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

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Specificity and polyreactivity of the antibody response during natural HIV-1 infection

Author: 
Date created: 
2006
Abstract: 

The specificity and polyreactivity of the antibody response in natural HIV-1 infection were studied. First, to investigate the overall antibody response, overlapping linear peptides were used to screen sera taken from HIV-1-infected individuals. The polyclonal antibody response was relatively stable during long-term infection, compared with acute infection, and mostly directed against immunodominant regions. Low level, transient antibody responses were detected against membrane proximal external region of gp41. To test if these antibodies are neutralizing, an affinity purification method was developed to isolate these serum antibodies. Second, in terms of polyreactivity of antibody response, we found that two broadly-neutralizing monoclonal antibodies bound only weakly to self antigen, cardiolipin. Moreover, sera were screened against cardiolipin; no significant reactivity was observed. We conclude that the antibody response in HIV-1 natural infection is relatively stable over time; the MPER is weakly immunogenic in vivo, and that broadly-neutralizing antibodies do not seem to be autoreactive.

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

The Role of Fa2p in Ciliary and Cell Cycle Regulation

Author: 
Date created: 
2006
Abstract: 

Cilia are microtubule based organelles with roles in motility and sensory perception. An emerging pattern suggests that various human diseases are caused by defects in the assembly, maintenance or function of cilia. Some ciliopathies, such as the polycystic kidney diseases and Bardet-Biedl syndrome, involve aberrant cell proliferation in conjunction with ciliary defects. Recent data suggests that the cilium serves as a highly conserved organizing center for early steps in signal transduction pathways that control cell growth and division. As such, signaling molecules important for growth, mitosis or differentiation have been localized to cilia. The relationship between cilia and cell cycle progression is poorly defined, but may involve regulation by the NIMA-family of kinases (Neks). Our discovery that the Nek Fa2p is important for ciliary function and cell cycle progression in Chlamydomonas provides a direct link between these two processes. Fa2p was originally identified from a screen for deflagellation-defective mutants in Chlamydomonas and shown to be defective in calcium-induced severing of the axonemal microtubules. We subsequently showed that fa2 mutants are delayed in transit through at least two points in the cell cycle: (1) G2/M transition; (2) assembly of flagella after exit from mitosis. In this study, we show that Fa2p localizes to a unique site at the proximal end of cilia in Chlamydomonas and kidney epithelial cells, suggesting a high level of conservation of this signaling complex. In both cell types, Fa2p localization is dynamic; when cells enter the cell cycle, Fa2p becomes reduced in the cilium and accumulates at the base of the basal bodies/centrioles. It remains associated with the spindle poles throughout the cell cycle and is assembled on cilia when they begin to regenerate after exit from mitosis. Importantly, Fa2p kinase activity is required for deflagellation, but does not appear to be essential for localization and efficient cell cycle progression. Furthermore, we show that two mammalian Nek homologs of Fa2p (mNek1 and mNek8), which are defective in murine models of polycystic kidney diseases, localize to primary cilia and centrosomes. Finally, biochemical analysis reveals the interaction of two proteins (~20 and ~60 kDa) with Fa2p in situ.

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

The PAAD domain OF IFI16 reveals a novel ssDNA binding function for the death domain super family

Author: 
Date created: 
2006
Abstract: 

The PAAD Death domain, involved in apoptosis and inflammation, shares a 6-helix bundle fold similar to other apoptotic sub-family members (DD/DED/CARD), but with a disordered region in helix 3. To investigate the structural basis for this difference I measured and compared thermodynamic folding parameters between PAAD and CARD members and show that PAAD domains have low stability and can undergo conformational changes when induced by mutagenesis or secondary structure promoting agents. The structural plasticity of the PAAD domain is consistent with an induced fit mechanism of ligand binding that may confer different protein-ligand interactions, contrasting with the common assumption that the Death domain is a protein-protein interaction domain. Finally, I challenged the above assumption by showing that the PAAD domain from the HIN-200 family member, IFI16, has all the characteristics of a single stranded nucleic acid binding protein, motivating further studies for the discovery of new PAAD-ligand interactions and functions.

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

In vitro selection of RNA aptamers against the dual conformations of a Photochromic Compound, and Construction of a Light-Sensitive Allosteric Hammerhead Ribozyme

Author: 
Date created: 
2006
Abstract: 

Using the photochemical properties of the dihydropyrene - cyclophanediene system, in vitro selection (SELEX) was carried out to obtain RNA aptamers that bind either closed or open form of a photochromic compound with high affinity and specificity. Among the isolated aptamers, the C8 aptamer, which bound to the closed form of the compound, showed the best affinity and specificity and was able to discriminate the open form of the compound. The C8 aptamer was used successfully to design an allosteric ribozyme by functionally combining the aptamer with a hammerhead ribozyme. This rationally designed allosteric ribozyme demonstrated effective light-dependent switching activity when shown to be catalytically activated by UV light irradiation and inhibited with visible light. In summary, this light-dependent allosteric ribozyme can be potentially used as a photo-regulated molecular switch to create new genetic control systems.

