Browsing by Author "Grewal, Savraj S."
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Item Open Access ATRX as a Regulator of Telomerase Activity in Cancer Cells(2020-03) Briggs, Sophie; Beattie, Tara L.; Cairncross, Gregory J.; Riabowol, Karl T.; Grewal, Savraj S.Telomere maintenance is the central process governing cellular immortality. The two distinct pathways – 1) telomerase activity and 2) the alternative lengthening of telomeres (ALT) – result in telomere elongation and allow cells to evade normal cellular ageing mechanisms. Both telomere maintenance pathways can become activated through gene mutations, leading to genetically unstable cells capable of unlimited proliferation. Expression of the chromatin remodeling protein ATRX is lost in almost all ALT+ cancers, suggesting a role for ATRX in ALT repression, however its exact role in telomere maintenance remains unclear. This thesis provides novel evidence suggesting ATRX acts to resolve telomeric G-quadruplexes (G4), thereby facilitating telomerase activity and indirectly repressing ALT. Using RNA interference in human cancer cell lines and a novel DNA ELISA-based technique, the data presented here establish that loss of ATRX expression results in increased G4 at the telomere. This G4 enrichment correlates with a reduction in telomerase activity in vitro which is exacerbated following treatment with the G4 stabilizing agent, Pyridostatin. Further, loss of the full-length ATRX isoform alone does not directly correlate with the ALT phenotype in the selected cell panel. However, a truncated ATRX isoform, ATRXt, was found to be expressed in all selected telomerase+ cells and shown to maintain key functions of the full-length protein. Therefore, alternative ATRX isoforms may act to facilitate telomerase activity through G4 resolution, even in the absence of full-length protein. Taken together, these data provide novel evidence identifying ATRX as an important factor facilitating telomerase-mediated telomere elongation through G4 resolution at the telomere.Item Open Access Characterizing the Genomic Heterogeneity of Pediatric Glioblastoma(2018-09-05) Hoffman, Mary; Gallo, Marco; Chan, Jennifer A. W.; Grewal, Savraj S.; Cobb, Jennifer A.Brain tumors are currently the most common cause of cancer-related deaths among children. With a 5-year survival rate of 20%, pediatric glioblastoma (pGBM) is a lethal brain tumor with no effective treatment options. Although pathologically indistinct from its adult counterpart, recent work has shown that pGBMs diverge at both the genetic and transcriptional level from the adult malignancy. Genomic analyses have identified a recurrent mutation in H3F3A, but this lesion is only present in a fraction of patients and has not contributed to the advancement of effective therapies. Using a longitudinal collection of primary and recurrent pGBMs with matched germlines, I have described a molecularly heterogeneous disease with extreme tumoral evolution. Perhaps my most striking finding is the presence of potentially deleterious structural variants in the patient germlines. Together, these findings suggest a novel hereditary component to tumor etiology which has not been previously described in this malignancy.Item Open Access Deducing the post-transcriptional role of the Pumilio protein Puf3 in fission yeast.(2019-05-29) George, Iain Fraser Scotney; Chua, Gordon; Muench, Douglas G.; Turner, Raymond Joseph; Grewal, Savraj S.; Kothe, UtePumilio family proteins (Pufs) are a highly-conserved group of post-transcriptional regulators in eukaryotes. Puf proteins contain an arc-shaped Pumilio homology domain with several coiled repeats that bind to potentially predictable sequences in the 3’-UTR of their mRNA targets. The binding of Puf proteins to mRNA targets can influence their stability, translation and cellular localization. In S. pombe, there are seven non-essential genes that encode classical Puf proteins. The biological function of these Puf proteins and their mRNA targets remain unknown. In this study, we investigated the biological role and mRNA targets of Puf3 in S. pombe. Over-expression of puf3+ with the nmt41 promoter results in reduced fitness and an enrichment of elongated cells arrested in mitosis. Phenotypic characterization of puf3 overexpression strains and genetic interaction screens identified a cell cycle regulatory role