Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

Esophageal squamous cell carcinoma (ESCC) is a common cancer in the northeast of Iran. In a series of studies we explored the genetic basis for this. First we showed the risk to age 75 of esophageal cancer in the first-degree relatives of patients with esophageal cancer was 34%, versus 14% for the first-degree relatives of the controls (hazard ratio = 2.3, 95%CI = 1.7-3.1; P = 3 x 10 – 8). Second, in a candidate-gene association approach, we showed that the ADH1B p.Arg48His mutation was associated with a significantly decreased risk of ESCC (OR = 0.41, 95%CI = 0.29-0.76; P = 4x10-4) under a recessive mode of inheritance, although our study subjects were not alcohol drinkers. Third, we showed the BRCA2 p.Lys3326X variant to be associated with increased risk of ESCC (OR = 3.38, 95%CI = 1.97-6.91; P = 2x10-4). Then, we hypothesized that the genes for Fanconi anemia may be candidate genes for ESCC and sequenced the entire coding regions of 12 Fanconi anemia genes in the germline DNA of 190 ESCC cases. We identified three heterozygous insertion/deletions in FANCD2, FANCE and FANCL. All three patients had a strong family history of ESCC. In addition, we found two homozygous patients for the deleterious FANCA p.Ser858Arg mutation. We found two more homozygotes in 556 more ESCC patients, but in none of 1373 matched controls (OR = 16.7, 95%CI = 6.2-44.2; P = 0.01). Finally, we implemented a pilot genome-wide association study in ESCC using 182 cases and 177 matched controls. None of the 1.2 M observed and imputed SNPs showed an association with ESCC at a genome-wide significance level. This showed that an ESCC susceptibility allele with OR>3 estimated from our familial risk study, is unlikely to be identified among common variants of the human genome and we should look for it among rare variants.

One of the most important risk factors in ovarian cancer is family history, and two well-studied tumour suppressor genes BRCA1 and BRCA2 have already been identified in “high-risk” families. However, alterations of other genes may also be important for ovarian cancer pathogenesis in individuals with family history of breast/ovarian cancer. In this thesis, I compared the gene expression profiles of tumours from patients with strong and weak family history of breast and/or ovarian cancer to identify genes that may be significant in the subset of patients with ovarian cancer predisposition. Based on this comparison, two genes of interest were selected for further investigations: hCDC4/FBXW7 (F-box and WD repeat domain containing 7) and PRKCZ (protein kinase C zeta). Through mutational analyses I identified one nucleotide alteration within exon 7 of hCDC4; however, overall I found that hCDC4 mutation is a rare event in ovarian tumours. Additional epigenetics analyses revealed that promoter methylation is not a significant mechanism responsible for repression of hCDC4 expression in ovarian cancer. Nevertheless, the variable expression of hCDC4 proteins observed in ovarian tumour tissues by immunohistochemical staining of tissue microarrays suggests that hCDC4 deregulation may potentially be important in a subset of ovarian cancers. Additionally, I observed that expression levels of PRKCZ are higher in ovarian tumours from patients with strong family history compared to patients with weak family history. PRKCZ has previously been shown to be involved in a variety of cellular processes; however its role in ovarian cancer remained elusive. To further understand the role of PRKCZ in ovarian tumourigenesis, including cell viability, cell migration, as well as relevant downstream signaling pathways, I performed functional assays using an in vitro ovarian cancer model. I observed that PRKCZ increases proliferation of the SKOV3 ovarian cancer cell line and participates in EGF-induced chemotaxis. Furthermore, I identified IGF1R (insulin-like growth factor 1 receptor) and ITGB3 (integrin beta 3) as downstream effectors of PRKCZ as expression of these genes is significantly altered when PRKCZ is over-expressed. Given their previously identified associations with familial ovarian cancer, the IGF1 and ITGB3 signaling pathways may therefore represent a possible link between PRKCZ and this disease.

