A main function of plant mitochondria is to generate energy by oxidizing carbon substrates as part of cellular respiration. What determines which substrate(s) a mitochondria uses to support respiratory ATP production? Catabolite repression is a concept originating from observations that microbes use a gene regulatory system to sequentially consume their favorite substrates one at a time rather than feasting on a bit of everything at once. Using a new high-throughput technique, which allows multi-hour leaf respiratory measurements following exposure to external metabolites, Brendan has determined that plant mitochondrial respiration operates in a way that is consistent with the existence of a catabolite repression regulatory network. Furthermore, this selective process is linked to the timed expression of transcription factors and specific downstream metabolic enzymes upon depletion of preferred substrates. Ongoing elucidation of catabolite repression signaling will advance our understanding of how the complex metabolic network encompassing respiration is regulated and how plants ensure that their cellular energy requirements are met under stressful environmental conditions.
 
Brendan is a former Queen’s student (BSc Biochemistry 2006 -> PhD Biology 2011) and captain of Queen’s Men’s Varsity Basketball team. After completing several years of post-doctoral research in the Dept. of Plant Sciences at the Univ. of Oxford (UK), he was awarded a prestigious Discovery Early Career Researcher Award (DECRA) from the Australian Research Council to work with Harvey Millar and his team at the Univ. of Western Australia in sunny Perth.   

​Overexpressing a mouse Bax cDNA in yeast cells induces cell death. When heterologously expressed in yeast cells overexpression of a mouse cDNA encoding Bax, human Thyroid Cancer-1 (TC-1) or annexin V (ANXA5) suppresses the Bax induced cell death. My goal was to characterize the anti-Programmed Cell Death (anti-PCD) potential of these human cDNA sequences. In addition to rescuing Bax-expressing cells, I found that TC-1 and ANXA5 are pro-survival sequences that protect yeast from other death-inducing stresses including copper sulfate. TC-1 also prevented the deleterious effects caused by over-expression of a number of endogenous yeast pro-apoptotic sequences, including the YCA1 encoded metacaspase; the YBH3 encoded Bcl-2 Homology domain 3 protein, the NUC1 encoded DNase, as well as the apoptosis-inducing factor encoded by AIF1. Even though the protective effects were more pronounced in rescued cells over-expressing YBH3 and YCA1, TC-1 could still protect yeast mutant strains lacking YBH3 and YCA1 from the growth inhibitory effects of copper sulfate. Our results demonstrate that TC-1 and  ANXA5 are pro-survival sequences that are able to attenuate stress-induced programmed cell death.

Insulin is a key hormone in the regulation of blood glucose levels in many animals.
Dysfunctional insulin signaling is one of the most studied and widely accepted theories in the pathogenesis of diabetes and Alzheimer’s disease (AD). Despite the claim of a link between diabetes and AD, the results in the literature have been incongruent. Some studies have claimed that insulin resistance is a major implication in AD, while others have reported that the risk is increased by excess insulin in the blood. Using the microscopic worm, Caenorhabditis elegans, we have developed a model to study the link between insulin signaling and AD pathologies, including changes in neuronal morphology and abnormal behaviour. Furthermore, we have examined the effects of hyperactive insulin signaling on the expression of the two characteristic proteins of AD, amyloid-beta and tau. By genetically modifying the key players of the insulin signaling pathway and the 40 insulin-like peptides in C. elegans, we may be able to identify novel targets for the prevention or treatment of AD. In this research, we tested whether the 40 insulin-like peptides are inhibitors or activators of the insulin signaling pathway, based on the aforementioned phenotypes associated with AD. Alternatively, some of these 40 insulin-like peptides may work through the non-canonical insulin-signaling pathway, with the potential of pointing us to novel genetic interactors. My work has shown that of the 40 insulin-like peptides, some exacerbated AD-like phenotypes, while others reduced them. Understanding the function of the components of the insulin-signaling pathway and the 40 insulin-like peptides in C. elegans may clarify the link between defective insulin signaling and AD and present novel treatment strategies for the disease. 

