Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Canterbury, 2009. / Typescript (photocopy). Includes bibliographical references (p. 172-183). Also available via the World Wide Web.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 155-170.

Thesis (Ph.D.)--York University, 2007. Graduate Programme in Physics and Astronomy. / Typescript. Includes bibliographical references (leaves 120-124). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR32065

<p>Every living cell faces a multitude of DNA threats in its lifetime because damage to DNA is intrinsic to life itself. A double-strand break (DSB) is the most cytotoxic type of DNA damage and is a potent inducer of chromosomal aberrations. Defects in DSB repair are a major driver of tumorigenesis and are associated with numerous developmental, neurological and immunological disorders. To counteract the deleterious effects of DSBs, organisms have evolved a homologous repair (HR) mechanism that is highly precise. The key to its error-free nature lies in its use of a homologous template in restoring the DSB and its preferential occurrence during late S and G2 phase of the cell cycle when identical sister chromatids are available as templates for repair. However, HR can also engage homologous chromosomes and ectopic substrates that share homology, resulting in mitotic loss-of-heterozygosity (LOH) and unwanted chromosomal aberrations. In this case, understanding of the underlying mechanisms and molecular factors that influence accurate sequence transfer and exchange between two homologous substrates becomes crucial. </p><p>The focus of this dissertation is examination of the genetic factors and molecular processes occurring at early intermediate steps (DNA end resection and DNA synthesis) of mitotic recombination in Saccharomyces cerevisiae. To model DSB repair, we established a unique plasmid-based assay with a small 8-base pair (bp) gap in the middle of an 800-bp plasmid substrate. To delineate the molecular structures of strand exchange intermediates during HR, we used a 2% diverged plasmid substrate relative to a chromosomal repair template to generate mismatch-containing heteroduplex DNA (hetDNA) intermediates. The assay was performed in a mismatch repair (MMR)-defective background allowing hetDNA to persist and to segregate into daughter cells at the next round of replication. Unexpectedly, even when MMR was inactivated, sequence analysis of the recombinants revealed patches of gene conversion and restoration reflecting mismatch correction within hetDNA tracts. We showed that, in this system, MMR and nucleotide excision repair (NER) correct mismatches via two different mechanisms. While mispairing of nucleotides triggers MMR, NER is recruited by the subtle 6-methyladenine mark on the plasmid substrate, leading to coincident correction of mismatches. The methylation marks on the plasmid were acquired from the bacterial host’s native restriction-modification system during plasmid propagation. </p><p>Formation of hetDNA occurs when a plasmid substrate engages the chromosomal template for repair, forming a D-loop intermediate. D-loop extension requires DNA synthesis by DNA polymerase/s. Translesion synthesis (TLS) polymerases have been implicated in HR in both chicken DT40 cells and fruit fly, but not in yeast. This class of polymerases is known for its low fidelity due to a lack of exonuclease domain and is commonly used for lesion bypass and in extending ends with mismatches. We reported for the first time a requirement of Polζ-Rev1 and Polη (TLS polymerases in S. cerevisiae) for completing gap repair. Moreover, gap-repair efficiency suggested that these two polymerases function independently. We concluded that TLS polymerases are involved in either extending the invading 3’ end and/or in the gap-filling process that completes recombination. </p><p>DNA resection of a DSB serves as a primary step to generate a 3’ single-stranded DNA (ssDNA) for subsequent homologous template invasion, but this process has mostly been studied in the absence of a repair template or when downstream HR steps are disabled. To analyze the individual contributions of identified nucleases to DSB resection in the context of repair, we established a chromosomal assay; the substrate size was increased to 4 kilobases (kb) and 85 SNPs were present at ~50 bp intervals. In this chromosomal assay, resection and DNA synthesis influence the length of hetDNA tracts in the final recombinants, allowing these two steps to be analyzed. We specifically focused on synthesis-dependent strand annealing (SDSA) events, where hetDNA reflects DNA synthesis and extent of resection. Our main conclusions are as follows. DNA end resection on the annealing end of NCO products generated by SDSA is not as extensive as one might expect from resection measured in single-strand annealing (SSA) assays. In addition, although the two long-range resection pathways (Sgs1-Dna2 and Exo1) can support recombination in a redundant manner, hetDNA was significantly reduced upon loss of either. End processing of DSBs is predominantly 5’ to 3’, but we also observed loss of sequences (greater than 8 nt but less than 40 nt) at the 3’ termini. We have tested and ruled out the involvement of Mre11 and Polε proofreading activity. Lastly, Pol32 functions as a subunit of Polδ to promote extensive repair synthesis during SDSA. hetDNA tract lengths were significantly shorter in the absence of the Pol32 subunit of Polδ, providing direct evidence that Polδ extends the invading end during HR. Together, this work advances our understanding of how resection nucleases and DNA polymerase/s function to regulate mitotic recombination outcome and influence the molecular patterns of NCOs.</p> / Dissertation

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNF&alpha; production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.