Human maternal responsiveness is a complex repertoire of infant-related behaviours and attitudes, which vary between and within mothers. Environmental and socioeconomic factors undoubtedly influence maternal care, but less is known about how genetic variation associates with maternal responsiveness. Where genetic variation has been examined in relation to mothering, the moderating effects of early life experiences in the mother have not been explored. Additionally, studies tend to focus on maternal sensitivity, neglecting to explore other dimensions of maternal responsiveness. The purpose of the present thesis was to explore genetic variation in three gene families – dopamine (DA), serotonin (5-HT), and oxytocin (OXT) – in relation to differences in maternal care in a Caucasian sample of new mothers recruited in early pregnancy from Hamilton, Ontario (N=187). Furthermore, interactions between early experiences in the mothers’ lives and their genetic polymorphic variants were examined. Early experience was a combined measure of the Parental Bonding Instrument and the Childhood Trauma Questionnaire. Maternal behaviour during mother-infant interaction was assessed at 6 months postpartum and derived from a video-recorded 30-minute mother-infant interaction. The behavioural outcomes of interest maternal sensitivity (assessed using the Ainsworth Maternal Care Scales) and maternal behaviors (vocalizing, orienting away from infant, instrumental caregiving). We also assessed maternal attitudes about parenting the infant at 6 months postpartum. Multiple polymorphisms on DA receptors D1 (rs686, rs4532, rs265981, rs265976, rs5326) and D2 (rs6277, rs1799732, rs1799978, rs1800497) associated with maternal vocalizing and orienting away from the infant, respectively. The OXT polymorphisms rs2740210 and rs4813627 associated with infant-directed vocalizing. A major polymorphism on the 5-HT transporter (5HTTLPR and a related rs25531 polymorphism) associated significantly with maternal sensitivity. There were gene-environment interactions between this 5-HT polymorphism and early adversity in association with maternal orienting away and maternal attitudes. Gene-environment interactions were also found between the OXT polymorphisms rs2740210 and rs4813627 and instrumental care of the infant. These results suggest that variations in genes encoding major brain neurotransmitters and neurohormones are related to observed maternal behaviour and self-reported maternal attitudes. These results showcase the importance of exploring multiple dimensions of complex behavioural phenotypes like mothering.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

Epithelial ovarian cancer is the leading cause of death by gynaecological malignancy. Elucidation of the driver genes of ovarian cancer will lead to treatment targets and tailored therapy for this disease. The Affymetrix Genome-Wide SNP Array 6.0 was used to study 100 serous ovarian samples and 10 normal ovarian samples to identify loci and driver genes. The ovarian cancer genome was found to have high overall genomic instability across all chromosomes and key known genes in this disease were identified in the dataset. Aberrant regions of copy number gain were located in “blocks” of constant copy number at 1p, 1q, 8q, 12p, 19q and 20q. The range in copy number for gains was 4.2 to 5.1. The “blocks” of genes were located at 8p and 5p for copy number losses. The range for copy number loss was 0.6 to 0.9.

Retinoblastoma, a rare childhood cancer of the retina, is characterized by loss of both alleles of the RB1 gene. However, additional mutational events are required for malignancy. CGH studies described common chromosomal changes indicating potential oncogenes and tumor suppressor genes. Previous work in the lab implicated Cadherin-11 (Cdh11) as a tumor suppressor after analysis in human retinoblastomas and in the simian virus 40 large T-antigen induced murine retinoblastoma model (TAg-RB) showed loss of Cdh11 expression. TAg-RB mice crossed with Cdh11-/- mice, revealed faster growing tumors in mice null for Cdh11. This thesis focused on defining the tumor suppressor role of Cdh11 in retinoblastoma progression. The results showed in vitro and in vivo evidence that Cdh11 was promoting apoptosis and not suppressing proliferation. We also observed an increase in invasion markers upon the loss of Cdh11. We conclude that Cdh11 acts as a tumor suppressor in retinoblastoma, through promotion of apoptosis.

Medicago sativa (Alfalfa) is an important agricultural plant for animal forage and nitrogen fixation, and has potential value in ligno-cellulosic energy production. In the quest to understand the plant, I generated a draft genome sequence of M. sativa via two reduced representation sequencing approaches: methylation-dependent filtration, and high CoT filtration. Libraries created from each approach were sequenced on an Illumina next-generation sequencing platform yielding approximately 2.5Gb of raw data. A combination of reference-based genome assembly approaches using the closely related species, Medicago truncatula as a reference, and de novo genome assembly approaches were performed to assemble the draft genome. The reference-based assembly generated 312,011 contigs with weighted median contig length (N50) of 247 bases, whereas de novo assembly produced 547,304 contigs with N50 of 275 bases. The creation of the M. sativa draft genome is vital for downstream functional analyses such as genome wide gene mining and gene expression profiling.

This study presents an integrated analysis of pancreatic ductal adenocarcinomas (PDACs) for identification of putative cancer driver genes in somatic copy number gains (SCNGs). SCNG data on 60 PDAC genomes was extracted to identify 756 genes, mapping to 20 genomic loci that are recurrently gained. Through copy number and gene expression analysis on a panel of 29 human pancreatic cancer cell lines, this gene catalogue was refined to 34 PDAC high-confidence candidate genes. The performance of these genes was assessed in pooled shRNA screens and only ECT2 showed significant essentiality to cell viability in specific PDAC cell lines with genomic gains at the 3q26.3 locus that harbor this gene. Targeted shRNA-mediated interference of ECT2, as well as pharmacological inhibition, are supportive of the pooled shRNA screen findings. These results favor ECT2 as a candidate target gene for further evaluation in the subset of PDACs presenting with 3q26 somatic copy number gains.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.