​Microalgae are photosynthetic microorganisms that can grow in a variety of environments. They have become a popular feedstock for the production of biofuels, high-value chemicals, and nutraceuticals. One of the main products obtained from microalgae is biodiesel: a mix of fatty acid methyl esters (FAMEs) obtained from the transesterification of triacylglycerol (TAG). However, microalgal biodiesel still cannot compete economically with petroleoum diesel. Therefore, the objective of this thesis was to explore alternatives to make microalgal cultivation for biodiesel more efficient.
Microalgal treatment of municipal wastewater has been discussed as a strategy for the simultaneous removal of excess nutrients and the generation of microalgal biomass. This thesis evaluated the performance of Scenedesmus sp. and Chlorella sorokiniana in the removal of nutrients from non-sterile, highly concentrated synthetic-wastewater. These microalgae removed up to 60% NH4+ and 44% PO43- in a semi-continuous cultivation mode without negatively affecting effluent quality, demonstrating that microalgal growth can be coupled with wastewater treatment.
Microalgal research is growing, but reliable genetic transformation protocols are still not available for many species. Such is the case of Chlorella vulgaris, whose transformation protocol has not been efficiently replicated. Therefore, work with the model Chlamydomonas reinhardtii is a practical alternative for proof-of-concept studies. The work in this thesis generated 5 Chlorella vulgaris starch mutants through UV-mutagenesis, and 4 Chlamydomonas reinhardtii transgenic lines overexpressing Chlorella vulgaris genes involved in glycerolipid metabolism. Starch-less mutant st27 produced up to 3.8-fold increase in TAG, while the transgenic lines increased more than 100-fold. Results from the starch-less mutants illustrate the importance of the organic carbon source in the medium to provide enough carbon precursors for TAG biosynthesis. Similarly, the increases in TAG content of the transgenic lines suggest that increasing glycerol-3-phosphate is crucial to generate oleaginous crops.
This thesis illustrates the possibility of generating microalgal strains rich in both starch and TAG, which would be a promising feedstock to implement in microalgal biorefineries. Additionally, two new gene candidates to improve TAG yields in microalgal strains are suggested, expanding on the knowledge available for metabolic engineering of microalgal crops for biodiesel production.

All organisms have evolved immune strategies to defend against infection. While animals have evolved both cell-autonomous and humoral immune responses, plants rely solely on the innate ability of each cell to detect and defend against infection. During infection, animal cells secrete MEMBRANE ATTACK COMPLEX and PERFORIN FORMING (MACPF) proteins that form pores in pathogen membranes resulting in their lysis and death. The model plant Arabidopsis thaliana encodes four proteins with some similarity to MACPF proteins, however the molecular function of these proteins is largely unstudied. Over the last two years, I have utilized functional genetic complementation, confocal microscopy, and genetic epistatic analysis to elucidate the role of one of these MACPF-domain containing proteins in the immune response of Arabidopsis. Through my work, I have been able to demonstrate that this protein plays a role in plant immune signaling, localizes to two sub-cellular localizations, and associates with at least one other member of the Arabidopsis MACPF family. 

​Plants have evolved sophisticated mechanisms for dealing with pathogen stressors. Conserved microbial patterns are perceived by receptors at the plant plasma membrane, inducing a signaling cascade which culminates in a robust immune response. Although essential for survival, uncontrolled immune responses can lead to severe fitness costs in the plant, and therefore immune signaling must be tightly regulated. One aspect of this regulation involves the maintenance of optimal levels of the immune signaling kinase, BIK1. The calcium-dependent protein kinase, CPK28, was previously found to contribute to its turnover via the 26S proteasome. We have since identified that the plant-Ubox proteins, PUB25 and PUB26 are the E3 ligases that contribute to BIK1 turnover through phosphorylation by CPK28. My MSc project has involved the use of various genetic and biochemical tools to further tease apart the fine mechanisms by which CPK28 and PUB25/26 function as a module to maintain optimal BIK1 levels, and thus plant immune homeostasis.  

