Mountain Scholar :: Browsing by Author "Thamm, Douglas, committee member"

Browsing by Author "Thamm, Douglas, committee member"

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Open Access
Applications of digital adaptive filters to time-resolved optical microscopy
(Colorado State University. Libraries, 2020) Gupta, Saurabh, author; Wilson, Jesse W., advisor; Pezeshki, Ali, committee member; Thamm, Douglas, committee member
Phosphorescence lifetime imaging is used on several fronts, such as, skin cancer or melanoma diagnosis, and estimation of tissue oxygenation among others. Oxygen profiling is critical for mapping brain activity, apart from its use to monitor several metabolic activities, and often employs oxygen tagging molecules/probes. In this work, we describe a novel technique to recover phosphorescence lifetime using a real-time digital adaptive filter running on a field-programmable gate array (FPGA) and conclude with an important takeaway. We also describe our strategy to mitigate relative intensity noise (RIN) in ultrafast fiber lasers, which are an attractive alternative to bulk lasers for non-linear optical microscopy due to their compactness and low cost. The high RIN of these lasers poses a challenge for pump-probe measurements such as transient absorption and stimulated Raman scattering, along with modalities that provide label-free contrast from the vibrational and electronic structure of molecules. Our real-time approach for RIN suppression uses a digital adaptive noise canceller implemented on a FPGA. We demonstrate its application to transient absorption spectroscopy and microscopy and show compatibility with a commercial lock-in amplifier. Lastly, we report the noise estimates specific to our current setup.
Open Access
Characterizations of chromosome aberrations, telomere dysfunction, and radiosensitivity signatures in canine cancer cell lines
(Colorado State University. Libraries, 2015) Maeda, Junko, author; Kato, Takamitsu, advisor; Bedford, Joel, committee member; Bailey, Susan, committee member; Thamm, Douglas, committee member
To view the abstract, please see the full text of the document.
Open Access
Clinical importance of autophagy dependency and inhibition in cancer treatment
(Colorado State University. Libraries, 2021) Van Eaton, Kristen M., author; Gustafson, Daniel L., advisor; Munsky, Brian, committee member; Thamm, Douglas, committee member; Thorburn, Andrew, committee member; Yao, TingTing, committee member
Autophagy, a lysosomal degradation recycling process, has a complex and context-dependent role in cancer. Certain cancers have been found to be inherently dependent on autophagy for survival regardless of the environment. Autophagy is also implicated as a mechanism of resistance to many chemotherapies. More autophagy dependent tumors are generally more sensitive to autophagy inhibition genetically and pharmacologically. Therefore, determining what tumors are autophagy dependent is important for selecting patients that are viable candidates for autophagy inhibition. Currently, autophagy inhibition is being tested in over 90 clinical trials using FDA- approved hydroxychloroquine (HCQ) alone or in combination with other therapies. However, responses have been variable, especially in trials where HCQ is used as a monotherapy. Further, the relationship between HCQ pharmacokinetics and pharmacodynamics is not well understood in patients. Pharmacokinetics of HCQ and one of its active metabolites DHCQ was assessed in non-tumor bearing mice. Both parent and metabolite were observed at clinically relevant concentrations after 72 hr and this corresponded with evident autophagy inhibition in various tissues, although autophagy inhibition was inconsistent across the mice. The pharmacokinetic data established 60 mg/kg as the human equivalent dose observed in patients based on HCQ exposure. Cellular responses to HCQ were assessed in 2D cell culture, 3D tumor organoids, and in vivo tumor xenografts using autophagy dependent and autophagy independent tumors. Overall, cellular responses were similar across the in vitro and in vivo methods. Autophagy was inhibited regardless of autophagy status, but autophagy dependent tumors had increased cell death and decreased cell proliferation at earlier time points and lower doses of HCQ, suggesting autophagy dependency matters for optimal results. Since autophagy inhibition was inconsistent in vivo, it is still important to determine better biomarkers and possibly consider using more potent autophagy inhibitors in the clinic. Since there have not been any major advancements in osteosarcoma survival over the past four decades, autophagy dependency was assessed in osteosarcoma. Osteosarcoma was found to be intermediately to very dependent on autophagy following a genetic screen. Further, initially autophagy dependent tumor cells were able to survive and adapt to autophagy loss. Not all tumor cells adapted in the same way nor were these autophagy deficient tumor cells more sensitive to standard osteosarcoma chemotherapy, highlighting the difficulty of determining what context autophagy inhibition should be used in the clinic. Since some autophagy inhibitors like HCQ are lysosomal inhibitors and do not specifically target autophagy alone, the results of these studies also emphasized the importance of understanding whether autophagy inhibition via lysosomal degradation or autophagy inhibition of the autophagic pathway itself is superior. Overall, these results indicate targeting autophagy in osteosarcoma is a promising therapy.
