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

Receive updates for this collection

The interferon-inducible protein 16 is a single-stranded nucleic acid binding protein with unwinding and endonuclease activities

Author: 
Date created: 
2010
Abstract: 

IFI16 belongs to a protein family (HIN200) that contains a characteristic 200-amino-acid protein domain with unknown function. Members of this family were initially described as transcriptional regulators but IFI16 is also involved in other processes such as DNA repair and programmed cell death. To gain further insights into the biochemical mechanisms underlying these functions we made bioinformatics predictions followed by biochemical validation. We predicted and verified experimentally that the 200-amino-acid domain of IFI16 is structurally and functionally similar to replication protein A (RPA), a protein that removes secondary structure on single-stranded DNA during DNA replication and repair. In addition, I found that IFI16 can unwind double-stranded DNA and has an endonuclease activity. This later unexpected discovery reveals a new function for IFI16. Finally, I further discuss the implications of this discovery in regard to the current knowledge about the function of this protein as well as new directions of studies.

Document type: 
Thesis
File(s): 
Supervisor(s): 
D
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Molecular modeling predicts helix tilting in HCN channels during cAMP binding

Date created: 
2010
Abstract: 

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are believed to regulate the heartbeat. Binding of cAMP to a cytoplasmic binding domain (BD) increases probability of HCN channel opening; a C-linker region (helices A’-F’ and A) links the BD to the channel pore. The C-linker was proposed to move when cAMP binds the BD; I hypothesized a movement of D’- F’. I created two models of a cytoplasmic fragment from an "apo" HCN channel (without cAMP), one by comparative modeling and one by molecular dynamics. Compared to the crystal structure of the fragment with cAMP, both apo models showed D’ tilting, such that N-terminus gets farther and C-terminus closer to helix A. E’ translates parallel to the long axis of A from C- to N-terminus. Helix A did not change relative to the BD. Therefore when cAMP binds the HCN channel, the N-terminus of D’ is likely tilting towards the BD.

Document type: 
Thesis
File(s): 
Supervisor(s): 
E
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Photo-induced charge flow through canonical DNA/RNA and non-canonical DNA containing trinucleotide repeats.

Author: 
Date created: 
2010
Abstract: 

The charge flow technique is used to study structure of nucleic acids. DNA-DNA duplexes and DNA-RNA heteroduplexes have been studied using charge transfer, yet little is known about RNA-RNA duplex conductivity. Both piperidine and aniline treatment on DNA-DNA duplexes with guanine-rich termini generated a large oxidative end-effect. Both DNA-DNA and RNA-RNA duplexes demonstrated similar findings. Sensitivity of the end-effect towards mismatches suggests its utility as sensitive reporter of mismatches. Various diseases are associated with polymorphic DNA trinucleotide repeats (TNR). Disease associated TNRs (CAG, CGG, CCG, CTG) can form alternative DNA structures in vitro with two Watson-Crick base pairs sandwiching single mismatches. Mismatches interrupt base stacking and hence are known to affect duplex conductivity. Charge flow through duplexes carrying TNRs did not show any statistically significant difference in damage. The increase in damage from 10 oC to 21 oC, was not statistically significant. Our approach could not be applied to TNRs.

Document type: 
Thesis
File(s): 
Supervisor(s): 
D
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Temperature dependence of the cardiac sodium calcium exchanger

Date created: 
2005
Abstract: 

Calcium (ca2') is a ubiquitous and highly versatile intracellular messenger as exemplified by the numerous mechanisms nature has evolved to facilitate ca2' transport. One of these mechanisms is ~ a ' / ~ a ~ ' exchange. The ~ a ' / ~ a ~ ' exchanger (NCX) is an integral membrane protein that catalyzes the counter-transport of Na' for ca2' ions. NCX is expressed in a wide variety of tissues throughout a diverse group of organisms. NCX expression is especially high in the heart where NCX mainly functions to extrude ca2' from the cytosol to allow relaxation. Cardiac function in ectotherms, such as the trout, is distinguished by its ability to maintain adequate contractility in temperatures that are cardioplegic to mammals. Using cloned trout NCX-TR1.O and mammalian NCX1.l expressed in Xenopus oocytes, we measured NCX mediated currents and showed that the NCX-TRl .O isoform is relatively insensitive to temperature compared with NCX1.l. Furthermore, our results indicate that this phenomenon is intrinsic to the NCX protein itself and is not due to differences in regulatory properties between isoforms. Using chimeras, we determined that the region of the NCX protein responsible for this differential temperature dependence is located within the N-terminal transmembrane domain of the protein. However, further chimeric studies within this region have produced equivocal results. To aid in generation of hypotheses as to which amino acids are likely to confer NCX temperature dependence, we employed bioinformatics and comparative analyses. Cloning of tilapia NCX-TL1.O provided a useful comparative model to NCX-TR1 .O, since tilapia share a close phylogenetic relationship with trout while adapted to live in warm waters. Despite high overall sequence identity with NCX-TR1.0, we found NCX-TL 1.0 to have mammalian like temperature dependence. Coupled with measurements using squid and fruit fly NCX isoforms and subsequent sequence analyses, these comparative studies provided insight into the relationship between NCX genotype and temperature phenotype. Finally, data mining of genomic data yielded 13 new NCX sequences, including a fourth NCX gene member that is present only in fish and amphibian genomes. Phylogenetic and evolutionary analyses indicate three serial gene duplication events occurred early in the evolution of vertebrates, giving rise to the four members of the NCX gene family.

