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Title: Characterizing the CRD-BP-RNA interaction in-vitro and in cells.
Contributors: Mark Barnes (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: The highly conserved family of RNA-binding proteins known as the VICKZ RNA-binding proteins play an integral role in the formation of cytoplasmic RNPs which leads to the stabilization, localization and translational control of many mRNA transcripts in the cell. The key investigation of this thesis was to analyse the binding ability of the VICKZ protein family member, the coding region determinant-binding protein (CRD-BP), both in-vitro and in cells. CRD-BP has four K-homology (KH) domains and two RNA-recognition motif (RRM) domains. Deletion studies in CRD-BP orthologs have shown that the KH domains, and not the RRM domains, are predominantly responsible for binding to RNA substrates. However, it is still unclear to what extent each of the KH domains play in their physical interaction with RNA molecules, nor is it known if each of the KH domains an play equal role in interacting with different RNA substrates. In an effort to address the above questions, we used site-directed mutagenesis to mutate the first glycine of the G-X-X-G motif in each KH domain separately, and in combinations. We mutated the glycine to an aspartate to introduce both physical and electrostatic hindrance for binding at the G-X-X-G motif. The goal was to determine if such a mutation can disrupt CRD-BP's ability to bind its RNA substrates both in-vitro and in cells. Our results showed that KG single mutants KH2, KH3 and KH4 did not disrupt the CRD-BP-c-myc CRD RNA interaction in-vitro. CRD-BP KH1 single mutant exhibited a modest reduction in binding to the c-myc CRD RNA substrate in-vitro. However, double KH domain mutations (KH1-2, KH1-3, and KH2-4) resulted in a complete abrogation of CRD-BP's ability to bind the c-myc CRD RNA substrate, suggesting these KH domains work in tandem to bind to the c-myc CRD RNA substrate in-vitro. Interestingly, the CRD-BP KH domain double mutant, KH3-4, showed only a modest reduction in the c-myc CRD RNA substrate binding, suggesting that the first glycine in the G-X-X-G motif of KH3 and KH4 doe
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2013

Title: The coding region determinant binding protein shields c-myc coding region determinant and MDR-1 RNAs from endonucleolytic attack by a novel mammalian endoribonuclease in vitro.
Contributors: Dan S. Sparanese (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2007

Title: Diastereoselective spiroannulation of phenolic derivatives: Effect of steric hindrance on the diastereoselectivity.
Contributors: Lyndia Susag (author), Guy Plourde (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: The objective of this study was to determine the effect, if any, that steric factors have on the oxidative spiroannulation of simple phenols bearing substituents that are increasing in size. Specifically, we wanted to determine whether or not the diastereoselectivity of the reaction would improve. To do so, the synthesis of five phenols was carried out from which four of the five spiroether targets were synthesized. Based on the [superscript]1H-NMR of these four compounds, it was found that the diastereoselectivity in these reactions did not improve it actually decreased. The fact that the diastereomeric ratios did not improve with increasing steric factors provides evidence that the spiroannulation is not affected by the size of the group attached to the 3-position. Instead, it gives supporting evidence that the stereoelectronic effect and location of the substituent has greater effect to the overall outcome of the reaction. --P.ii.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2010

Title: Characterization of a novel endoribonuclease capable of degrading c-myc mRNA in vitro.
Contributors: Joel Christopher Urquhart (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2006

Title: Evaluation of potassium stress responses and identification of novel RT-qPCR reference genes in the haloarchaeon, Haloarcula marismortui .
Contributors: Matthew W. Jensen (author), Andrea Gorrell (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Growth characteristics and stress responses in the halophilic archaeon, Haloarcula marismortui, have been poorly investigated and knowledge of the effects of extracellular potassium concentration on halophilic growth is limited. We report the evaluation of cellular generation times across a range of extracellular potassium concentrations to assess the organisms responses to extreme potassium stress. Our results show Haloarcula marismortui exhibits an optimal generation time of 4.19 ± 0.14 hours at an extracellular KC1 concentration of 100mM. This corresponds to an intracellular K⁺ concentration of 2.02M as determined through the use of Induction-Coupled Plasma Mass Spectrometry. Additionally, the validation of several candidate reference genes for use with RT-qPCR studies is reported. Five reference genes (16S rRNA, rpoB, pykA, polA, and rpoA) have been confirmed as being stably expressed in accordance with the Minimal Information for the Publication of Quantitative PCR Experiments (MIQE Guidelines) across several unique halophilic growth conditions.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2013