Open Access
Computational approaches to predict drug response to cytotoxic chemotherapy
(Colorado State University. Libraries, 2020) Mannheimer, Joshua D., author; Gustafson, Daniel, advisor; Prasad, Ashok, advisor; Krapf, Diego, committee member; Thamm, Douglas, committee member
Cancer is the second leading cause of death in the United States. Statistically, within a lifetime there is slightly above a one-third chance of developing some form of cancer and a one in five chance of dying from the disease. Thus, it is no hyperbole that the understanding and treatment of cancer is one of the most pressing issues in medical research of the current era. Cytotoxic chemotherapies are a class of anti-cancer drugs that are widely used to treat a number of cancers. While cytotoxic chemotherapies are extremely effective in treating a subset of individuals for some cancers, drug resistance resulting in failure of treatment is a prominent obstacle in many cancer patients. Precision medicine, a novel concept to the 21st century, is the application of disease treatments that are specifically tailored to an individual and the specific attributes of their disease. In oncology, precision medicine particularly refers to the use of gene expression and other biological factors to inform an individual's treatment. Because cancer and its response to treatment result from many complex biological interactions, computational methods have become an essential tool to identify the molecular signatures that are the basis for precision treatment. In this thesis, a systematic analysis of the computational approaches is performed to gain insight necessary for the development of novel computational approaches in precision medicine in cancer. Statistical learning models are a class of computational modeling methods that identify and extrapolate complex patterns from large amounts of data. Specifically, this involves applying statistical learning approaches on in vitro data from cell lines and patient tumor data to predict drug response, particularly for cytotoxic chemotherapies, with an emphasis on understanding the fundamental modeling principles and data attributes driving model performance. The first chapter serves as an introduction to chemotherapy and the advancements that have driven computational approaches to precision applications in cancer. The second chapter serves as a technical introduction to statistical learning models and approaches. In the third chapter a systematic assessment of linear and non-linear modeling approaches are applied to in vitro cell lines panel including the National Cancer Institute's 60 cancer cell lines (NCI60) and cell lines of Genomics of Drug Sensitivity in Cancer (GDSC) to predict drug response in several cytotoxic chemotherapies. With in-depth analysis it is shown that the relationship between tumor tissue histotype and drug response is the major driver of model performance and can be maintained in as little as 250 random genes. The fourth chapter utilizes statistical models to explore the influence of drug induced gene perturbations on drug response models in comparison with basal gene expression. The findings indicate that drug induced changes in gene expression are superior predictors of drug response. Second, it is demonstrated that Boolean network representation of gene interactions show distinct topological differences between drug induced changes in gene expression and basal gene expression. Finally, in the fifth chapter, drug induced gene changes demonstrating high levels of connectivity in the previously developed networks are applied to derive a basal gene expression signature to predict response to combined gemcitabine and cisplatin chemotherapy treatment in patients with bladder cancer. These models show that this derived signature performs better than a random cohort of genes and in some situations genes derived directly from basal gene expression.
Open Access
Correlation of HAS2-associated gene duplications with biological aggressiveness of mast cell tumors in Chinese Shar-Pei dogs
(Colorado State University. Libraries, 2013) Garner, Alana Pavuk, author; Avery, Anne, advisor; Thamm, Douglas, committee member; Basaraba, Randall, committee member
Cutaneous mucinosis in Shar-Pei dogs is the result of excessive dermal hyaluronan, a protein associated with angiogenesis and tumor cell motility in multiple human and canine neoplasms. Cutaneous mucinosis in Shar-Pei has been associated with gene duplications upstream of the hyaluronic acid synthase 2 gene (HAS2). The objective of this study was to evaluate the relationship between HAS2, cutaneous mucinosis, and features of mast cell tumor (MCT) aggressiveness in Shar-Pei dogs. Biopsies of cutaneous MCTs from 149 Shar-Pei and 100 non-Shar-Pei were graded according to two schemes for canine cutaneous MCTs. Biopsies of the Shar-Pei MCTs were also evaluated for degree of cutaneous mucinosis, depth of invasion, and microvessel density (MVD). Shar-Pei and non-Shar-Pei MCTs were evaluated via qPCR for relative copy number of the gene duplication upstream of HAS2. The proportion of grade III tumors was significantly higher in Shar-Pei than the general canine population (p=1.044e-11), with no difference in average age at diagnosis. Shar-Pei biopsies had significantly higher HAS2-associated gene segment duplications than non-Shar-Pei (p=1.128e-11), and copy number was significantly associated with the development of grade III tumors (p=0.0077), mitotic index > or = 7 (p=0.022), and tumoral MVD (p<0.05). Relative copy number was not significantly associated with the degree of cutaneous mucinosis or depth of invasion. Our data suggest a relationship between HAS2 gene duplications and features of MCT aggressiveness in Shar-Pei dogs.