Document type: 
Thesis
File(s): 
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)

Developmental changes in the immunolocalization of the sodium calcium exchanger and the ryanodine receptor of ventricular cardiac myocytes

Author: 
Date created: 
2005
Abstract: 

Excitation of adult mammalian heart plasma membrane (sarcolemma, SL) triggers the opening of L-type voltage-gated ca2+ channels (DHPR), allowing ca2+ to cross the SL and initiate release of ca2+ stores from the sarcoplasmic reticulum (SR) via activation of the ryanodine receptor (RyR), a process termed calcium-induced-calcium release (CICR). The functional proximity of DHPR and RyR, which is essential to CICR, is not exhibited in neonate hearts. It has been proposed that NCX may contribute to ca2+ influx via reverse mode activation during excitation-contraction (EC) coupling in neonate myocytes. To investigate if RyR activation from ca2+entry via reverse mode NCX is possible, we examined the colocalization of these two proteins during development. Changes in colocalization were examined using NCX and RyR immunolabelling and confocal microscopy. Deconvolved images of cardiomyocytes collected from rabbits 3- 56-days old suggests the percentage of NCX colocalized with RyR increases in myocytes of 20- and 56-day old rabbits.

Document type: 
Thesis
File(s): 
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

A role for the sterol-binding protein Osh4/Kes1 in polarized growth

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

In the budding yeast Saccharomyces cerevisiae, seven OSBP-homologues (Osh) proteins are implicated in the transfer of sterol lipids between membranes within cells. To test whether Osh4p acts as a sterol transfer protein, a sterol-binding deficient mutant was analyzed. The mutant Osh4 protein was hyperactivated despite its inability to bind sterols, contrary to what is predicted for a sterol transporter. OSH genes also have an established role in vesicle docking during polarized exocytosis. To determine how Osh4p might affect vesicle docking, genetic interactions between OSH4, actomyosin-related genes, and regulatory genes involved in vesicle docking were analyzed. Genome-wide screens were performed to identify novel OSH genetic interactions and to better understand the functional interrelationship between the seven OSH genes. These results suggested that a primary role of the OSH genes is to regulate vesicle docking during polarized exocytosis, and not necessarily for the direct transfer of sterols between membranes.

Document type: 
Thesis
File(s): 
Supervisor(s): 
C
Department: 
Dept. of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Characterization of let-765/nsh-1 and its role in RAS signalling in Caenorhabditis elegans

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

The broad diversity of cell types generated during development of a multicellular organism result from cell-fate decisions. Many fate decisions are directed by the actions of signalling pathways such as the RAS/map kinase (MAPK) pathway via the transcriptional regulation of target genes. The differential expression of target genes is, in turn, responsible for the determination of cell fate. In C. elegans, a RAS/MAPK pathway acts downstream of the epidermal growth factor (EGF) receptor to regulate cell fates in several tissues. In particular, development of the vulva has provided an opportunity to dissect the function of RAS pathway components as well as positive and negative regulators of the pathway. I have identified the essential gene, let-765, as notch signalling pathway homolog-1 (nsh-1), a DExD/H box protein. To investigate the role of let-765 with respect to C. elegans development, I performed a genetic and molecular analysis of the gene. let-765 was found to be broadly expressed throughout development and an assessment of reduction-of-function phenotypes suggests that it is likely required for RAS pathway-directed processes, including larval viability, vulval induction, and development of the male tail and posterior ectoderm. By investigating genetic interactions between the EGFR/RAS/MAPK pathway and let-765, I have demonstrated that it promotes RAS pathway activity and is required for vulval induction. These studies indicate that let-765 acts upstream of the ligand and antagonizes the repressive activity of the synthetic multivulva (synMuv) genes. Specifically, LET-765 appears to positively regulate the transcription of lin-3 EGF, which is required to specify vulval cell fates. The variety of phenotypes generated by a loss or reduction of let-765 function is consistent with a role for LET-765 regulating multiple processes through interactions with factors in addition to the EGFR/RAS pathway. A novel role for DExD/H box proteins in transcriptional regulation has been proposed recently and, together with the data demonstrating a role for LET-765 in the regulation of lin-3 transcription, I propose that LET-765 may interact with transcriptional regulatory complexes to promote gene expression. In summary, this study has provided novel insight into the control of LIN-3/EGF expression and the EGFR/RAS pathway during C. elegans development.

