Browsing by Author "Thamm, Douglas H., committee member"
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Item Open Access Autophagy modulation: role in anti-cancer therapy(Colorado State University. Libraries, 2015) Barnard, Rebecca A., author; Gustafson, Daniel L., advisor; Thamm, Douglas H., committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee memberAutophagy is a conserved lysosomal degradation process characterized by cellular self-digestion. Autophagy results in turnover of the cytoplasm allowing for metabolic maintenance and organelle quality control, particularly during cell stress. These aspects of autophagy can facilitate tumor cell survival and resistance. As such, autophagy inhibition is being explored in clinical trials as a novel approach to chemosensitization. However, there are still a number of unresolved concerns in regards to the use of autophagy inhibition as a therapy. It is still unclear how autophagy functions in metastasis development. Therefore, we investigated the role of autophagy in metastasis by modulating autophagy in different mouse models and cell based assays that reflect the steps of metastatic development. We found that autophagy was not required for tumor cell colonization within the site of metastasis nor did autophagy alter the metastatic capabilities of the cells. Rather, autophagy appeared to impact the pre-metastatic environment through effects on bone marrow derived cell number which mediate the establishment of the metastatic niche. Stimulating autophagy, before tumor cells disseminated, could speed metastatic development and increase the number of these cells within circulation and eventual sites of metastasis. Correspondingly, inhibiting autophagy could delay metastasis and reduce circulating bone marrow derived cells. These studies suggest that autophagy is most critical in the stages prior to tumor cell arrival at the site of metastasis, by influencing the metastatic microenvironment. While increased autophagy is often considered to be a common tumor adaptation, it is now apparent that some tumor types are more dependent on autophagy than others. However, it is not well understood which tumors these are. Triple negative, Stat3 activated breast cancers were identified as autophagy dependent by collaborator Dr. Paola Maycotte. We tested the efficacy of autophagy inhibitor chloroquine (CQ) in xenograft models of triple negative and estrogen receptor positive breast cancer. CQ was only efficacious in the triple negative tumors. As some canine osteosarcomas also have constitutive Stat3 activity, we assessed the relationship of Stat3 activity and CQ sensitivity. Unlike in breast cancer, Stat3 phosphorylation did not indicate increased sensitivity to CQ in canine osteosarcoma. However, all the osteosarcoma cell lines responded to treatment. Using microarray analysis we identified potential compensatory pathways that have been previously reported to work in concert with autophagy in other cell types and may serve as useful combinational therapies. Currently, the only autophagy inhibitor available clinically is CQ or derivative hydroxycholorquine (HCQ). It is still uncertain whether these drugs can actually achieve autophagy inhibition in patients. Dogs serve as a good model for human cancer and there is an unmet need for novel therapies in the treatment of canine lymphoma. Thus we conducted a phase I clinical trial in canine lymphoma patients with the goals of finding a maximum tolerated dose in combination with doxorubicin (DOX) and the relationship of HCQ concentration and autophagy inhibition. We found that this combination can be well tolerated with a 20% reduction in DOX. HCQ can achieve autophagy inhibition in patients, but not consistently. There appears to be a threshold requirement of HCQ needed in order to effectively inhibit autophagy. There was a suggestion of efficacy as response rate was superior to historical data employing DOX alone. Therefore autophagy inhibition warrants further clinical study as an anti-cancer therapy.Item Open Access Biomarkers of disease progression and chemotherapeutic resistance in canine osteosarcoma(Colorado State University. Libraries, 2011) O'Donoghue, Liza E., author; Duval, Dawn L., advisor; Thamm, Douglas H., committee member; Bouma, Gerrit J., committee member; Weil, Michael, committee memberOsteosarcoma is the most common primary bone malignancy in both humans and dogs. Over 10,000 canine patients develop this highly aggressive cancer annually and many succumb to metastatic disease in less than a year. In recent years, canine osteosarcoma has been increasingly recognized as an excellent model for the disease in humans, especially with regard to the molecular biology of the disease. Thus, research targeted at canine osteosarcoma benefits not only dogs but the field of human oncology as well. Research into the genetic and molecular derangements of osteosarcoma in both species has identified a number of oncogenes and tumor suppressor genes that may