for Puf3, principally at the G2/M transition and possibly at the G1/S control point. Genetic interaction screening, fluorescence microscopy and tandem mass spectrometry analysis of immunoprecipitated Puf3-HA identified Puf3-interacting proteins in the post-transcriptional machinery of 3’-deadenylation of mRNA and localized to stress granules. Bioinformatic analysis of the Puf proteins in S. pombe identified the Puf repeat domains and these were used to enumerate the predicted Puf binding element in the transcriptome. The enumeration data and annotation data were used to identify putative mRNA targets from microarray, RNA-seq and RNA-immunoprecipitation sequencing experiments. These identified several components of the kinetochore and mitotic checkpoint as putative mRNA targets of Puf3.Item Open Access Effects of Heat and Cold Shock on Drosophila larval growth and metabolism(2013-11) Samantray, Sikta; Ghosh, Abhishek; Grewal, Savraj S.Item Open Access Examining how ribosomal proteins affect growth and body size(2014-12-05) Samantray, Sikta; Grewal, Savraj S.An important aspect of growth is ribosome biogenesis. The ribosome is the molecular machine that is responsible for protein synthesis hence growth. What still remains unclear is the contribution of rRNA and ribosomal proteins to tissue and body growth in developing animals.Using Drosophila as a model system, the experiments described showed that mutations in certain ribosomal proteins result in no change or an increase in overall body size of Drosophila pupae.
The results showed a link to delayed development to the pupal stage, suggesting an explanation to the increased body size.
Item Open Access FOXO as a Regulator of Hypoxia Tolerance in Drosophila(2020-12) Barretto, Elizabeth; Grewal, Savraj S.; Childs, Sarah J.; Brook, William J.Animals often live in conditions where environmental oxygen levels fluctuate. When oxygen is abundant, growth is promoted, but when oxygen is scarce, metabolic processes are altered to limit growth and promote survival. One important mechanism controlling responses in hypoxia is the regulation of metabolic gene expression. The conserved hypoxia-inducible factor (HIF) is a transcriptional regulator of genes important for hypoxia-responses. However, less is known about other transcription factors important in hypoxia adaptation. We have explored this question in Drosophila melanogaster. Drosophila can tolerate low oxygen conditions. At 5% oxygen, larvae slow their growth and development, but viability remains unaffected. At 1% oxygen, larvae and adults exhibit a state of ‘suspended animation’ but can survive for up to 12 or 30 hours respectively. We found that upon switching larvae from normoxia to hypoxia, the transcription factor Forkhead Box O (FOXO) was rapidly relocalized from the cytoplasm to the nucleus in larval tissues even though animals maintain normal feeding. Moreover, we saw that many known FOXO target genes were induced in animals exposed to hypoxia. FOXO is important for regulating starvation and stress responses and can regulate aging in Drosophila and Caenorhabditis elegans. We found that foxo null mutant larvae and adults show reduced survival in hypoxia. We also identified two downstream FOXO target genes involved in hypoxia tolerance. The first is the translational repressor 4e-bp. Upon exposure to hypoxia, 4e-bp expression is increased in a FOXO-dependent manner, while 4e-bp mutants showed a modest decrease in hypoxia survival. The second factor is the NF-kB transcription factor relish, a mediator of innate immune responses. We found that expression of relish and antimicrobial peptides was increased in a FOXO-dependent manner in hypoxia, and that relish null mutant animals have reduced survival in hypoxia. To better understand the breadth of FOXO responses, we examined metabolites and found that FOXO is necessary for maintenance of glycogen and lipid stores during hypoxia. foxo null mutants also have deregulated expression of lipases and glycolytic genes. Transcriptomic analyses further revealed that FOXO may modulate expression of genes involved in Notch and Hippo signaling, oocyte development, and translational effectors such as Ribosomal Proteins. Together, these data indicate that FOXO is a hypoxia-inducible factor that mediates tolerance to hypoxia by controlling protein synthesis, immune signaling, and