Most immortal human cells maintain their telomeres by up-regulating the enzyme telomerase. Approximately 10-15% of immortal cells maintain their telomere lengths by a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Human ALT cells are characterized by ALT associated promyelocytic bodies (APBs) that contain proteins involved in DNA damage response and repair. Our lab has found significant colocalization of several components of the Fanconi Anemia (FA) pathway with telomeres and demonstrated that knockdown of FANCD2 leads to ALT-specific increase in the amount of telomeric DNA as well as increased aneuploidy and cell death. In this study, we examined the role of FANCM in telomere maintenance in ALT cells. We found a significant colocalization of FANCM with telomeres in two ALT cell lines. Knockdown of FANCM was associated with reduced growth, increases in the size of TRF2 foci and in the amount of telomeric DNA. These data suggest that FANCM plays a role in telomere length regulation and maintenance.

Most immortal human cells maintain their telomeres by up-regulating the enzyme telomerase. Approximately 10-15% of immortal cells maintain their telomere lengths by a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Human ALT cells are characterized by ALT associated promyelocytic bodies (APBs) that contain proteins involved in DNA damage response and repair. Our lab has found significant colocalization of several components of the Fanconi Anemia (FA) pathway with telomeres and demonstrated that knockdown of FANCD2 leads to ALT-specific increase in the amount of telomeric DNA as well as increased aneuploidy and cell death. In this study, we examined the role of FANCM in telomere maintenance in ALT cells. We found a significant colocalization of FANCM with telomeres in two ALT cell lines. Knockdown of FANCM was associated with reduced growth, increases in the size of TRF2 foci and in the amount of telomeric DNA. These data suggest that FANCM plays a role in telomere length regulation and maintenance.

Tubby mice succumb to blindness, deafness, and obesity. Vision and auditory deficits are attributed to neurodegeneration and tubby-associated obesity has been postulated to result from neuronal deficits in brain regions controlling weight regulation. TUB has been implicated in Gq signaling and 2 isoforms of TUB, found exclusively in the brain, may have opposing effects on transactivation. Toward this end, I developed several cell culture assays to interrogate TUB function and found that TUB directs neuronal outgrowth in an isoform-specific manner. One isoform directs stable and polar outgrowth while the other directs multiple process outgrowths and branching. These effects can occur via Gq signaling and require nuclear localization. Furthermore, I have found that the serotonergic system of tubby mice displays morphological and innervation deficits. Since the serotonergic system is implicated in modulating moods and behaviours, including appetite, these deficits may result in the obesity and motivational issues observed in tubby mice.

Genomic evolution is a continuous process that involves the accumulation of neutral and adaptive variation within DNA sequences. Duplication, a mechanism that introduces new genetic material into a genome, is thought to be the primary source of new genes that have arisen during vertebrate evolution. This hypothesis, popularized by Susumu Ohno in 1970, has transformed the field of evolutionary biology. Consequently, many evolutionary studies have concentrated on identifying examples of gene duplication and assessing their impact on the evolution of genomes. This thesis presents the identification and analysis of three examples of gene duplication involved in shaping mammalian genomes. Through these analyses, I investigate the fate of duplicated genes and discuss the potential impact of duplication on genomic evolution. The fates depicted within these studies range from the pseudogenization of recent gene duplications to the preservation of ancient duplications for over 100 million years in multiple mammalian genomes. The consequences of these fates include neofunctionalization, subfunctionalization, and gene relocation. In additional, the analyses in this thesis demonstrate different rates and directions of evolution following gene duplication. Some duplicated genes are shown to diverge gradually over time throughout mammalian evolution, while others exhibit an accelerated evolutionary rate within a specific lineage. In other rare cases, divergence is impeded such that duplicated genes evolve in synchronization, under a process known as concerted evolution. This can lead to examples showing mosaic evolution, where both divergent and concerted evolutionary signatures are observed within a single duplicated gene. Through the analyses presented in this thesis, I illustrate some of the different evolutionary histories that result from gene duplication and examine the variety of forces that influence the evolution of duplicated genes. These studies examine the role of duplication in mammalian evolution and represent a significant contribution to the growing body of knowledge in the field of evolutionary biology.

