Molecular Biology and Biochemistry - Theses, Dissertations, and other Required Graduate Degree Essays

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The NIMA-related kinases as regulators of ciliary assembly, disassembly, and length

Date created: 
2013-09-18
Abstract: 

Cilia are microtubule-based, membrane-enclosed organelles that project from the surface of most eukaryotic cells. Cilia perform important signalling functions in development and homeostasis, and disruption of these functions in humans and other metazoans is associated with diseases known as ciliopathies. Previously, it was hypothesized that the NIMA-related kinases (Neks) evolved to coordinate ciliogenesis and ciliary resorption with the cell cycle. In this work, I examine the events surrounding pre-mitotic resorption in Chlamydomonas, and the roles of two Neks in ciliary regulation, thereby advancing our understanding of the mechanisms that regulate ciliogenesis, ciliary resorption, and ciliary length. Pre-mitotic ciliary resorption occurs to free the basal body to act as a spindle pole during mitosis, although little is known of the mechanisms or signals that regulate this event. Here, I show that pre-mitotic resorption culminates in a severing event that separates the basal body from the transition zone in Chlamydomonas. This severing may be essential for cell cycle progression in Chlamydomonas and other organisms. Mutations in mammalian Nek1 are associated with defective ciliogenesis and severe ciliopathies, and signalling to the nucleus may be important for the etiology of these ciliopathies. Here, I show that Nek1 cycles through the nucleus. Nek1 is therefore a candidate to transduce signals between the cilium and nucleus. Previous work demonstrated that RNAi of the Chlamydomonas Nek CNK2 caused a slight increase in flagellar length. I characterized a new cnk2-1 null mutant and discovered that it contributes to the regulation of flagellar resorption and length control by regulating the rate of flagellar disassembly. My work with cnk2-1 and a mutant strain defective in a second ciliary kinase, lf4-7, revealed that flagellar length is controlled by a feedback system, wherein rates of both flagellar assembly and disassembly are modulated when flagella are too long.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Lynne Quarmby
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

O-linked N-acetylglucosamine protein modification in mouse models of neurodegenerative diseases

Author: 
Date created: 
2011-08-15
Abstract: 

The O-linked addition of β-N-acetylglucosamine to proteins (O-GlcNAc) is a form of intracellular glycosylation that has gained increasing attention for its potential involvement in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD). ALS has several causes including abnormal phosphorylation of neuronal proteins as well as several gene mutations including those in the sod1 gene, which encodes superoxide dismutase 1, as well as the TARDBP gene, which encosed TDP-43. Abnormally elevated phosphorylation of proteins including TDP-43, neurofilaments, and tau are all implicated in neurodegeneration. Tau, for example, forms intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. O-GlcNAc transferase (OGT) catalyzes the installation of GlcNAc onto specific serine and threonine residues of target proteins, while O-GlcNAcase (OGA) removes the modification. It is known that O-GlcNAc modification of tau and other proteins is reciprocal to phosphorylation. The objective of this thesis was to improve our understanding of the role that O-GlcNAc has on proteins implicated in neurodegeneration in animal models of ALS and AD. The main findings are (1) O-GlcNAc levels were reduced in spinal cord tissue from the mSOD mouse model of ALS specifically in motor neurons; (2) mislocalization of TDP-43 occurs in aged mSOD mice; (3) mouse brain TDP-43 was found to be O-GlcNAc modified and four O-GlcNAc modification sites were mapped on recombinant full-length human TDP-43; (4) OGA inhibitor Thiamet-G treatment to JNPL3 mouse model of AD increased tau O-GlcNAc modification, hindered tau aggregation, and protected mice against neuronal cell loss. These results suggest that the neurodegeneration found in mSOD mice might be associated with a reduction of O-GlcNAc levels in motor neurons, and O-GlcNAc modification might influence the abnormal phosphorylation of TDP-43 in ALS. These studies also provide new insight into the potential association of SOD1 and TDP-43, and offer support for OGA as a viable therapeutic targets that might provide an opportunity to alter disease progression in AD and offer benefits in other diseases characterized by the aggregation of proteins that can be O-GlcNAc modified.

