphasizing the necessity for novel therapeutics. Uniquely, most NB cells highly express disialoganglioside (GD2) a cell surface glycolipid that can provide a target for tumour-specific delivery. This study demonstrates a comprehensive evaluation of silver nanoparticles (AgNPs) and the first preliminary evaluation of anti-GD2-AgNP antibody-drug conjugates (ADCs) against NB in vitro. This present study validates the potential for AgNPs as an anti-cancer agent against NB as AgNPs demonstrated preferential toxicity towards NB cells through metabolic inhibition and indicative morphological alterations, while a less tumorigenic cell line demonstrated resistance to AgNP treatment. Therefore, this work identified an AgNP cell-type-dependent cytotoxicity effect. Low conjugation efficiency of the anti-GD2 monoclonal antibody, 14.G2a, to NHS-activated AgNPs failed to exert greater toxicity than the AgNPs alone. Collectively, this thesis provides novel information regarding the anti-cancer effects of AgNPs against NB with recommendations for anti-GD2-AgNP ADCs.

Author Keywords: ADC, Chemotherapy, GD2, Neuroblastoma, Silver nanoparticles

Understanding the maintenance and spread of invasive diseases is critical in evaluating threats to biodiversity and how to best minimize their impact, which can by done by monitoring disease occurrences across time and space. I sought to apply existing and upcoming molecular tools to assess fluctuations in both presence and strain variation of frog virus 3 (FV3), a species of Ranavirus, across Canadian waterbodies. I explored the temporal patterns and spatial distribution of ranavirus presence across multiple months and seasons using environmental DNA techniques. Results indicate that ranavirus was present in approximately 72.5% of waterbodies sampled on a fine geographical scale (<10km between sites, 7,150 km2), with higher detection rates in later summer months than earlier. I then explored the sequence variability at the major capsid protein gene (MCP) and putative virulence gene (vIF-2α) of FV3 samples from Ontario, Alberta, and the Northwest Territories, with the premise of understanding pathogen movement across the landscape. However, a lack of genetic diversity was found across regions, likely due to a lack of informative variation at the chosen genetic markers or lack of mutation. Instead, I found a novel FV3-like ranavirus and evidence for a recombinant between FV3 and a ranavirus of another lineage. This thesis provides a deeper understanding into the spatio-temporal distribution of FV3, with an idea of how widespread and threatening ranaviruses are to amphibian diversity.

Keywords: ranavirus, frog virus 3, amphibians, environmental DNA, phylogenetics, wildlife disease, disease surveillance, major capsid protein, vIF-2α

Author Keywords: amphibians, environmental DNA, frog virus 3, phylogenetics, ranavirus, wildlife disease

Iridoviruses are large (120-200nm) double stranded DNA viruses that contain an icosahedral capsid. The iridoviridae family is composed of five genera that infect a wide range of poikilothermic vertebrates (Lymphocystivirus, Ranavirus and Megalocyivirus) and invertebrate hosts (Iridovirus, Chloriridovirus). Frog virus 3 (FV3) is a member of the Ranavirus genus, and is commonly used as a model system to study iridoviruses. I was interested in understanding virus-host interaction in FV3. I studied two viral genes, FV3 97R and FV3 75L. Here I demonstrate that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane This study found that 97R possess a unique phenotype and that its localization to the ER is mediated through its C-terminus transmembrane domain. FV3 75L encodes an 84 amino acids protein. I showed that FV3 75L localizes to the early endosomes, while its cellular binding partner, LITAF, localizes to late endosome/lysosome. Interestingly, when FV3 75L and LITAF are co-transfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosome/lysosomes. A physical interaction between LITAF and FV3 75L was demonstrated through a pull-down assay and that a highly conserved domain found in both proteins may mediate the interaction. LITAF has been proposed to function in protein degradation, but there is still uncertainty on LITAF's specific role. I was interested in further characterizing LITAF and its implications in protein degradation and a neurodegenerative disorder. At least 9 mutations of LITAF are associated with Charcot-Marie-Tooth disease type 1C (CMT1C), which belongs to the group of most common heritable neuromuscular disorders, affecting approximately one in 2500 people. We show that LITAF mutants G112S and W116G mislocalize from the late endosome/lysosome to the mitochondria while the T49M and P135T mutants show partial mislocalization with a portion of the protein present in the late endosome/lysosome and a portion of the protein localized to the mitochondria. Since LITAF is believed to play a role in protein degradation, it is possible that the specific characteristics of CMT1C may occur though impaired degradation of Schwann cell membrane proteins, such as PMP22. I was able to show that when WT LITAF is present, there is a decrease in the PMP22 intracellular levels, which suggest that LITAF plays an important role in protein degradation, and also in other types of CMT. Insight into how mutations in LITAF cause CMT1C may not only help better understand cellular pathways, but also further elucidate the role LITAF's viral homolog FV3 75L during viral infection.

Author Keywords: 75L, Charcot-Marie-Tooth, CMTC1, ER, FV3, LITAF

The aim of this work was to identify the DNA sequences recognized by the Giardia TBP (gTBP) in vivo by using a chromatin immunoprecipitation assay (ChIP). Since a specific antibody for the protein of interest is required for this assay, a company was contracted to produce and purify a custom polyclonal antibody from the immunization of rabbits. Recombinant GST-gTBP was produced at a suitable yield and purity and used as the immunogen. The antibody was then tested for reactivity to the native protein in our laboratory. By Western blot analysis, it was possible to observe the enrichment of the gTBP within the nuclear fraction compared to a cytoplasmic fraction extracted from Giardia cells. However, the antibody could not be successfully used in an immunoprecipitation assay - suggesting that the antibody is unable to bind to the native structure of gTBP. Therefore, the focus of this work was changed to analyse gTBP via multiple sequence alignments, homology modelling and BLAST to identify any unique regions that may contribute to its unusual binding characteristics. These techniques were also used to identify specific regions of gTBP that may be used to generate synthetic peptides as immunogens for future antibody production.

Author Keywords: ChIP, Giardia intestinalis, Homology modelling, Immunoprecipitation, TATA-binding protein, Western Blotting