contribute to tumorigenesis. Additionally, some mediators of invasion and metastasis have been recognized (e.g. Ezrin, matrix metallopeptidases). Despite this, only a limited number of studies have been performed that examine the molecular genetics of osteosarcoma in the context of patient outcome. Thus, with the aim of identifying new target genes and pathways that contribute to disease progression and chemoresistance in osteosarcoma, we first performed transcriptomic and genomic analyses of primary tumors from dogs that had experienced good or poor outcomes following definitive treatment for osteosarcoma. These broad survey experiments yielded a selection of targets for future investigation. To further focus in on the genes that were most deranged from "normal" expression patterns, we compared gene expression patterns from tumors to those of normal bone. This study provided valuable perspective on genes that were identified in the outcome-based experiments, allowing selection of four promising gene targets to pursue. We next set out to validate in vitro models of canine osteosarcoma so that mechanistic studies could be pursued. Assays to test species and short tandem repeat identity were adapted to cell lines in use in our facility and presumed osteosarcoma cell lines were verified to be bone-derived via PCR testing of a bone-specific marker. Additionally, four anti-human antibodies were validated for use in canine samples. Two genes whose expression progressively altered with increased tumor aggressiveness where chosen for further study: insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and n-Myc downstream regulated gene 2 (NDRG2). IGF2BP1 has been identified as an oncofetal protein and its mRNA was strongly overexpressed in patients with the worst outcome while it was virtually undetectable in normal bone. We identified one possible mechanism for dysregulation of this gene in OSA and we also discovered that knock down of this gene in a canine osteosarcoma cell line inhibited cell invasion. NDRG2 has been dubbed a tumor suppressor in a number of different tumor types yet had not been previously investigated in osteosarcoma. We found NDRG2 mRNA to be underexpressed in all tumors relative to normal bone; patients with poor outcomes had the lowest expression levels. Multiple isoforms of the gene were found to be expressed in canine samples: these were cloned and transfected into a low-NDRG2-expressing cell line. Exogenous expression of NDRG2 in this in vitro system enhanced sensitivity to doxorubicin, one of the drugs most commonly used to treat osteosarcoma. Additionally, three possible mechanisms of dysregulation of this gene were identified. The studies presented herein progress from fact-finding surveys to in-depth functional examination of two genes that likely contribute to osteosarcoma invasion and chemoresistance. Furthermore, additional genes identified in our survey experiments offer promise for future studies into molecular mechanisms of osteosarcoma metastases and chemotherapeutic resistance. Finally, these studies have laid the groundwork for the development of gene-expression-based prognostic screens for dogs with osteosarcoma.Item Open Access Epidithiodioxopiperazines: synthetic studies of (+)-chetomin and (-)-sporidesmin A(Colorado State University. Libraries, 2012) Welch, Timothy R., author; Williams, Robert M., advisor; Rovis, Tomislav, committee member; Wood, John L., committee member; Ackerson, Chris, committee member; Thamm, Douglas H., committee memberThis dissertation documents efforts toward the asymmetric total syntheses of the natural products (+)-chetomin and (-)-sporidesmin A. Synthetic methods have been developed to efficiently construct the dioxopiperazine core of both molecules. Additionally, a simple epidithiodioxopiperazine has been synthesized to demonstrate a general method for the addition of a sulfur bridge to a dioxopiperazine ring. The work described herein, while not totally successful, provides a basis for future completion of the asymmetric total syntheses of these two epidithiodioxopiperazines and other related fungal metabolites.Item Open Access Flavonoids hydroxyl group position effect on inducing ssDNA damage mediated by cupric ions(Colorado State University. Libraries, 2020) Elmegerhi, Suad Ahmed, author; Kato, Takamitsu, advisor; Bailey, Susan, committee member; Thamm, Douglas H., committee member; Boss, Mary-Keara, committee memberTo view the abstract, please see the full text of the document.Item Open Access Global analysis reveals differential regulation of mRNA decay in human induced pluripotent stem cells(Colorado State University. Libraries, 2013) Neff, Ashley T., author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., advisor; Thamm, Douglas H., committee member; Weil, Michael, committee memberInduced Pluripotent Stem (iPS) cells are able to proliferate indefinitely while maintaining the capacity for unlimited differentiation and these properties are reflected by global changes