metabolism.Item Open Access The Role of Adipose HIF1a in Drosophila Hypoxia Tolerance(2021-01-06) Polan, Danielle; Grewal, Savraj S.; Shutt, Timothy E.; Brook, William J.Animals often develop and live in environments where environmental factors are constantly fluctuating. One of these factors is oxygen, and low oxygen, or hypoxia, is quite harmful to most animals. However, some have developed mechanisms that allow them to alter their physiology and metabolism in order to survive these hypoxic conditions. Drosophila are one such animal, making them an ideal model organism for hypoxia research. In this study, I examined the role of the transcription factor HIF-1α/ Sima in adult hypoxia tolerance in Drosophila. I found that Sima expression in the fat body controls the systemic response to hypoxia. The systemic effects are exerted independently of insulin signaling. Instead, my findings suggest that a key role of fat body Sima is to restrict the induction of the IMD and JAK/STAT signaling in hypoxia, two pathways involved in the Drosophila immune response. Further, I found that over activating the JAK/STAT pathway decreased hypoxia survival. Additionally, I found that fat body Sima plays a role in metabolic alterations that are likely important for hypoxia tolerance. I found that adults accumulate lipid stores in hypoxia and these lipid stores are even further increased in Sima knockdown adults. Therefore, Sima in the fat body has a role in properly regulating lipid metabolism. Fat body Sima knockdown impacted the level of glycogen and glycogen metabolism genes in normoxia, suggesting that Sima also has important roles in controlling metabolism at basal levels Overall, these results increase our understanding of the mechanisms underlying hypoxia tolerance in Drosophila adults, emphasizing the importance of the fat body in the non-autonomous response. Sima/ HIF-1α, as well as the immune and cytokine pathways I found it to regulate are conserved in humans. Additionally, adipose tissue often becomes hypoxic, leading to HIF-1α accumulation in obesity. Several other diseases are also associated with hypoxia and HIF-1α, making my findings relevant to our understanding of human disease.Item Open Access Sema6d Signaling in the Developing Visual System(2018-06-28) Cechmanek, Paula Bernice; McFarlane, Sarah; Grewal, Savraj S.; Schuurmans, CarolThe retinal pigment epithelium (RPE) is a highly specialized monolayer of epithelial cells that arise from retinal progenitors and form a tight barrier between the neural retina (eye) and the brain. Although only a thin monolayer, RPE cells perform numerous important functions and are indispensible for mature photoreceptor renewal and survival. When RPE dysfunction occurs, in diseases like Retinitis Pigmentosa and Age-related Macular Degeneration (AMD), devastating vision loss can occur. While much work has focused on understanding RPE function in adult life, little is known about how the RPE arises during early embryonic life or how it spreads to wrap around the neural retina. In this thesis I use zebrafish (Danio rerio) as a model to a) characterize early RPE development, and b) implicate the classical axon guidance cue Semaphorin6d (Sema6d), and its receptor Plexina1 (Plxna1), in RPE and optic cup morphogenesis in the developing zebrafish embryo. I show that markers of RPE differentiation turn on in RPE progenitors prior to optic cup morphogenesis, and that RPE morphogenesis occurs in two phases: First an antero-posterior expansion of the RPE progenitor domain, followed by a second phase coupled to optic cup morphogenesis, where the RPE domain is stretched to form a thin, single-cell epithelium around the eye. Through Sema6d loss-of-function analysis I show that this second phase depends on signals between the Sema6d-expressing progenitors of the temporal neural retina and RPE progenitors expressing Plxna1. This is the first signaling pathway to be identified as a driver for optic cup morphogenesis in any model system. Developing zebrafish as a model system to study early RPE development and retinal cell migration may provide insights to understanding human RPE/retinal cell migration during development and disease.Item Open Access Thyroid Stimulating Hormone Receptor Mutations in Non-Autoimmune Hyperthyroidism(2020-08-24) Stephenson, Alexandra; Paschke, Ralf; Robbins, Stephen M.; Grewal, Savraj