Document type: 
Thesis
File(s): 
Supervisor(s): 
D
Department: 
Dept. of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)

Deciphering the role of a microtubule severing protein and a protein kinase in cell cycle and ciliogenesis in Chlamydomonas Reinhardtii

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

Many types of eukaryotic cells are ciliated – they project a microtubule-based structure extending from the cell surface called a cilium, which plays roles in cellular motility and sensing the environment. Cilia are anchored at the cell surface by microtubule-based structures called basal bodies or centrioles. In addition to nucleating cilia, centrioles act as spindle organizing centres during cell division. We are beginning to understand that several proteins are involved in simultaneously regulating cilia and centrioles with cell division. My research focused on the function of two proteins in the ciliated alga, Chlamydomonas. The first protein, katanin, severs microtubules and we predicted that it is required during the disassembly of cilia prior to mitosis. I used a genetic approach to repress expression of the gene encoding katanin in Chlamydomonas , and thereby demonstrated that katanin in fact severs microtubules at a distinct location between basal bodies and the cilium. The other gene that I studied encodes a cyclic GMP-dependent kinase type 2 (PKG2). Mutant cells deficient in PKG2 had either no cilia, or cilia of unequal length. I discovered that PKG2 is expressed in the cytoplasm as well as the flagella and may interact with other proteins to regulate ciliary length and structure. Taken together, this research identified novel mechanisms that help explain the coordinated regulation of cilia during the cell cycle, and a novel gene with roles in ciliary assembly was also identified.

Document type: 
Thesis
File(s): 
Supervisor(s): 
L
Department: 
Dept. of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)

Mapping the deflagellation-defective ADF1 gene in Chlamydomonas reinhardtii.

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

Cilia function in motility, chemo- and mechanosensation and have been linked to the cell cycle and human disease. Stress triggers the calcium-dependent severing of axonemal microtubules at a site near the base of the cilium, resulting in deciliation (also known as deflagellation). Originally isolated as a spontaneous mutation in Chlamydomonas reinhardtii, the adf1-1 mutant is defective in Ca2+ influx that triggers deflagellation. Five new alleles of adf1 were recovered from UV and insertional mutagenesis screens, but none of the alleles were tagged with insertional DNA. I have used PCR based recombination mapping to place ADF1 in a 394 kb region of linkage group IX. Six BACs spanning the region have been used to transform mutant adf1 strains, with the goal of rescuing the phenotype. BAC transformations have not yielded a rescue; therefore, several candidate genes have been subcloned and are being used in attempts to identify the elusive gene.

Document type: 
Thesis
File(s): 
Supervisor(s): 
L
Department: 
Dept. of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Examining the cell biology of the NIMA-related kinases

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2009
Abstract: 

The NIMA-related kinases are the least well characterised of the so-called cell cycle kinases. Mounting evidence suggests some members of the family have roles in centrosome splitting, chromosome segregation, DNA damage response and regulation of cilia. Other members of the family are implicated in cystic kidney diseases, polycystic ovary syndrome and cancer. In this thesis I addressed specific questions related to three Neks: (i) NEK8 due to its role in cystic kidney diseases (ii) NEK4 because of its unusual sub-cellular localisation and (iii) CNK6 a potential link between cilia and the cell cycle. I developed the tools necessary for the identification of NEK8 substrates. I showed NEK4 localises to the distal end of the mother centriole. Neither kinase activity, nor the phosphorylation state of thr526 are necessary for this localisation. CNK6, initially identified in the flagellar proteome, does not localise to flagella and may be essential for Chlamydomonas reinhardtii survival.

Document type: 
Thesis
File(s): 
Supervisor(s): 
L
Department: 
Dept. of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)