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

The chemical synapse is an asymmetric structure consisting of presynaptic and postsynaptic terminals in direct apposition to each other. Synapses function to mediate the transmission of signals between neurons and their targets. The formation of synapses is a tightly regulated process requiring the interaction of many genes and molecular pathways. I am interested in identifying genes and signaling pathways that are required for proper synapse formation. Using the GABAergic neuromuscular junctions of C. elegans as a model system, I have identified fsn-1 (F-box synaptic protein), a gene required for the control of synaptic growth. fsn-1 mutants exhibit a synaptic defect characterized by both synaptic over differentiation and under differentiation. FSN-1 is an F-box protein with a SPRY (SPla and RYanodine receptor) domain that functions cell-autonomously in neurons to regulate synaptic growth. I have shown that it functions in an E3 ubiquitin ligase-like complex with the RING-H2 finger protein RPM-1 (Regulator of presynaptic morphology), SKR-1 and Cullin. The composition of this complex is similar to SCF (Skp1, Cullin, F-box) E3 ubiquitin ligases. We hypothesize that this complex controls synapse formation by down regulating synapse promoting factors through an ubiquitin mediated process. We have identified two receptor tyrosine kinases that genetically interact with fsn-1, the Anaplastic Lymphoma Kinase homolog SCD-2 (Suppressor of Constitutive Dauer) and the C. elegans insulin receptor DAF-2 (abnormal DAuer Formation). Loss of function mutations in scd-2 or daf-2 partially suppress the synaptic differentiation defects of fsn-1 mutants, suggesting that they participate in signaling pathways whose activities are normally inhibited by FSN-1 during synapse formation. Unlike FSN-1 that functions in GABAergic neurons, I found that SCD-2 and DAF-2 have cell non-autonomous functions at GABAergic neuromuscular junctions. SCD-2 is required in the nervous system in the RID interneuron where it could modulate synapse formation through ligands present on the motoneuron cell surface. The DAF-2/insulin pathway functions in postsynaptic muscle cells to regulate FSN-1 dependent presynaptic growth likely through a retrograde or feedback mechanism. I propose a model where FSN-1 regulates synapse formation by attenuating signals that converge upon the presynaptic terminal to stimulate or inhibit synaptic growth.

