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This thesis describes a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. Each of a series of 16 synthetic oligonucleotide primers was annealed to single-stranded Ml3 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations were assayed using 5'-labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template nucleotide and dNTP was important; although purine:pyrimidine (dNTP substrate: template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this the results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity. The recovery of point mutants arising from reverse transcriptase-directed misincorporation of single dNTPs onto single oligonucleotide primers is described and discussed. Misincorporation of dNTPs is a form of in vitro mutagenesis which facilitates the generation of a library of point mutations spread throughout a gene. Conditions have been established in this study for the production of a bank of primers with 3' termini distributed over a region of a gene to be mutated. The misincorporation of single dNTPs onto the termini of such a bank of primers should allow the generation of a library of point mutants.

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Objective: To determine whether genotype at the catechol-O-methyltransferase rs4680, dopamine D2 receptor rs1800497, serotonin receptor 1A rs6295 or serotonin transporter 5-HTTLPR single nucleotide polymorphisms is associated with response to citalopram and escitalopram treatment in patients with major depressive disorder and generalized anxiety disorder. Methods: Twenty one patients with depression or anxiety who were treated with citalopram or escitalopram for greater than one year, and who stopped the medication for a period of time during which their symptoms returned, and upon re-commencing the medication their symptoms were again reduced, were classified as responders. Patients were assessed using the Sheehan Disability Scale and the Quick Inventory of Depressive Symptomology- self report. The control group consisted of 146 healthy participants. Genotype was determined at each of the candidate genes studied: catechol-O-methyltransferase, dopamine D2 receptor, serotonin receptor 1A and serotonin transporter. Chi squared tests were used to compare genotypic and allele frequencies between responders and controls. Results: There was no significant difference in genotypic or allele frequencies between responders and controls at each of the genes analyzed. Conclusions: This pilot study suggests that genotype at the catechol-O-methyltransferase, dopamine D2 receptor, serotonin receptor 1A and serotonin transporter genes is not associated with response to citalopram and escitalopram treatment in patients with depression and anxiety. A larger sample size, along with a genome-wide scan are needed to identify genetic variants that predict medication response in future patients. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-08-31 12:26:21.402

Introduction: The purpose of this study was to examine phenotypic expression of craniofacial form, shape, and size as it relates to the genotype of an individual. Shape analyses were completed on 3-D images of each subject's craniofacial structure by landmarking 45 points of interest on the cranial base, facial bones, and upper and lower jaws. A candidate gene analysis was undertaken focusing on specific genes in the Hippo Signaling Pathway to examine genotype-phenotype correlations that play a role in craniofacial development. This study is a continuation of a larger project aimed at the identification of candidate genes associated with human dento-skeletal bite problems led by Dr. Lina Moreno-Uribe. Methods: The sample size for our study included 166 individuals who had never been treated orthodontically at the time of records. Each individual was genotyped and a CBCT of the craniofacial complex was captured. Each CBCT image was landmarked by a single observer using 45 points to mark points on the cranial base and facial bones including the maxilla and mandible. General Procrustes superimposition was used to find correlations with phenotype and genotype. Size analysis was completed with average Euclidean Distances and ANOVA analysis. Results: 2 SNP's from the FOX03 gene had significant associations with size. The AA genotyped individuals appeared larger in overall size than AB genotyped individuals. 3 SNP's had statistically significant associations with facial form. The FOX06 SNPs had significant associations with increased anterior-posterior growth of the maxilla. The AJUBA SNP had significant associations with increased overall craniofacial breadth. Conclusion: Genes in the Hippo signaling pathway have specific roles in the development of facial form and size.

The aim of the project was to identify biologically relevant novel gene clusters by using combined genomic data instead of using only gene expression data in isolation. The clustering algorithm based on self-organizing maps (Kasturi et al., 2005) was extended and implemented in order to use gene location data together with the gene expression and the motif occurrence data for gene clustering. A distance function was defined to be used with gene location data. The algorithm was also extended in order to use vector angle distance for gene expression data. Arabidopsis thaliana is chosen as a data source to evaluate the developed algorithm. A test data set was created by using 100 Arabidopsis genes that have gene expression data with seven different time points during cold stress condition, motif occurrence data which indicates the occurrence frequency of 614 different motifs and the chromosomal location data of each gene. Gene Ontology (http://www.geneontology.org) and TAIR (http://arabidopsis.org) databases were used to find the molecular function and biological process information of each gene in order to examine the biological accuracy of newly discovered clusters after using combined genomic data. The biological evaluation of the results showed that using combined genomic data to cluster genes resulted in new biologically relevant clusters.

<p>The aim of the project was to identify biologically relevant novel gene clusters by using combined genomic data instead of using only gene expression data in isolation. The clustering algorithm based on self-organizing maps (Kasturi et al., 2005) was extended and implemented in order to use gene location data together with the gene expression and the motif occurrence data for gene clustering. A distance function was defined to be used with gene location data. The algorithm was also extended in order to use vector angle distance for gene expression data. <em>Arabidopsis thaliana</em> is chosen as a data source to evaluate the developed algorithm. A test data set was created by using 100 Arabidopsis genes that have gene expression data with seven different time points during cold stress condition, motif occurrence data which indicates the occurrence frequency of 614 different motifs and the chromosomal location data of each gene. Gene Ontology (http://www.geneontology.org) and TAIR (http://arabidopsis.org) databases were used to find the <em>molecular function</em> and <em>biological process</em> information of each gene in order to examine the biological accuracy of newly discovered clusters after using combined genomic data. The biological evaluation of the results showed that using combined genomic data to cluster genes resulted in new biologically relevant clusters.</p>

16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the alpha-, beta- and gamma-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the delta-proteobacteria and epsilon-proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB's sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores.

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