Document type: 
Thesis
File(s): 
Supervisor(s): 
David Vocadlo
Charles Krieger
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Sex Determination in Tasmanian Atlantic Salmon

Date created: 
2013-07-26
Abstract: 

Although male heterogamety controls Atlantic salmon sex, hormone treatment can induce sex reversal. In Australia where Atlantic salmon males are unmarketable, sex reversed females (neo-males) are crossed with females to produce all female stock. However, neo-males are indistinguishable from males making early male culling difficult. Therefore, a sex-specific genetic marker was needed to make this distinction. With no such marker available offspring sex was predicted via familial microsatellite analysis. Markers from Chromosome 2 (Ssa02), where the sex locus (SEX) previously mapped, predicted test family offspring sex inaccurately. A 64 SNP genome-wide scan suggested Chromosome 6 (Ssa06) housed SEX instead. Analysis of 38 male lineages revealed three sex loci on Ssa02, Ssa06 and Ssa03 with 34, 22 and 2 representative families respectively. An exon PCR test for the rainbow trout master sex-determining gene (sdY) was consistent with a single sex-determining gene that jumps around the genome in Atlantic salmon.

Document type: 
Thesis
File(s): 
Supervisor(s): 
William Davidson
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Discovery of a Suppressor of ADF1 and the Mapping of a new ADF gene, ADF2, in Chlamydomonas reinhardtii

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2013-07-24
Abstract: 

Eukaryotic cilia are evolutionary conserved microtubule-based organelles that play important roles in cell signalling, development and motility. Cilia can be shed through a process known as deflagellation, in which the cilium is severed at a specific site at its base in response to a stress signal, through calcium signalling. Fifteen years ago three genes were uncovered in a screen for deflagellation mutants, two of which were cloned and their roles in the microtubule-severing event characterized. The third gene, named ADF1 has to date eluded identification. A previous graduate student genetically mapped ADF1 to Linkage Group (Chromosome) IX to identify the ADF1 gene. I tried, unsuccessfully, to rescue the adf1 mutation with wild type DNA from the gene locus. In the rescue attempts I recovered a potential suppressor of the adf1-3 mutant. Despite the many attempts I was unable to rescue adf1 or identify the flanking DNA of the adf1-3 suppressor. Since no MT severing protein or calcium sensor were recovered in the original genetic screen a second genetic screen is being carried out, this time for conditional deflagellation mutants. From this screen I characterized a new acid induced deflagellation gene, ADF2, and mapped it to Linkage Group III. The work in this thesis thus contributes new information about two previously unknown deflagellation genes, ADF2 and ADF1-Suppressor.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Lynne Quarmby
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Identification of hcf-1 in a genetic screen for dsh-2 suppressors in c. Elegans

Date created: 
2013-06-18
Abstract: 

Asymmetric cell division is an essential process to generate cell diversity during development. In C. elegans, many asymmetric cell divisions are regulated by Wnt signalling. We have identified a Wnt/CWN-1, a Frizzled/MOM-5 and a Dishevelled/DSH-2 that function to control asymmetric neuroblast division. Loss of both maternal and zygotic dsh-2 function results in asymmetric neuroblast division defects and embryonic/early larval lethality, while loss of zygotic dsh-2 function disrupts asymmetric cell division of the somatic gonadal precursor cells (SGPs), Z1 and Z4. Through a DSH-2 domain analysis, we found that the DEP domain of DSH-2 is required for viability and asymmetric neuroblast division, while neither the DIX or DEP domains were essential for SGP cell division. To identify genes that function with dsh-2 in asymmetric division, we undertook a genetic screen to isolate suppressors of dsh-2 lethality and isolated over 60 dominant suppressors. We focused our characterization on Sup305 and Sup245 which we demonstrated were also strong suppressors of both asymmetric neuroblast and SGP division defects. In SGP division, reciprocal asymmetric localization of SYS-1/β-catenin and POP-1/TCF in Z1/Z4 daughter cells regulate correct cell fate. This asymmetric localization is lost in dsh-2 mutants. Both suppressors partially restored the asymmetric localization of SYS-1/β-catenin and POP-1/TCF in dsh-2 mutants. Genetic mapping experiments placed Sup305 on the middle of chromosome IV and Sup245 on the right arm of chromosome I. Both suppressor strains were sent for whole genome sequencing and the resulting sequence analyzed to identify potential candidates. In combination with additional mapping experiments, we determined that Sup305 was a G to A in mutation in hcf-1 resulting in a Proline to Serine amino acid substitution. Loss of hcf-1 rescued dsh-2 phenotypes suggesting that Sup305 is a dominant negative mutation in hcf-1. hcf-1 is a transcriptional cofactor that bridges transcription factors to the chromatin modifying machinery. In C. elegans, hcf-1 has been previously shown to modulate cell cycle, stress and lifespan. Our results indicate that it is also a novel Wnt pathway interactor. Further work will determine the mechanism of hcf-1 suppression of dsh-2.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Nancy Hawkins
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Modulation of Neuronal Insulin Signaling Rescues Axonal Transport Defects in an Alzheimer’s Disease Model