S.Non-autoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) germline mutation. Germline mutations in TSHR lead to sporadic and familial NAH (SNAH, FNAH) whereas somatic mutations lead to hot thyroid adenoma (HTA). The role and prevalence of TSHR mutations in NAH have been reported to vary significantly. Furthermore, the result of these mutations appears to vary across different reports. Most interestingly, there is also a proposed role for TSHR in thyroid carcinoma. This thesis seeks to determine the true prevalence of TSHR mutations in HTA (the subset of NAH where the most samples are available), explore the phenotype of germline NAH, provide an overview of all TSHR associated disorders and begin to unravel the role of TSHR in carcinoma. This is done in 4 chapters. The first uses targeted NGS technology to determine the true prevalence of TSHR mutations in NAH (specifically HTA). This found that TSHR is the sole gene responsible for the development of HTA (96% mutation positive in an optimal subset of samples). The second chapter explores the phenotype of germline NAH, the variability of presentation, the consequences of late diagnosis, and the possible role of TSHR in bone through literature review and two novel case reports. The third chapter is an all-encompassing look at disorders associated with the TSHR including thyroid carcinoma, as documented by the TSHR mutation database. Thyroid carcinoma is further explored in the fourth chapter which outlines preliminary results and background for a plan to further evaluate TSHR’s role in thyroid carcinogenesis. This thesis concludes that TSHR signaling is solely responsible for HTA, that NAH can have variable presentations and requires early total thyroidectomy, and that TSHR undeniably plays a role in thyroid carcinoma that warrants further exploration.Item Open Access Vascular Smooth Muscle Cell Development in Zebrafish(2018-09-20) Whitesell, Thomas Richard; Childs, Sarah Jane; Grewal, Savraj S.; Biernaskie, Jeff A.During the formation of blood vessels, the endothelium becomes progressively covered by vascular smooth muscle cells and pericytes, known collectively as vascular mural cells. The vascular mural cells of the head arise from migratory neural crest and lateral mesodermal populations, and differentiate into mural cells which surround the endothelium of blood vessels, providing support and contraction. Using zebrafish as a model, we created transgenic reporter lines for acta2/αsma (α-smooth muscle actin) that label mature vascular and visceral smooth muscle. Using these reporters, we study the cellular dynamics of mature vascular smooth cells along the ventral aorta and in the brain. These reporter lines only begin robust expression of vascular mural cells at 4 days post fertilization (dpf); however, there are peri-vascular cells present around the endothelium by 2 dpf. Therefore, there is a need to identify an earlier marker of the vascular mural cell lineage. One such marker is the forkhead box transcription factor foxc1b. Utilizing a foxc1b:EGFP reporter line, foxc1b labels groups of mesenchymal cells which undergo a morphological change to associate with the endothelium, and then later co-express the mature smooth muscle marker acta2. These vascular smooth muscle cells on the ventral aorta are the earliest identified smooth muscle cells in the zebrafish embryo. foxc1b:EGFP continues to label smooth muscle cells through the lifespan of zebrafish, but is not co-expressed with pdgfrβ (platelet derived growth factor β) and therefore likely does not label pericytes. Thus, foxc1b is an early smooth muscle marker, but not a pericyte marker. To assess if there are transcriptomic differences between mural cell populations, fluorescence-activated cell sorting and RNA Sequencing were used to identify differentially expressed genes. Further examination of genes from the RNA Sequencing datasets reveals novel vascular mural cell markers. One highly differentially expressed gene was Ras-like family protein member 12, rasl12. rasl12 is expressed in both smooth muscle cells and pericytes. rasl12 knockout mutants were created, but there were large phenotypic differences between two mutant alleles, requiring further study. Overall, this thesis addresses the development of vascular smooth muscle cells, by labelling and visualizing cellular behaviors in zebrafish.