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

Characterization of drosophila homologues of activated Cdc42 associated kinase (ack)

Author: 
Date created: 
2005
Abstract: 

Cdc42 is a member of the Rho GTPase family of proteins, which act as molecular switches to turn signal transduction pathways on or off by alternating between active GTP-bound and inactive GDP-bound states. Cdc42 is involved in diverse biological processes such as actin cytoskeletal organization, microtubule dynamics, axonal guidance and epithelial wound repair. The ACK family of non-receptor tyrosine kinases function downstream of Cdc42. The intent of this study was to characterize Drosophila homologues of ACK, DACK and DPR2. A P element excision approach was used in an attempt to create mutations in the DACK and DPRZ genes. DACK mutants were successfully created, characterized at the molecular level, and a preliminary phenotypic characterization done. The results suggest that DACK is largely redundant in development, but may have some role in axonal guidance. DACK may have overlapping functions with DPR2, and knocking out both ACKs will likely reveal more roles.

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

Characterizing the roles of Memo in TGFߠSignalling

Date created: 
2004
Abstract: 

Drosoplzila nemo encodes a serine threonine MAP kinase that is involved in patterning and cell fate determination. nemo participates in crosstalk with several pathways; with studies linking the vertebrate homologue, nlk to the TGFP pathway. TGFPs are structurally related extracellular polypeptides including the Bone Morphogenetic Proteins (BMPs) that are potent regulators of development. In Drosophila the BMP molecules Decapentaplegic (Dpp) and Glass bottom boat (Gbb) promote vein formation while Nemo promotes intervein fates. Genetic studies revealed that Nemo counteracts the effects of components of the BMP pathway; and nemo mutant pupal wings show high levels of BMP signalling activity in ectopic veins, supporting an inhibitory role for Nemo on BMP activity. These studies show that nemo may act as a negative regulator of TGFP signalling; and supports the emerging roles of nemo as an important regulator of signalling in different pathways.

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

Functional analysis of ATP binding cassette (ABC) transporters in Caenorhabditis Elegans

Author: 
Date created: 
2005
Abstract: 

This thesis deals with functional analysis of ABC transporter genes in Caenorhabditis elegans. ABC transporters constitute one of the largest gene families in C. elegans. Sixty ABC genes have been identified and classified into eight different subfamilies. Fifty-seven orthologous pairs can be found between C. elegans and C. briggsae. As an initial step toward understanding the functions of the ABC genes, I generated transgenic animals containing prornoter::GFP/DsRed fusion constructs for the whole family in order to address when and where these genes are turned on in vivo. I observed frequent expression of ABC genes in intestine, pharynx and excretory cell, especially for those in subfamilies B and C. Sixteen of the 60 ABC genes are tandemly duplicated genes, forming two 4-gene and four 2-gene clusters. Interestingly, the majority of the promoters from the same clusters drive temporally andfor spatially differential expressions, suggesting active function of these duplicated genes in different tissues or stages rather than their being inactivated, as is typical for duplicated genes. Currently 18 single ABC gene mutants have been created. All of them are wild type under normal laboratory conditions. I used one of the ABC genes, pgp-12, an excretory cell-expressing gene, to characterize its transcriptional regulation. A 10 base pair (bp) cis-element, Ex-1 and its binding protein C26C6.5a, were isolated. C26C6.5a is also expressed in the excretory cell and binds Ex-l in vitro. RNAi analysis revealed that the C26C6.5a protein functions as a trans-activator for excretory cell specific expression. The only member of subfamily E is the ABCE gene that has been annotated as an RNase L inhibitor. This annotation is unlikely to be correct because of the lack of the inhibitor gene in C. elegans. Functional analysis of the gene reveals that it is essential for life, possibly involved in transcription and translation, and may function as a nucleocytoplasrnic transporter. RNAi data implies that the protein functions in both germline and vulvae.

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

A light-harvesting catalytic DNA for thymine dimer repair

Date created: 
2005
Abstract: 

Since the discovery of ribozymes in the 198O's, there have been many examples of catalytic DNA or RNA that can catalyze a variety of reactions, including phosphodiester bond cleavage, porphyrin metallation and nucleotide synthesis. A novel catalytic DNA (DNAzyme) was generated by in vitro selection that could perform a photochemical reaction, the photoreversal of thymine dimers upon irradiation of light. Photolyase enzymes found in nature catalyse the photorepair of thymine dimers with the aid of sensitizers, such as FADH and tryptophan. Curiously, one DNAzyme, UVlC, could repair thymine dimers without the aid of photosensitizers. UV 1 C was found to be active when using light that was significantly red-shifted relative to that of normal DNA absorbance. Using spectroscopic techniques, it was determined that the red-shifted action spectrum and rate enhancement profile were caused by the presence of a guanine quadruplex in the catalytic core. Further characterization of UV1 C revealed interesting information on the folding, substrate specificity and enzyme mechanism. Using sequence mutant constructs, it was determined that the 3'-binding arm of the enzyme could be removed with very little effect on repair rates. Cross-linking reactions determined that the thymine dimer was situated in close proximity, or stacked with the putative guanine quartet. Interestingly, substrates containing uracil dimers with deoxyribose sugar rings were efficiently repaired by UVl C, whereas thymine dimer substrates containing ribosyl sugar rings were not. Oxidative damage to guanines was not detected, which implied that a fast rate of back electron transfer may have occured after the thymine dimer photosplitting process. DNA "aptamers" (binders) were generated by in vitro selection that could simultaneously bind to the electron transfer protein cytochrome c, and to the small metalloporphyrin, hemin. Aptarners selected contained guanine-rich sequences located centrally with respect to the overall sequence. Chemical probing analysis determined that a central guanine-quadruplex formed that was responsible for binding to hemin. Through footprinting analysis, the binding site for cytochrome c on the DNA was determined. The ternary complex represents a potential model for the study of electron transfer between artificial and naturally-occurring heme electron transfer systems.

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