Date created: 
2013-07-19
Abstract: 

Defective brain insulin signaling contributes to the cognitive deficits in Alzheimer's disease (AD). Oligomeric amyloid-β peptides (AβOs), the neurotoxin implicated in AD, induce a variety of cellular insults, including dysregulation of intracellular signaling cascades and disruption of fast axonal transport. I show that modulation of insulin signaling prevents AβO-induced defects of brain-derived neurotrophic factor (BDNF) transport in wild type (tau+/+) and tau knockout (tau-/-) primary hippocampal mouse neurons. Tideglusib, an inhibitor of glycogen synthase kinase-3β (GSK3β), an insulin signaling intermediate implicated in AD, rescues BDNF transport in tau+/+ and tau-/- neurons. Furthermore, Exendin-4, an anti-diabetes agent, activates the insulin signaling pathway through glucagon like peptide-1 receptor stimulation to also rescue BDNF transport defects similarly to Tideglusib. These results indicate a protective link between insulin signaling and tau-independent transport regulation. By establishing links between insulin signaling and AβO action, my results allow for establishing novel directions for AD therapeutics.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michael Silverman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Evolution and characterization of the fatty acid-binding proteins (fabps) in Atlantic salmon (Salmo salar)

Author: 
Date created: 
2012-04-20
Abstract: 

It is suggested that gene or genome duplication is the driving force in evolution that leads to speciation. Two models, the classical model and the duplication- degeneration-complementation (DDC) model, have been proposed on the fates of gene duplicates resulting from either a gene or a genome duplication event. The classical model suggests that one of the gene duplicates might result in loss of function (non- functionalization) or gain of a new function (neo-functionalization) depending on whether the accumulated mutations over the years are deleterious or beneficial to the organism. In the DDC model, it is proposed that each of the gene duplicates might accumulate different deleterious mutations in the regulatory region of the gene, such that these genes partition the ancestral gene function (sub-functionalization). Combinations of the phylogenetic analysis of many gene families support that salmonids have undergone two additional whole genome duplications compared to the mammals, one occurred in the common ancestors of teleosts and another happened in the common ancestor of salmonids approximately 25-120 million years ago. In this thesis, the evolution of the fatty acid-binding protein (fabp) family in fish and salmonids was examined. I have characterized eighteen unique fabp genes in Atlantic salmon. These include the seven fabp sub-families described previously in fish. Phylogenetic analyses and conservation of synteny support the two whole genome duplication events in the common ancestors of teleosts and salmonids and indicate when gene losses occurred. Genetic mapping of fabp gene duplicates to homeologous chromosomes in Atlantic salmon also support that they arose by the 4R genome duplication. I also searched for the signatures of neo-functionalization and sub-functionalization by calculating dN/dS ratios, examining the nature of amino acid substitutions and expression patterns, and suggested the fates of fabp gene duplicates in Atlantic salmon. Overall, the findings of this project provide insight into the evolutionary processes at play in salmonid genomes.

Document type: 
Thesis
File(s): 
Supervisor(s): 
William Davidson
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

Integration of transport pathways in Yeast

Author: 
Date created: 
2012-03-22
Abstract: 

Cell polarity is maintained via a balance of exocytosis and endocytosis; the protein machinery that mediates these transport processes must be co-ordinated with membrane lipid signals. This lipid signalling is, in part, dependent on the establishment of membrane domains through lipid transport. Cholesterol is transported via a poorly defined route that is independent of vesicle-mediated secretory protein transport. This “non-vesicular” sterol transport is postulated to involve the conserved family of Oxysterol binding protein (OSBP) Related Proteins (ORPs), which are proposed to be sterol lipid transport proteins (LTPs). To test if ORPs primarily act as sterol LTPs or alternatively as sterol-responsive signalling proteins, the function of Saccharomyces cerevisiae OSBP Homologues (OSH1-OSH7) were analyzed. Depletion of all Osh proteins in yeast cells inhibited growth, and defects in endocytosis, polarized exocytosis, and sterol homeostasis, were observed. Consistent with a direct role in exocytosis, Oshdepletion disrupted the polarized localization of vesicle transport regulators (Rho- and Rab-GTPases, and exocyst complex subunits) and the Osh protein Osh4p was observed to travel on exocytic vesicles to sites of polarized growth. Osh4p also formed complexes in vivo with specific Rho- and Rab-GTPases, and exocyst complex subunits. Contrary to the postulated role of ORPs as LTPs, a designed mutation in Osh4p that disrupts its ability to bind and thereby transport sterols, did not inactivate the protein but caused a gain-of-function phenotype affecting exocytosis. Our experiments suggested that ORPs are not sterol LTPs and implied that sterols act as signalling ligands that repress Osh4p, and potentially other ORPs. To understand how Osh proteins might simultaneously affect both exocytosis and endocytosis, I tested whether the regulation of the exocytic and endocytic machinery are directly coupled. I found that the Rab GTPase Sec4p, which is an integral component of exocytosis, directly interacted with specific endocytic proteins at actin patches. SEC4 was required for proper endocytic site assembly and actin patch polarization, indicating that Sec4p links exocytosis and endocytosis to maintain cell polarization. Because these novel mechanisms involving sterol signalling and cell polarization are likely to be conserved, I propose these studies have broader medical implications applicable to cancer cell growth and metastasis.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Christopher Beh
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

