The function and evolution of the response regulator CtrA in Rhodobacter capsulatus and Alphaproteobacteria

Rhodobacter capsulatus is a model organism for studying gene transfer agents (GTAs). GTAs are a unique facilitator of gene transfer in prokaryotes. The DNA binding response regulator CtrA plays a key role in modulating GTA activity in R. capsulatus, as well as flagellar biosynthesis and cell motility. CtrA is an OmpR/PhoB response regulator with an N-terminal receiver domain and a C-terminal transcriptional regulator domain. One unusual aspect of CtrA function in R. capsulatus is that it regulates gene expression in both the phosphorylated and nonphosphorylated forms. Using overlap extension PCR, the constructs for expression of three of different versions of ctrA in R. capsulatus were prepared: wild type, phosphomimetic, nonphosphorylatable. These constructs place the genes under the control of the R. capsulatus puf promoter for high level of expression and the encoded proteins have 6×-histidine tags for purification in studies aimed at determination of the DNA binding sites of the different versions of CtrA. Horizontal gene transfer is an interesting way that bacteria can increase their genetic diversity. In this work, the distribution of ctrA in the Alphaproteobacteria was examined and evidence of horizontal gene transfer of this gene was found. Using phylogenetic analyses, several instances of apparent misclassification of alphaproteobacteria to the wrong orders were found and one candidate ctrA horizontal gene transfer event that may have occurred in an ancestral bacterium that subsequently evolved into one lineage within the order Sphingomonadales was found

Investigation of the treatment of epilepsy with cannabinoids

Cannabis has been consumed by humans for millennia, and is currently used in Canada for the treatment of a variety of medical conditions including anxiety, PTSD, and chronic pain. The medical community is hesitant to accept the use of Cannabis and cannabinoids to treat epilepsy due to inadequate information on mechanism of action and long-term effects. Cannabidiol (CBD) is approved to treat pediatric patients with severe epilepsies such as Dravet Syndrome and Lennox-Gastaut Syndrome in the US and some European countries, but there are many individuals with less severe epilepsies whose quality of life is affected by negative side-effects from current anti-epileptic drugs. This research aims to globally evaluate which of the 6 most prevalent cannabinoids show seizure reduction and to investigate the mechanism of action of cannabinoids in an epilepsy model. Using a chemical model of epilepsy, zebrafish larvae were treated with phytocannabinoids, and their seizures measured through an optimized behaviour tracking method. Unique to this study, cannabinoid uptake was measured in larvae with a novel HPLC method developed in this project. This accomplishment is superior to previous attempts to quantify cannabinoid uptake by measuring losses in the water used to deliver cannabinoid to fish, which assumes that all losses are due to uptake and metabolism by the study organisms. CBD induced seizure reduction is partially mediated by the Gprotein coupled receptor GPR55 and potentially through CB1R. Treatment with cannabinol (CBN) and cannabichromene (CBC) decreased seizure intensity at lower concentrations than CBD. Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC), and cannabigerol (CBG) only showed antiepileptic effects at a high concentration, but when concentrationd in combination with CBD reduced seizures more than either treatment alone. RT-qPCR showed changes in expression of endocannabinoid system (napepld, gde1, faah, ptgs1, ptgs2a) and neural (fosab, pyya) genes in response to phytocannabinoid treatment. The data reported in this thesis supports the hypothesis that phytocannabinoids are promising anti-epileptics and could be used in combination therapies for more effective seizure relief

Mutation of foxl1 Results in Reduced Cartilage Markers in a Zebrafish Model of Otosclerosis

Bone diseases such as otosclerosis (conductive hearing loss) and osteoporosis (low bone mineral density) can result from the abnormal expression of genes that regulate cartilage and bone development. The forkhead box transcription factor FOXL1 has been identified as the causative gene in a family with autosomal dominant otosclerosis and has been reported as a candidate gene in GWAS meta-analyses for osteoporosis. This potentially indicates a novel role for foxl1 in chondrogenesis, osteogenesis, and bone remodelling. We created a foxl1 mutant zebrafish strain as a model for otosclerosis and osteoporosis and examined jaw bones that are homologous to the mammalian middle ear bones, and mineralization of the axial skeleton. We demonstrate that foxl1 regulates the expression of collagen genes such as collagen type 1 alpha 1a and collagen type 11 alpha 2, and results in a delay in jawbone mineralization, while the axial skeleton remains unchanged. foxl1 may also act with other forkhead genes such as foxc1a, as loss of foxl1 in a foxc1a mutant background increases the severity of jaw calcification phenotypes when compared to each mutant alone. Our zebrafish model demonstrates atypical cartilage formation and mineralization in the zebrafish craniofacial skeleton in foxl1 mutants and demonstrates that aberrant collagen expression may underlie the development of otosclerosis