in gene expression required for reprogramming of differentiated cells. Although the rate of transcription is an important regulator of steady-state mRNA levels, mRNA decay also plays a significant role in modulating the expression of cell-specific genes. The contribution of regulated mRNA decay towards establishing and maintaining pluripotency is largely unknown. To address this, we sought to determine global mRNA decay rates in iPS cells and the genetically-matched fibroblasts (HFFs) they were derived from. Using a microarray based approach, we determined half-lives for 5,481 mRNAs in both cell lines and identified three classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs. We found that replication-dependent histone mRNAs are more abundant and more stable in iPS cells, resulting in increased histone protein abundances. This up-regulation of histone expression may facilitate the unique chromatin dynamics of pluripotent cells. A large set of C2H2 ZNF mRNAs are also stabilized in iPS cells compared to HFFs, possibly through reduced expression of miRNAs that target their coding regions. As many of these mRNAs encode transcriptional repressors, stabilization of these transcripts may support the overall increased expression of C2H2 ZNF transcription factors in early embryogenesis. Finally, we found that mRNAs containing C-rich elements in their 3'UTR are destabilized in iPS cells compared to HFFs and many of these mRNAs encode factors important for development. Interestingly, we also identified the Poly(C)-Binding Protein (PCBP) family as differentially regulated in iPS cells and investigated their possible involvement in regulation of the mRNAs in our dataset identified as destabilized in iPS cells and having C-rich 3'UTR elements. Thus, we identified several interesting classes of mRNAs whose decay is differentially regulated in iPS cells compared to HFFs and our results highlight the importance of post-transcriptional control in stem cell gene expression. Coordinated control of mRNA decay is evident in pluripotency and characterization of the mechanisms involved would further contribute to our limited understanding of pluripotent gene expression and possibly identify additional targets for reprogramming.Item Open Access Regulatory T cells in canine cancer(Colorado State University. Libraries, 2011) Burton, Jenna Hart, author; Biller, Barbara J., advisor; Avery, Anne C., committee member; Dow, Steven W., committee member; Thamm, Douglas H., committee memberNumbers of regulatory T cells (Treg) are increased in some human malignancies and are often negatively correlated with patient disease-free interval and survival. Canine Treg have previously been identified in healthy and cancer-bearing dogs and have found to be increased in the blood of dogs with some types of cancer. Moreover, some preliminary research indicates that increased numbers of Treg and decreased numbers of CD8+ T cells in the blood of dogs with osteosarcoma (OSA) are associated with decreased survival. The aim of this project was to examine Treg distributions in dogs with cancer compared to normal dogs and to determine any association between Treg numbers and patient outcome. Additionally, we sought to determine if treatment with daily low-dose (metronomic) chemotherapy would decrease Treg in dogs with cancer. Pre-treatment Treg populations in blood, tumors, and lymph nodes of dogs with OSA were assessed and compared with Treg in blood and lymph nodes of a healthy, control population of dogs. No significant changes in Treg numbers in the blood or lymph nodes of the OSA-bearing dogs compared to the control population were identified. The dogs with OSA with treated with amputation of the affected limb followed by adjunctive chemotherapy. Treg numbers in the blood or tumor were not associated with outcome but an elevated ratio of CD8+ T cells to Treg in the tumor was associated with a prolonged disease-free interval and increased survival. These findings suggest that changes in number of Treg and effector T cell subset may provide prognostic information for canine OSA. In mice and humans with advanced cancer, the administration of metronomic (daily low dose) cyclophosphamide (CYC) selectively decreases circulating Treg numbers and inhibits their function. Protocols containing metronomic CYC likely have anti-tumor properties in dogs as well. Despite wide use of metronomic CYC in veterinary medicine, its effects on canine Treg have not previously been reported. Dogs with soft tissue sarcoma (STS) were administered CYC at 12.5 mg/m2 or 15 mg/m2 orally once daily for 28 days. Whole blood samples and tumor biopsies were obtained on days 0, 14, and 28 to assess changes in T lymphocyte subsets, circulating endothelial cells, and tumor microvessel density (MVD). Administration of CYC at 12.5 mg/m2/day significantly decreased the number of Treg from day 0 to 28, but there was no change in the percentage of Treg or tumor MVD. In dogs that received CYC at 15.0 mg/m2/day, both the number and