Title: Inhibition of pre-mRNA splicing by small molecules.
Contributors: Kamalprit Kaur Chohan (author), Stephen Rader (Thesis advisor), Kerry Reimer (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Cellular function is dependent upon the correct translation of genomic information encoded in DNA into functioning products, usually proteins. Prior to protein translation, DNA is transcribed into messenger RNA (mRNA). In eukaryotes such as human, this process must almost always undergo an intermediate step, termed pre-mRNA splicing, in which non-protein-coding regions (introns) are removed from the mRNA, yielding mature message (exons). Defects in pre-mRNA splicing are responsible for various human disorders including retinitis pigmentosa, spinal muscular atrophy, and myotonic dystrophy. In order to work towards a cure for these diseases, it is necessary to understand how pre-mRNA splicing works normally. One potentially useful tool for this is small molecule inhibitors of splicing, which have previously been shown to inhibit catalytic RNAs the additional benefit of being candidates for therapeutics. Only two papers (Hertweck et al., 2003; Kaida et al. 2007) have explored the effects of small molecules on nuclear splicing. Previous work has investigated inhibition of human splicing. In this work, I have examined the effect of small molecules on yeast splicing in order to make use of the powerful genetic and biochemical tools available for yeast. The main goal of this thesis was to identify small molecule inhibitors of yeast pre-mRNA splicing and to characterize the step at which they exert their inhibitory effects. Thirty-two different small molecules were tested. Ten of them were found to completely inhibit pre-mRNA splicing. IC₅₀ values were measured for each of the inhibitory small molecules, and neomycin was found to be the strongest inhibitor with an IC₅₀ of 80~M, while cefoperazone was the weakest inhibitor with an IC50 of 6.1 mM. Native gel analysis was used to establish the step at which splicing was inhibited. Four of the ten inhibitors showed a complete block in spliceosome assembly with accumulation of spliceosomal complex H; one accumulated spliceosomal complex A; two accumulated both spliceosomal complexes A and B; and three accumulated spliceosomal complexes B and C. I anticipate that these inhibitors will be useful tools for the studying the mechanism of pre-mRNA splicing. By characterizing the splicing complexes that accumulate in the presence of these inhibitors it will be possible to map out the path by which splicing complexes assemble. Furthermore, several of the inhibitors are previously uncharacterized, and consequently have potential to be useful in a variety of other context. In the long term, these inhibitors may lead to novel therapies for splicing related diseases.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2008

Title: Characterizing the endoribonuclease activity of APE1.
Contributors: Wan Cheol Kim (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Recent evidence shows that mRNA stability and turn-over is an integral control point in the regulation of gene expression. The stability of various mRNAs within a eukaryotic cell can differ and this results in a magnitude of difference in mRNA abundance. An enzyme known as APE1, apurinic/apyrimidinic DNA endonuclease 1, has recently been discovered to possess an endoribonuclease activity against c-myc messenger RNA (mRNA) in vitro. The identification of APE1 as an endoribonuclease warranted this research to further characterize this novel activity both in vitro and in vivo. Previous studies have discovered the residues constituting an active site for apurinic/aprymidinic [sic] DNA (AP-DNA) incision activity of APE1. Whether these residues are shared in the RNA-cleaving activity of APE1 was unknown. The first objective of this thesis was to assess the role of these amino acid residues in contributing to the endoribonuclease activity of APE1. Our results revealed that APE1 indeed shared these residues to cleave both RNA and AP-DNA. However, we also discovered certain differences in the activities of one mutant (D283N) in carrying out AP-DNA and RNA incisions. This suggested that the roles of active site residues in each reaction are not entirely identical. In addition, we have assessed the RNA-cleaving activities of APE1 variants identified in the human population. For a few variants, RNA-cleaving activities were severely reduced while its AP-DNA incision activities were functional. These results suggested a possible unrecognized link between the reductions in the RNA-cleaving activity of the variants and their reported association in certain diseases. The second objective of this thesis was to establish the RNA secondary structures and sequences that are preferentially cleaved by APE1. Our results revealed that APE1 has preference for cleaving the single stranded regions or weakly base paired regions of the RNA. Also, preferred sequences of cleavage were determined to be UA, UG, and CA dinucleotides. Prevalent A
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2010