Regulation of Dlg-containing adhesion complexes during epithelial and synaptic plasticity in Drosophila

Author: 
Date created: 
2013-02-28
Abstract: 

Discs large (Dlg) is a multi-PDZ domain-containing protein belonging to the MAGUK superfamily of scaffolding proteins. In epithelia, Dlg serves as an apicobasal polarity determinant, where its disruption leads to tumor formation. Dlg also plays roles in maintaining synaptic structure and function, and has been implicated in neurodegeneration. Thus, understanding Dlg regulation can provide insights into human diseases including cancers and neurological disorders. In this thesis, we characterize novel Dlg regulators during epithelial and synaptic plasticity events in Drosophila development through two separate but overlapping projects. Project 1: Dorsal closure, which is a wound healing model, occurs when a hole in the embryonic epidermis is closed due to surrounding epidermal flanks that migrate towards each other over the amnioserosa occupying the hole. During migration, the leading edges of the epidermal cells abutting the hole exhibit a breakdown in apicobasal polarity as adhesions are severed with the amnioserosa. At the end of migration, however, apicobasal polarity is re-established as adhesions must form between the opposing epidermal flanks in order to seal the hole shut. Mammalian studies indicate that the Dlg-containing Scribble complex recruits p21-activated kinases (Paks), effectors for Rho GTPase signalling, to the leading edge during cell migration. We show that this interaction can act in the opposite direction as Paks can recruit the Scribble complex back to the leading edge upon dorsal closure completion. We propose that the bidirectional relationship between Paks and Dlg may allow epithelia to toggle between migratory and adhered states. Project 2: Previous studies have shown that adducin, a membrane cytoskeletal protein that regulates actin, is hyperphosphorylated in spinal cord tissue taken from patients who died with the motor neuron disease, Amyotrophic Lateral Sclerosis. To further explore the roles of adducin in the nervous system, we decided to study the Drosophila orthologue encoded by hu-li tai shao (hts). We show that Hts regulates larval neuromuscular junction morphogenesis by controlling Dlg postsynaptic targeting via indirect phosphorylation. This process is partially supressed when Hts phosphorylation in the MARCKS domain is blocked. We propose that Hts is a signalling-responsive cytoskeletal protein that contributes to synaptic growth through Dlg-mediated adhesion.

Document type: 
Thesis
File(s): 
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
Supervisor(s): 
Nicholas Harden
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Comparative Genome Analysis of Malaria Parasite Species

Author: 
Date created: 
2013-04-11
Abstract: 

With over 200 million infections and up to one million deaths every year, malaria remains one of the most devastating infectious diseases affecting humans. Over the last few years, complete genome sequences of both human and non-human malaria parasite species have become available, adding comparative genomics to the toolbox of molecular biologists to study the genetic basis of human virulence. In this thesis, I computationally compared the published genomes of seven malaria parasite species with the aim to gain new insights into genes underlying human virulence. This comparison was performed using two complementary approaches. In the first approach, I used whole-genome synteny analysis to find genes present in human but not non-human malaria parasites. In the second approach, I first clustered virulence-associated genes into gene families and then examined these gene families for species-specific differences. Both comparisons resulted in interesting gene lists. Synteny analysis identified three key enzymes of the thiamine (vitamin B1) biosynthesis pathway to be present in human but not rodent malaria parasites, indicating that these two groups of parasites differ in their ability to synthesize vitamin B1 de novo. My gene family classification exposed within the largest and highly divergent surface antigen gene family pir a group of unusually well conserved orthologs, which should be considered as high-priority targets for experimental characterization and vaccine development. In conclusion, this thesis highlights genes and pathways that are different between human and non-human malaria parasites and therefore could play important roles in human virulence. Experimental studies can now be initiated to confirm virulence-associated functions and to explore their potential value for drug and vaccine development.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Nansheng Chen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.