percent of Treg as well as tumor MVD were significantly decreased over 28 days. These findings suggest that metronomic dosing of CYC may have immunomodulatory and antiangiogenic effects in dogs with cancer. Taken together the work described in this thesis support the theory that Treg are altered in dogs with cancer and that these changes may have prognostic value. Additionally, Treg can be decreased in dogs with cancer though administration of metronomic doses of CYC; the therapeutic benefit of Treg depletion has yet to be evaluated in cancer bearing dogs.Item Open Access Studies on the biosynthesis of prenylated indole secondary metabolites from Aspergillus versicolor and Aspergillus sp.; and A novel approach to tumor specific drug delivery: use of a naphthyridine drug linker with a DNA hairpin(Colorado State University. Libraries, 2011) Finefield, Jennifer M., author; Williams, Robert Michael, advisor; Rovis, Tomislav, 1968-, committee member; Kennan, Alan J., committee member; Elliott, C. Michael, committee member; Thamm, Douglas H., committee memberHerein are documented our efforts in two projects, beginning with studies toward elucidating the biosynthesis of prenylated indole alkaloids from two different Aspergillus species. Marine-derived Aspergillus sp. and terrestrial-derived Aspergillus versicolor were found to produce antipodal metabolites, in which we have developed several putative biosynthetic pathways to determine the enantio-diverging point of these fungal cultures. Through the synthesis of several potential intermediates, both with and without isotopic labeling, as well as through bioinformatics analysis of both the (-)- and (+)-notoamide biosynthetic gene clusters, significant progress has been made toward identifying a single biosynthetic precursor that serves as an intermediate to the postulated enantio-diverging event, the intramolecular hetero Diels-Alder cycloaddition. In the second project discussed, through collaboration with Dr. James Berenson at the University of California, Los Angeles, we have developed a novel tumor specific drug delivery system. Two naphthyridine-drug derivatives were synthesized and conjugated to a modified DNA oligonucleotide specifically targeted for multiple myeloma cells. The oligonucleotide-drug conjugate was successfully delivered and activated specifically within RMI8226 multiple myeloma cells.Item Open Access Total synthesis of hapalindoles J and U, formal synthesis of haplaindole O, synthesis of the proposed biosynthetic precursor to hapalindole K and work towards the ambiguine family of alkaloids(Colorado State University. Libraries, 2011) Rafferty, Ryan J., author; Williams, Robert M., advisor; Shi, Yian, committee member; Crans, Debbie C., committee member; Prieto, Amy L., committee member; Thamm, Douglas H., committee memberHerein I discussed the total synthesis of hapalindoles J and U, the formal synthesis of hapalindole O, the proposed biosynthetic precursor to hapalindole K and efforts towards other hapalindole and ambiguine families of alkaloids. The hapalindoles and ambiguines both possess a highly functionalized 6:6:6:5, which I accessed over six synthetic steps via a developed silyl strategy with an overall 54% yield. Hapalindole J was synthesized in an overall 11% yield over eleven synthetic steps and hapalindole U in an overall 25% yield over thirteen synthetic steps from commercially available materials utilizing the silyl strategy developed. A formal synthesis of hapalindole O, intercepting Natsume's total synthesis, was accomplished as well via the developed silyl strategy. In addition, the synthesis of the proposed biosynthetic precursor to hapalindole K was accessed. Currently, this newly developed silyl strategy is being employed in accessing some of the more functionalized hapalindoles (such as K) as well as the complex ambiguine core.Item Open Access Towards the total synthesis of 14-acetoxygelsenicine and synthesis of largazole analogs(Colorado State University. Libraries, 2009) Newkirk, Tenaya L., author; Williams, Robert M., advisor; Stermitz, Frank R., committee member; Ferreira, Eric M., committee member; Rickey, Dawn, committee member; Thamm, Douglas H., committee memberHerein are documented our efforts in two projects, beginning with studies towards the total synthesis of 14-acetoxygelsenicine. We have developed different strategies towards this complex natural product, wherein we have developed routes towards an appropriate substrate for a novel, intramolecular hetero Diels-Alder cyclization. The developed route would also lead to related members of this family of alkaloids, and helps set the stage for future efforts. In the second project discussed, we have successfully pursued the synthesis of numerous, biologically active analogs of the natural product (+)-largazole. Synthetic efforts have led to the design of inhibitors with unprecedented biological activity, as well as providing information regarding the structure-activity relationship of these molecules.