Title: Isolation and characterization of the U4 snRNP in Saccharomyces cerevisiae.
Contributors: Heath de la Giroday (author), Stephen Rader (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Pre-messenger RNA (pre-mRNA) splicing is a cellular phenomenon common to all eukaryotes in which non-coding sequences are removed from pre-mRNA transcripts and the coding sequences spliced together yielding mRNA. The spliceosome is the entire complement of proteins and snRNAs responsible for pre-mRNA splicing and forms around a core of five small nuclear ribonucleoprotein particles (snRNPs). There is evidence suggesting that the RNA component of the spliceosome is responsible for the two transesterification reactions that produce the spliced mRNA, highlighting the importance of understanding the behaviour of spliceosomal RNAs and the relationship between RNA and proteins in the spliceosome. With the goal of identifying the major structures of the spliceosome and their organization, there has been an effort to isolate the discrete snRNPs, identify the associated proteins and structurally characterize the particles. While the U1, U2, U4/U6, U4/U6.U5 and U2/U6 snRNPs have been isolated and studied, there are other snRNPs that have been hypothesized to exist. The U4 snRNA is believed to exist as a discrete particle prior to incorporation into the U4/U6 snRNP however, the free U4 snRNP has not been isolated as it does not accumulate in cellular lysates. This investigation utilized both genetic and biochemical means to generate U4 snRNP from Saccharomyces cerevisiae for the purposes of isolation and characterization. I identified the 1sub/4sub allele of prp24 ...as a mutation set that causes a shift to almost 100% of U4 RNA in the cell occurring in the free form. Analysis of lysates from this strain on cesium chloride gradients provided evidence that the accumulated U4 RNA is associated with a mass of protein and that it is indeed an RNP. In addition, I successfully modified Raghunathan & Guthrie's ...method of generating Brr2-released U4 snRNA species with the goal of creating a preparative system for generating U4 snRNA/P and confirmed that the released species is not composed of naked RNA and is als
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2008

Title: A quasiclassical trajectory study of H2 + H2 energy transfer: a survey of applicability of detailed balance
Contributors: Mosammat Hasina Akter (author), Margot Mandy (thesis advisor), University of Northern British Columbia College of Science and Management (Degree granting institution)
Abstract: State-to-state cross sections and a survey of detailed balance for transitions among the vibrational and rotational (v; j) states of H2 1Pg+ below 37 mEh as the result of collisions in the H2 + H2 system were determined by using the quasiclassical trajectory (QCT) method. Study of this system is necessary for an improved under-standing of the kinetics in the interstellar medium to model processes occurring in the molecular clouds. The potential energy surface of Boothroyd et al. (J. Chem. Phys. 116, 666, 2002) was used for trajectory calculations. The Discrete Variable Explicit Runge-Kutta (DVERK) method was used for numerical integration. State-to-state cross sections were examined for agreement with microscopic reversibility. In the majority of cases the agreement was poor. Consequently the conclusion is that QCT is not a viable method for the evaluation of state-to-state rate coefficients in the H2 + H2 system.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2016

Title: Assessing the role of Syntaxin18 in controlling c-myc mRNA expression and growth in human breast cancer cells.
Contributors: Tyler A. Bassett (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Messenger RNA stability is critical in the control of gene expression. Enzymes capable of cleaving RNA, termed ribonucleases, are gaining appreciation for their role in regulating gene expression. Our lab has discovered a novel protein which has been shown to cleave c-myc mRNA within the coding region determinant (CRD). This protein was tentatively identified as Syntaxin18, a known t-SNARE protein. Recombinant Syntaxin18 has been shown to possess endoribonucleolytic activity against c-myc mRNA in vitro. In order to determine whether Syntaxin18 functions as an endoribonucleae in cells, the effect of altering its expression on the proliferation, levels and stability of c-myc mRNA was examined. The expression of Syntaxin18 in various cell lines and tissues were also compared. Through Western analysis, it was discovered that the expression of Syntaxin18 was similar in all immortalized cancer cell lines examined. However, higher Syntaxin18 expression was observed in cancerous breast tissue when compared to the normal breast tissue. MTT assays revealed that modulation of Syntaxin18 was able to affect the proliferation of MCF-7 cells, and this effect was observed only upon incubation with 17-β-estradiol and insulin. Finally, ribonuclease protection assay analysis showed that modulation of Syntaxin18 may have an effect on the steady-state levels of c-myc and β-actin mRNAs; however, down-regulation of Syntaxin18 did not impact the stability of the c-myc transcript. Based on results obtained from this study, it remains unclear whether Syntaxin18 can function as an endoribonuclease in MCF-7 cells.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2008