We previously reported that in inherited photoreceptor degenerations (IPDs), the mutant photoreceptors (PRs) are at a constant risk of death (Pacione, Szego et al, 2003). Using microarrays to identify genes that may mediate the constant risk, I identified 145 differentially expressed transcripts in the Rds+/- mouse model of IPD at a time when 90% of the PRs were alive. A major finding was the up-regulation, quantified by qPCR, of four components of a putative IL-6 cytokine signaling pathway: Oncostatin M (Osm) (2-fold increased), Oncostatin M receptor (Osmr)(2.6-fold increased), Stat-3 (2.3-fold increased), C/EBP delta(3.2-fold increased). Similarly, I found increases in the cognate proteins Osmr (3-fold), Stat-3 (2.6-fold), and the phosphorylated, transcriptionally active form of Stat-3, pStat-3 (5.8-fold)(all p<0.01). Other Il-6 cytokine signaling molecules were largely unchanged, but the mRNA of leukemia inhibitory factor (Lif), was increased (3.0-fold). Comparable increases of most transcripts were also present in the Rd1-/- and mutant rhodopsin P347S transgenic (P347S) IPD models. The increases in cytokine signaling molecules occurred predominantly in Müller glia, although C/EBP delta transcript was increased in PRs. Because exogenous IL-6 cytokine treatment slows PR death in IPDs, I asked whether the endogenous increases in IL-6 pathway proteins in IPD retinas were a survival response, and generated IPD models with Osmr, Lif or C/EBP delta loss-of-function (LOF) mutations. Osmr LOF decreased PR survival in the retinas of Rds+/-;Osmr-/- mice, which had 12.5% fewer PRs than those of Rds+/-;Osmr+/+ mice (n=9, p<0.05) at 4 month of age, and Tg-RHO(P347S);Osmr-/- mice had 13.5% fewer PRs (n=6, p<0.01) at 31 days of age. Unexpectedly, Osmr LOF had no effect on pStat3 levels in Rds+/-;Osmr-/- retinas, indicating that retinal Stat3 activation may be predominantly regulated by other molecules. In contrast to the Osmr LOF, Lif or C/EBP delta LOF unexpectedly increased mutant PR survival. Rd1-/-;Lif -/- mice at 13 days had 14% more PRs than Rd1-/-;Lif+/+ mice (n=6, p<0.003) and a 1.7 fold decrease in pStat-3 (n=4, p<.05). Similarly, 8 month-old Rds+/-; C/EBP delta-/- mice had 18% more PRs than Rds+/-; C/EBP delta+/+ mice (n=5, p<0.005). These findings suggest that in mutant PRs: 1) up-regulation of the Osmr receptor is protective; 2) the presence of Lif or C/EBP delta is pathogenic, and therefore 3) Osmr, Lif and C/EBP delta act either in different pathways or different cells, to account for the differing effects of their LOF on PR cell death; and 4) the partial effects of Osmr, Lif and C/EBP delta LOF indicate that other genes also mediate the constant risk of death of mutant PRs in IPDs.

In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

Vascular smooth muscle cells (VSMC) serve as key constituents of the developing vasculature, ensuring stability of the nascent vessels, and are essential for the proper performance of the mature cardiovascular system. Pathological alterations in SMC biology, manifesting as perturbations in the differentiation state of SMC, play a pivotal role in the development and progression of various vascular diseases. Thus an understanding of the mechanisms and factors that control SMC differentiation underlies the potential for diagnostic and therapeutic advances. As such, this thesis aimed to implicate the c-Myb transcription factor in the process of SMC differentiation. For this purpose, in vitro assays were developed using the embryoid body (EB) model system, suitable for clarifying the molecular pathways and extrinsic factors that promote or obstruct SMC lineage specification and maturation to the contractile phenotype. Contractile activity was observed in the developing EBs, and further characterized as smooth muscle and cardiac contractions. Temporal induction of SM-specific markers preceded and paralleled contractile SMC appearance and their mRNA levels predicted the relative ability of distinct mouse ES cell (mESC) lines to generate EBs with contracting SMC. Using fluorescent SM-specific promoter-reporter gene strategy it was shown that spontaneous SM-contractions coincide with SM-myosin-heavy-chain (SM-MHC) promoter activity. Moreover, this technology was employed to generate SMC (SM-MHC+) in a scalable stirred-suspension culture system. This thesis addressed the effects of c-myb ablation on SMC differentiation both in vitro and in vivo. c-myb-/- mESC were unable to produce EBs with spontaneously contracting SMC, but gave rise to contracting cardiomyocytes unimpaired. Temporal patterns of myogenic and SM-specific gene expression levels during the course of differentiation revealed that, unlike wild-type EBs, c-myb-/- EBs lacked robust induction of these critical markers. Accordingly, c-myb-/- EBs exhibited significantly reduced SMC numbers. Additionally, in chimaeric EB model substantial contribution by c-myb-/- mESC was detrimental to the development of SM-contractions. To corroborate the in vitro data, chimaeric embryos and adult mice were used to demonstrate significantly reduced c-myb-/- cell contribution to vascular and visceral SMC lineages in vivo. Collectively, this thesis assigns a novel role for the c-Myb transcription factor and advances knowledge of in vitro SMC differentiation.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.