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

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HIV-Associated Modulation of Serological and Immunogenetic Features of Humoral Immunity

Date created: 
2013-10-24
Abstract: 

An effective vaccine against HIV/AIDS will likely need to induce broadly (b) neutralizing (Nt) antibodies (Abs). Such Abs are produced rarely during natural infection, take years to develop, and have unusual genetic and reactivity features. The research presented here is part of continuing investigations into the origins of Abs bearing these features within HIV-infected hosts. First, the reactivity profiles of monoclonal bNt Abs were analyzed and compared with those of pathogenic autoAbs. Unlike pathogenic autoAbs, HIV bNt Abs were minimally self-reactive and modestly polyreactive only at high concentrations, supporting a fundamental distinction between their binding behaviour. Moreover, the polyreactivity of HIV bNt Abs, but not of pathogenic autoAbs, was abrogated in assays using complex blocking proteins, which more closely reflect in vivo conditions. Second, HIV serum cohorts were assayed for reactivity to envelope glycoproteins and self-antigens, and for idiotypic composition. No HIV sera were significantly self-reactive, but most displayed a novel form of ‘conditional’ non-specific polyreactivity, and all contained high titers of IGHV1-69-encoded IgGs. Multiple lines of evidence supported a division between the serum Abs of cross-Nt and slow progressor sera, which were focused against particular sites on Env and displayed the lowest degree of self-reactivity and polyreactivity, and those from recent seroconverters and rapid progressors, which were cross-reactive against diverse Env epitopes, mildly self-reactive and highly polyreactive. Finally, novel methods were developed to interrogate expressed VH gene repertoires of rare B-cell subsets in HIV+ and healthy individuals. Immunogenetic analyses revealed that Ab repertoires of HIV+ individuals accrue somatic mutations indiscriminately, primarily in resting memory B-cells but also in other compartments. Other genetic features of bNt Abs, including long CDR-H3s and biased VH gene usage, were not observed in the overall repertoire, and so presumably emerge via antigen selection by HIV Env. Taken together, our data indicate that HIV infection induces fundamental changes in the reactivity and immunogenetic features of Abs. In addition to providing a plausible genetic and immunological mechanism for the development of highly mutated, polyreactive Abs, HIV-associated immunomodulation could significantly enhance secondary Ab repertoire diversification, potentially contributing to the development of bNt specificities.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Jamie Scott
Felix Breden
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Application of RNA-Seq in Ecotoxicogenomics: Exploring the Effects of Ibuprofen Exposure on Rainbow Trout and C. elegans

Date created: 
2013-12-16
Abstract: 

RNA-Seq was applied in this ecotoxicogenomics study to investigate the effects of ibuprofen in two species, rainbow trout (Oncorhynchus mykiss), a fish routinely used in ecotoxicology tests, and Caenorhabditis elegans, a well-studied nematode with immense genomics information. Exposure to environmentally relevant levels of ibuprofen resulted in gene expression changes relating to stress, prostaglandin synthesis, reproduction and development in both species. In fish, we observed sex-dependent differences in vitellogenin and prostaglandin synthase gene expression, highlighting the importance of genetic sex determination of juvenile fish used in bioassays. In worms, we saw a decrease in progeny production count. Our results suggest that ibuprofen may have negative impacts on reproduction in both species but requires further investigation. This study demonstrated the benefits and challenges of using RNA-Seq in ecotoxicology and the value of studying diverse species, including traditional models and more microscopic organisms, to better understand toxicant impact on an entire ecosystem.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Fiona Brinkman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

The Wntome: A genome-wide analysis of kinases and phosphatases that regulate Wnt signaling

Author: 
Date created: 
2013-09-20
Abstract: 

Evolutionarily conserved cell signaling pathways regulate diverse and indispensable processes during metazoan development. A precise control of both the state and threshold of signaling is required for normal development to occur. Cells predominantly utilize the kinase- and phosphatase-mediated reversible phosphorylation of proteins to control signaling. The canonical Wnt signaling pathway has homologous roles during the processes of axis polarization and stem cell maintenance in diverse metazoans. Wnt signaling is regulated through reversible phosphorylation both in its silent and active state. We have identified and characterized the function of Drosophila Hipk, a novel kinase component of the pathway. Genetic and biochemical analyses suggest that Hipk stabilizes the pathway effector Armadillo in a process that is dependent on its catalytic activity. Additionally, Hipk acts to promote pathway activity independent of its effect on Armadillo stability. We find that Hipk2 has a functionally conserved role to regulate Wnt signaling in mammalian cells. Hipk and a limited number of other kinases (and phosphatases) have thus far been implicated in the regulation of Wnt signaling. We have performed an in vivo loss-of-function RNAi screen to identify additional enzymes that regulate this pathway through reversible phosphorylation in Drosophila. Our analyses have identified novel pathway components at all levels of the Wnt signaling relay.

Document type: 
Thesis
File(s): 
Senior supervisor: 
ESTHER VERHEYEN
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Characterization of colonization factors from enteric pathogens Vibrio cholerae and enterotoxigenic Escherichia coli

Date created: 
2012-06-04
Abstract: 

Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) cause severe gastrointestinal diseases and are a significant cause of mortality in developing countries. These bacteria colonize the gut mucosa and secrete exotoxins resulting in severe diarrhea. Colonization of the small intestine by V. cholerae requires the toxin coregulated pilus (TCP), a type IV pilus that self-associates to hold the bacteria in microcolonies. The TCP assembly apparatus is responsible for secreting a soluble colonization factor, TcpF, which is encoded on the tcp operon along with all genes necessary for TCP assembly. Its function is unknown, but is critical for V. cholerae colonization in the infant mouse model and antibodies against this protein are protective. The ETEC colonization process is less well-characterized, but some ETEC express CFA/III, a type IV pilus that is homologous to TCP, encoded by the cof operon. This operon has a gene cofJ, at a position syntenic to tcpF in the tcp operon, which encodes a putative soluble protein, CofJ, that has similar size to TcpF. We showed that CofJ, like TcpF, is secreted by its type IV pilus system. Although CofJ and TcpF share no amino acid sequence homology with each other or with any other known protein, we hypothesized that they may nonetheless have similar structures and roles in pathogenesis. We solved the TcpF crystal structure to 2.4 Å resolution, revealing a novel bilobed protein with two domains joined by a flexible linker. The N-terminal domain resembles a C-type lectin-like domain and the C-terminal domain has a fibronectin type III fold. We solved the CofJ structure at 2.55 Å resolution. CofJ is very different from TcpF, composed primarily of β-strands forming a large β-sandwich. Structural homology searches revealed CofJ has very limited similarity to the C-terminal domain of perfringolysin O, a pore-forming protein secreted by Clostridium perfringens. As both CofJ and TcpF have patches of surface-exposed hydrophobic residues, we hypothesized that they may interact with epithelial cell membranes. Both proteins associated with synthetic lipid vesicles and CofJ associates with cultured epithelial cells and oligomerizes in their presence. Thus, our preliminary data suggest that CofJ and TcpF bind to epithelial cell membranes.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Lisa Craig
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

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): 
Senior supervisor: 
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): 
Senior supervisor: 
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): 
Senior supervisor: 
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): 
Senior supervisor: 
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): 
Senior supervisor: 
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): 
Senior supervisor: 
Michael Silverman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.