Title: Investigating the Cellular Localization of APE1.
Contributors: Sang Eun Kim (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Apurinic/apyrimidinic endonuclease 1 (APE1) is a well-known DNA repair enzyme with multiple functions which include redox-activation, 3'-DNA phosphodiesterase, 3'-5' DNA exonuclease, and RNase H activities. Recently the novel function of APE1 in RNA metabolism has been demonstrated. APE1 was shown to possess the ability to cleaving single-stranded RNA as well as abasic RNA. It was also shown to physically interact with proteins involved in RNA metabolism. Previous studies have discovered the altered distribution and expression of APE1 in different cell types including cancer cells. The cytoplasmic distribution of APE1 has been shown to have correlation with aggressive carcinomas and poor prognoses for patients. This has led to an increasing number of studies on the undefined extra-nuclear roles of APE1 and APE1's potential implications in cancer development. In this thesis, I aimed (i) to confirm the importance of nuclear localization signal (NLS) of APE1 at the N-terminus, (ii) to discover the role of single nucleotide change of APE1 human population variants in its sub-cellular distribution, (iii) to assess the cellular localization of APE1 with processing bodies and stress granules under cellular stresses, and (iv) to initiate immune-fluorescence analysis of APE1 in breast cancer tissue. Our results revealed that APE1 indeed has an important NLS of 1-20 amino acids at the N-terminus and APE1 human population variants showed nuclear localizations identical to the wild-type APE1. There was no co-localization of APE1 with PBs and/or SBs implying that APE1 is unlikely to be involved in mRNA processing that is carried out in PBs and SGs. We observed redistribution of the ND20-APE1-GFP upon cellular stresses in HepG2 cells and this phenomenon has highlighted possible degradation of the cytoplasmic APE1 upon cellular stress. This finding is also consistent with previous discovery of the five critical lysine residues that are responsible for ubiquitination and APE1 degradation upon cellular stress. In summary, this
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2012

Title: Investigating the effects of expressing APE1 human population variants in cellular systems.
Contributors: Conan Ma (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Apurinic/apyrimidinic endonuclease 1 (APE1) is a multi-functional mammalian protein which has recently been shown to possess the ability to endonucleolytically cleave single-stranded RNA and abasic RNA. Several population variants of APE1 (L104R, E126D and D148E) are known to exist in the human population. L104R and E126D have been linked to Amyotrophic Lateral Sclerosis while D148E has been linked to various cancers. The exact molecular mechanisms which correlate these variants with human disease are currently unknown. Recent evidence has shown that the in vitro endoribonuclease activities of these variants are different from the wild-type APE1 protein. Here, we hypothesize that the altered endoribonuclease activity of APE1 population variants may be associated with phenotype changes leading to disease pathogenesis. The goal of this thesis was to determine whether APE1 population variants can cause an altered phenotype when expressed in prokaryotic (Origami™ (DE3) cells) and eukaryotic systems (HeLa cervical cancer and HepG2 hepatoma cancer cell lines). Subsequently, these changes were to be linked to altered endoribonuclease activity of these variants. Using two separate assays, it was shown that the L104R and E126D variants possess enhanced cytotoxicity to Origami™ (DE3) cells. This correlates with their distinct endoribonuclease activity demonstrated in vitro. The D148E variant, which had lost endoribonuclease activity, had no effect on colony formation and growth of Origami™(DE3) cells. Interestingly, this study also showed that, when over-expressed, the L104R and E126D variants are capable of causing enhanced growth in the mammalian HepG2 cells. Preliminary microarray and quantitative real time polymerase chain reaction experiments were conducted in an attempt to understand the mechanism for the L104R-induced cell growth in HepG2 cells. Unfortunately, the results were inconclusive. In summary, this thesis has demonstrated a solid correlation between having distinct endoribonuclease act
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2012