INTRODUCTION: For many years, stress and anxiety disorders have taken a heavy toll on the American population. Affecting approximately 40 million individuals over the age of 18, the discovery of treatment options is very important. Ever since the 1950s, a wide variety of compounds have been discovered and proven to have antagonistic properties for such disorders. For the last three decades, however, researchers have focused on a specific peptide that was discovered in 1981 by Dr. Wylie Vale and his colleagues at the Salk Institute in San Diego, California, corticotropin releasing factor (CRF). CRF is a 41 amino acid peptide that has been shown to play a very important role in an organism's endocrine response to stress through the activation of the hypothalamic&ndash;pituitary&ndash;adrenal (HPA) axis. Ever since its discovery, the identification and characterization of the CRF receptors and family members have allowed for the development of novel peptide and non&ndash;peptide antagonists. Unfortunately, these compounds have been unsuccessful in the progression to later stage clinical trials that could lead to promising therapeutics. There are two receptor subtypes for this family of peptides known as CRFR1 and CRFR2. While there have been many compounds identified that can block CRFR1, currently, there are no known selective non&ndash;peptide antagonists for the CRFR2 subtype. As the two receptor subtypes share 70% sequence identity, close observation of the functional properties of antagonist ligands for CRFR1 may lead to the development of such ligands for CRFR2. METHODS: In our current study, we focused on two residues in transmembrane domains (TMD) 3 (His199) and 5 (Met276) of CRFR1 that have proven to be important for the function of the highly selective small molecule antagonist antalarmin. In order to further prove the importance of these sites, we have mutated the two corresponding amino acids in CRFR2β to those of CRFR1: V215H in TMD 3 and V292M in TMD 5. In addition, we mutated a third amino acid residue, M293I, in order to avoid the positioning of two adjacent methionine amino acids. With this mutant construct, CRE–luciferase and cyclic AMP radioimmunoassay methodologies were used to observe the function of antalarmin on CRFR1, the mutant and wild type CRFR2β. The accumulation of cAMP was measured intracellularly following stimulation by the CRF receptor peptide agonists sauvagine, isolated from frog, and urocortin 1, isolated from rat. RESULTS: For the initial CRE–luciferase functional assay, we used the CRF receptor agonist sauvagine on our mutant CRFR2β to indirectly measure the accumulation of intracellular cAMP through the enzyme luciferase. In the presence or absence of the antagonist antalarmin, there were no significant changes on the function of the mutant CRFR2β. On the other hand, when directly measuring the accumulation of intracellular cAMP via radioimmunoassay, antalarmin successfully showed a functional inhibitory effect on the mutant CRFR2β receptor. As expected, Ucn1 stimulation of CRFR1 in the presence of antalarmin indicated a decrease in the EC50 for the peptide agonist, and thus an inhibitory effect by antalarmin. Compared to CRFR1, we observed a similar effect for Ucn1 stimulation of the mutant CRFR2β receptor in the presence of antalarmin. While the presence or absence of antalarmin did not have a significant inhibitory effect on the wild type CRFR2β, it can be concluded that the mutant CRFR2β receptor possessed similar properties to the CRFR1 receptor with respect to antalarmin antagonist activity. CONCLUSION: In our study, we were able to further support the importance of the two amino acid residues in TMD 3 and 5 of CRFR1 for the function of small molecule antagonists. In addition, we were able to show that antalarmin, a small molecule antagonist known to be highly selective for CRFR1, can have a functional inhibitory effect on the mutant CRFR2β. The progressive study of these discrete differences between the two CRF receptor subtypes may enable the discovery of novel selective non–peptide CRFR2β receptor antagonists.