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. As one of the most predominant nosocomial pathogens, it tends to preferentially colonize immunocompromised patients such as those with cystic fibrosis, cancer, or AIDS. It can also cause pneumonia, urinary tract infections, and gastrointestinal infections. Clinically, P. aeruginosa is particularly difficult to treat. With the increasing incidence of antibiotic-resistant P. aeruginosa infections, in 2017 World Health Organization declared this bacterium a “Priority 1” pathogen that is in urgent need of new antibiotics.
Pseudomonas aeruginosa utilizes Type II secretion system (T2SS) to translocate protein virulence factors and facilitate infection in the host. We have solved three-dimensional structures of T2SS core complexes and designed structure-based inhibitors to target the protein-protein interaction interface of the T2SS core complex. The inhibitors not only disrupted the complexes in vitro, but also inhibited the secretion of T2SS substrates in P. aeruginosa. Most importantly, they also attenuated Pseudomonas aeruginosa infection in the animal model of Caenorhabditis elegans. Our work represents the first demonstration of successful T2SS inhibition in any bacterium.

​Plants rely on innate immunity to perceive and respond to pathogenic microbes. Developmental age can also affect a plant’s ability to defend itself from pathogens. Although plants do not possess moving immune cells, they possess the ability to alert systemic tissues to ongoing localized infections. In the Cameron lab we are interested in understanding systemic and developmental resistance in plants with the long-term goal of enhancing disease resistance in crop plants. Systemic Acquired Resistance (SAR) allows a plant to perceive an initial infection in one leaf and transmit a signal to inform systemic leaves about the pathogen threat. We use Arabidopsis, cucumber and tobacco to understand how SAR long-distance signals move from initially infected leaves to distant healthy leaves. We are investigating how DIR1, a lipid transfer protein (LTP) moves to distant leaves during SAR. We also study developmentally regulated Age-Related Resistance in Arabidopsis. Young plants are highly susceptible to the bacterial pathogen, Pseudomonas syringae, but when mature, Arabidopsis displays ARR and responds with a super immune response to these same pathogens. Our studies indicate that during ARR, mature  plants defend themselves by limiting  bacterial growth and biofilm formation. Although both SAR and ARR pathways allow plants to respond in a resistant manner to normally virulent pathogens, our evidence to date indicates that the mechanisms responsible are unique to each pathway.

​Transcriptional-regulatory networks establish the gene expression programs responsible for normal growth and disease states.  They are composed of interactions between transcription factors and their target genes by direct binding to the promoter.  The complete mapping of these networks in model systems by identifying the binding specificities and target genes of each transcription factor remains an important goal to achieve a comprehensive understanding of cellular and organismal systems.  The transcriptional-regulatory network of the fission yeast Schizosaccharomyces pombe consists of approximately 100 sequence-specific DNA-binding transcription factors regulating about 5000 genes in the genome.  Despite being an extensively-studied model organism, its transcriptional-regulatory network remains substantially incomplete.  We have carried out systematic genetics combined with functional genomic approaches such as transcriptomics, genome-wide chromatin immunoprecipitation and synthetic genetic array analysis to functionally characterize S. pombe transcription factors and identify their target genes.  These studies have elucidated a novel transcriptional-regulatory network that governs flocculation, implicated new transcription factors and metabolism genes in cell cycle regulation and estimated the functional redundancy of transcription factor gene pairs in S. pombe.

Dr. Gary Armstrong is Assistant Professor in the Department of Neurology and Neurosurgery at the Montreal Neurological Institute, McGill University. Prior to joining the MNI he was a Post-Doctoral Research Fellow at the Research Centre of the Centre Hospitalier de l’Université de Montréal in the Department of Neurosciences in the laboratory of Pierre Drapeau. His Ph.D. and M.Sc. were completed at Queen’s University in the Department of Biology under the guidance of Dr. Mel Robertson, where he investigated neuronal circuit dysfunction arising during hyperthermia and hypoxia, and the protective mechanisms that preserve neural function during exposure to extreme stress. His current research interests lie in gaining a better understanding of the physiological and molecular mechanisms which culminate in neuronal dysfunction resulting from mutations in genes associated with amyotrophic lateral sclerosis (ALS). Studying this disease requires a model system which can facilitate detailed manipulations of the genes and proteins involved and a nervous system accessible to electrophysiological investigations of neuronal and synaptic activity. The zebrafish larva offers the rare opportunity to combine cutting edge genetic techniques (such as the use of the CRISPR/Cas9 mutagenic system) with in vivo neurophysiological (patch-clamp) recordings of identified neurons, allowing for thorough investigations into the physiological nature of defects arising as a result of mutant protein expression.