Title: Quasiclassical trajectory study of D+HH and H+HD.
Contributors: Glen J. McNamara (author), Margot Mandy (thesis advisor), University of Northern British Columbia (Degree granting institution), Erik Jensen (committee member), Peter Jackson (committee member)
Abstract: An examination of the dynamical behavior of H + HD and D + H\u2082 in the ground electronic state is performed on the BKMP2 potential energy surface using the quasiclassical trajectory method. The complete set of state-to-state energy transfer and state-specific dissociative cross sections and thermal rate coefficients has been obtained for both systems. Comparisons are made to the H + H\u2082 system to investigate isotopic effects on reactivity and energy transfer. Collision-induced dissociation, exchange reactions, and non-reactive energy transfer are analyzed and compared to previous results on this system, when such are available. As a prototypical three-body system, H + H\u2082 and its isotopic analogues are interesting as benchmarks for both theoretical and experimental methods. The results of this work can be applied to the general field of molecular reaction dynamics, to interstellar physics and chemistry, to models of planetary atmospheres and stellar system formation, and to studies of the effects of isotopes on reaction rates. The work is motivated by astrophysical applications: in particular, the data may be used as inputs for master equation calculations for interstellar gases.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2011

Title: Investigating the 3' RNA Phosphodiesterase and 3'-5' Exoribonuclease Activities of APE1.
Contributors: Manbir Kaur Chohan (author), Chow H.M. Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Messenger RNA degradation is a major control point in determining the abundance of mRNA, and hence in the regulation of gene expression. Until recently, endoribonucleases, enzymes that cleave the phosphodiester bond within a polynucleotide chain, have been dismissed as the key enzymes responsible for mRNA turnover. The human apurinic/apyrimidinic endonuclease 1 (APE1) was recently identified as an endoribonuclease that is capable of cleaving c-myc mRNA in vitro and regulate c-myc mRNA level and half-life in cells. The ability to subject any target mRNA for degradation is a powerful therapeutic intervention, and the technologies currently in use have employed activities of endoribonucleases. Hence, in addition to increasing our basic knowledge on its role in the regulation of gene expression, a better understanding on the RNA-degrading enzyme such as APE1 has the potential in leading to the development of novel gene inactivation technology. The first objective of this thesis was to determine whether APE1 possesses 3'RNA phosphodiesterase and 3'-5' exoribonuclease activities. Using three difference RNA substrates, we confirmed that APE1 indeed has 3'RNA phosphodiesterase activity which is clearly absent in the common Ribonuclease A. We also found that Myricetin, a known inhibitor of AP-DNA activity, is capable of inhibiting 3'RNA phosphodiesterase activity of APE1. Our results also revealed that APE1 has weak to negligible 3'-5' exoribonuclease activity against unstructured poly-ribonucleotides. However, as with its known 3'DNA exonuclease activity, we found that APE1 has preferential in removing one nucleotide at 3' overhang of RNA. The second objective of this thesis was to determine the critical amino acids responsible for the 3' RNA phosphodiesterase activity of APE1. Our results revealed that APE1 uses several similar critical residues tin carrying out both its endoribonuclease and 3' APE1 uses several similar critical residues in carrying out
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2013

Title: Identification and Validation of the U2, U4, U5, and U6 Spliceosomal snRNAs in Cyanidioschyzon merolae.
Contributors: William St. Clair Dunn (author), Stephen Rader (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Pre-mRNA splicing is an essential step in eukaryotic gene expression, and introns have been found in nearly all eukaryotic genomes sequenced to date. The red alga Cyanidioschyzon merolae is found in acidic thermal springs, and its recently sequenced genome revealed a surprising paucity of intron-containing genes, raising the question of whether the normal complement of splicing machinery is maintained to splice so few introns. To address this I searched for snRNAs computationally, successfully identifying C. merolae homologues for four of the five snRNAs. I experimentally confirmed their expression, found that their structural elements are similar to those known from other organisms, and demonstrated that U4 and U6 base pair to each other, as expected. My data support the proposed switch in U6-5's splice site base pairing between the two catalytic steps, as well as a recent model for free U6. --P. ii.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2011