The cyclical interaction between the force-generating protein myosin and actin is the mechanism responsible for muscle contraction among all muscle types. Cardiac muscle contraction is tightly controlled to ensure that blood pumps effectively and efficiently from the heart to peripheral organs. Mutations in various cardiac proteins can lead to cardiac dysfunction and a number of cardiomyopathies. The first part of this dissertation studies two disease-linked mutations in the regulatory light chain of the cardiac myosin molecule, D166V and K104E, and assesses the kinetic and mechanochemical effects of the mutations via the in vitro motility assay. The data show that D166V mutant myosin force generation is reduced compared to wild type, and exogenous phosphorylation of the mutant light chain rescues force generation. In contrast, the K104E mutation showed no deficit in force production but exhibited increased calcium sensitivity of activation. These results are consistent with contractile defects associated with cardiomyopathies caused by various mutation-induced changes to protein function and mechanism of interaction. The second part uncovers the actin-binding mechanism of one of the chief muscle regulatory proteins tropomyosin. In cardiac and skeletal muscle, tropomyosin and troponin modulate muscle contraction. Tropomyosin binds along the length of actin filaments and blocks myosin-binding sites. Following an excitatory stimulus, calcium binds troponin and causes tropomyosin to shift its position on actin, allowing myosin to bind. The precise mechanism of how tropomyosin monomers with low actin affinity bind to form a stably bound, high affinity chain is unknown. By directly observing fluorescently labeled tropomyosin binding to actin filaments, it was shown that tropomyosin molecules bind randomly along the actin filament. Subsequent monomer binding, and formation of tropomyosin end-to-end bonds, increases the probability of sustained chain growth by decreasing the probability of detachment prior to additional monomer binding. Tropomyosin molecules added to the growing chain at approximately 100 monomers/(μM*s). Different tropomyosin isoforms segregate to distinct functional and structural regions of cells. The last chapter presents data that show spatial segregation of two different tropomyosin isoforms on actin filaments. This suggests that tropomyosin sorting in cells is, at least partly, an intrinsic property of the binding mechanism.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in women and fourth in men globally. While expansions in preventative measures have increased the detection of CRC at the early stages of disease, only 40% of CRC patients are diagnosed when the disease is at a local stage. Moreover, many anti-cancer drugs fail to significantly improve the life expectancy of patients due to innate and acquired resistance, underscoring a need for better diagnostic and therapeutic strategies for CRC. Immunoglobulin-containing and proline-rich receptor-1 (IGPR-1) is a novel cell adhesion molecule (CAM) that was recently identified in our laboratory. IGPR-1 is expressed in epithelial and endothelial cells and promotes cell-cell adhesion. Expression of IGPR-1 in endothelial cells regulates angiogenesis; however, its role in epithelial cells, particularly cancer cells with an epithelial origin, remains unknown. The overall goal of this study was to investigate the possible function of IGPR-1 in CRC tumor cell growth and response to chemotherapeutic agents. Specifically, we aimed to test the hypothesis that increased expression of IGPR-1 in CRC tumor cells promotes cell survival and contributes to the resistance of tumor cells to doxorubicin. Human CRC tumor cell lines, HCT116 and HT29, were transduced via a retroviral system to express IGPR-1 or empty retroviral vector pQCXIP. The effect of overexpression of IGPR-1 in HCT116 and HT29 cells was measured by MTT assay in non-adherent 24-well plates. In addition, cells were viewed under a light microscope, and images were taken to assess multicellular aggregation. Results demonstrated that expression of IGPR-1 in HCT116 and HT29 tumor cells promoted CRC tumor cell growth, increased multicellular aggregation, and stimulated resistance to the conventional chemotherapeutic agent doxorubicin in non-adherent cell culture conditions in vitro. Intriguingly, treatment of cells with doxorubicin promoted phosphorylation of IGPR-1 at serine 220 (Ser220), suggesting a critical role for phosphorylation of IGPR-1 in the development of resistance to chemotherapeutics. In addition, non-adherent cell culture conditions promoted activation of the key pro-apoptotic kinase, p38 MAPK in CRC tumor cells. Ectopic expression of IGPR-1 reversed this activation. This data suggests that IGPR-1, by suppressing p38 activity, in part, promotes tumor cell survival and increases the resistance of tumor cells to the killing effects of doxorubicin. Our findings are the first to demonstrate that IGPR-1 promotes CRC tumor cell growth and increases the resistance of CRC tumor cells to the cytotoxic effects of chemotherapeutic agents. The data suggests that IGPR-1 plays an important role in CRC by inhibiting the cellular apoptotic response and promoting chemotherapeutic resistance. Finally, IGPR-1 phosphorylation at Ser220 in response to doxorubicin may account for the IGPR-1-mediated development of resistance to doxorubicin in CRC.