Title: Synthesis of Spirolactones Substituted at the Benzylic Position: Studies Toward the Formation of Manumycin Structural Analogs.
Contributors: Thomas Wayne Scully (author), Guy Plourde (Thesis advisor), Stephen Rader (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: This study was designed to create a series of spirolactone compounds produced through an oxidative spiroannulation reaction, each with a different substituent at the benzylic position. The effect, if any, on the diastereoselectivity of the spiroannulation reaction caused by the benzylic substitution would then be determined by ¹H-NMR. Several synthetic methodologies were employed in attempts to produce these compounds but only a single compound was successfully produced in 27% yield in 5 steps. This compound had a hydroxyl function at the benzylic position and showed total enantioselectivity in the spiroannulation reaction. Several unique properties of the intermediate molecules created in these studies were discovered while attempting to synthesize the target molecules. --P. iii.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2012

Title: Dissection of U4 snRNA functional domains using in vitro reconstitution.
Contributors: Amy Hayduk (author), Stephen Rader (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Splicing, the removal of non-protein-coding introns from pre-mRNA, is a critical step in eukaryotic gene expression, yet this process is still far from being fully understood. Investigation of the splicing mechanism has been hindered by its complexity. The spliceosome, the dynamic ribonucleoprotein complex which catalyzes splicing, contains over 100 molecular components, some of which are organized into small nuclear ribonucleoprotein particles (snRNPs). Each snRNP is composed of a small nuclear RNA (snRNA U1, U2, U4, U5, or U6) and its associated proteins. While the general roles of U1, U2, U5, and U6 are reasonably well understood, the function of U4, though essential for splicing, remains much less clear. It has been demonstrated that U4 forms an extensive base-pairing interaction with U6, but the function and mechanism of formation of this interaction have not been established. One reasonable hypothesis is that interaction of U4 and U6 allows U4 to activate U6 for assembly onto the pre-mRNA. Furthermore, there is only a limited knowledge of the proteins associated with U4, where they bind, and what functions they may perform during snRNP and spliceosome assembly and splicing catalysis. Determination of the structure of the U4 snRNP and its role is splicing has been hindered by lack of an in vitro system allowing U4 reconstitution analyses. To facilitate molecular dissection of the structure and function of U4 in splicing, I have developed an in vitro assay for reconstitution of functional U4 snRNPs. Endogenous U4 was depleted from yeast splicing extract through oligonucleotide-directed RNase H degradation, and optimal conditions for this reaction were identified. The most important factor for efficient U4 degradation was to perform the reaction under conditions of active pre-mRNA splicing, as the splicing process was found to increase accessibility of the targeted region of U4. Depletion of U4 resulted in an early block in spliceosome assembly, strongly inhibiting splicing activity. Subsequent addition of
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2010

Title: Investigation into class II P-glycoprotein messenger RNA decay in normal liver and liver tumours.
Contributors: Janis Alexandra Shandro (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2004

Title: RNA sequences and structures cleaved by a novel mammalian endoribonuclease in vitro.
Contributors: Alaeddin Walid Tafech (author), Chow Lee (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: No abstract available.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2006

Title: Artificial weathering reveals reduced elemental leachability from hardened biomass ash.
Contributors: Erwin Rehl (author), Kerry Reimer (Thesis advisor), Michael Rutherford (Thesis advisor), University of Northern British Columbia (Degree granting institution)
Abstract: Biomass ashes are potential soil amendments that reduce soil acidity and provide plant nutrients, but trace elements in ash may be leached from the solid phase, thereby posing environmental concerns. We determined the leachability of major and trace elements as influenced by ash pre-treatments, the presence of soil, and the pH of the receiving environment. Weathering was simulated by serial batch extraction where pH was uncontrolled, and by single extraction under controlled pH conditions. We found that hardening reduced the solubility of ash, and reduced the leachability of Al, Ba, Ca, Cu, Mo, Sr, and V, as determined by ICP-MS. In a separate experiment, extractions of ash samples showed that when pH was lowered the leachability of most elements increased while a few decreased. The results of the weathering experiments support the use of ash as a soil amendment. --Leaf ii.
Discipline: Chemistry
Content Model: info:fedora/ir:thesisCModel
Date added: 2014