Open Access
Design and synthesis of biologically active Largazole derivatives, including development of improved syntheses of Largazole analogs
(Colorado State University. Libraries, 2018) Dunne, Christine E., author; Williams, Robert M., advisor; Shi, Yian, committee member; Prieto, Amy, committee member; Thamm, Douglas, committee member
Natural product histone deacetylase inhibitor, Largazole, has been developed into a streamlined synthetic pathway for the development of a complex library of analogs. The library developed within the Williams laboratory encompasses an array of derivatives, including but not limited to: thiazole modification and macrocycle substitutions. The cap group of Largazole, portion of the molecule extending outside of the enzyme binding pocket, was successfully modified to install new chemical handles for biologic and dual therapeutic conjugation. Biological conjugates of Largazole, as well as its derivatives, aid in increasing selectivity and potency of the compound. Largazole has been conjugated to both biotin and folic acid for further studies. Additionally, a streamlined synthesis towards Wnt inhibitor 3289-5066 and a developed path for conjugation have been explored. Modified procedures were developed to aid in scale up and improvement of synthetic pathways. Scale up is crucial for development of sufficient material for biological testing and further development of conjugative therapeutics. One main impediment in the synthesis of Largazole peptide isostere is towards the southern fragment, specifically the Grubbs olefin metathesis. Multiple routes were explored to combat this low yielding step. Further exploration of these synthetic routes are underway.
Open Access
Determining the cancer risks presented by space radiation: genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion and γ-ray induced tumors
(Colorado State University. Libraries, 2016) Edmondson, Elijah F., author; VandeWoude, Sue, advisor; Weil, Michael, advisor; Thamm, Douglas, committee member; Olver, Christine, committee member; Kamstock, Debra, committee member
To view the abstract, please see the full text of the document.
Open Access
Establishing canine osteosarcoma as a solid tumor model for the evaluation of B7-H3 CAR T cell therapy
(Colorado State University. Libraries, 2023) Cao, Jennifer, author; Dow, Steven, advisor; Avery, Anne, committee member; Schenkel, Alan, committee member; Thamm, Douglas, committee member
Osteosarcoma (OS) is a highly aggressive primary bone cancer that mainly affects children and young adults. OS is the third most common childhood cancer, after lymphoma and brain tumors. Major advances in the 1980's in neoadjuvant chemotherapy has increased 5-year survival rates in OS from 30% to 70%. Unfortunately, for patients that do not respond to standard therapy or that have metastatic disease the 5-year survival rate is still 20% with no major improvements in the last 4 decades. Approximately 15-20% of patients have metastatic lesions at the time of diagnosis and 25-30% of all OS patients will develop metastatic disease. For this subset of patients advances in treatment options are desperately needed. OS also occurs in high rates in large breed dogs with an estimated 10,000 cases in dogs per year in the United States compared to 1,000 cases per year in humans. The dog has been a well-established translational model for OS due to the similar clinical presentation, cell origin, histological features, and disease progression between canine and human OS. Development of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment for advanced and relapsed B-cell lymphomas and leukemias. CAR T cell targeting of B cell marker CD19 has shown up to 90% complete remission in patients with advanced B cell leukemia. However, efforts to expand CAR T cell therapy to solid tumor types have not seen the same clinical success as with blood cancers. Major barriers unique to solid tumor CAR T cell therapy are A) selection of tumor associated antigen target, B) CAR T cell trafficking to tumor sites from the circulation and C) immune suppressor cells within the tumor microenvironment (TME). To develop more effective CAR T cell therapies against solid tumor, we utilized canine OS as a translational animal model. To establish canine osteosarcoma as a platform for evaluating B7-H3 CAR T cell therapy, first we validated B7-H3 as an antigen target in canine OS. We found differential expression of B7-H3 with high levels of B7-H3 expression on OS cell lines and FFPE biopsies, whereas normal canine tissues were B7-H3 negative or low. Next, we optimized generation of canine B7-H3 CAR T cells from whole blood isolated from tumor bearing dogs to maximize both T cell expansion and CAR transduction efficiency. We also found that the addition of cytokines IL-7 and IL-15 minimize CAR T cell exhaustion due to ex vivo activation and expansion. We next determined that canine B7-H3 CAR T cells exerted antigen specific killing and cytokine activity against B7-H3+ canine OS cell lines. To address issues with CAR T cell trafficking we evaluated the addition of chemokine receptor CXCR2 to B7-H3 CAR T cells. To assess the utility of the B7-H3-CXCR2 CAR we determined that canine OS cell lines secreted high levels of ligand chemokine CXCL8 at baseline. To further evaluate functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the B7-H3-CXCR2 CAR construct had significantly greater anti-tumoral activity than the single B7-H3 CAR construct in inhibiting tumor growth and achieving complete tumor elimination. Studies were also designed to determine if modifying the TME with combination drugs losartan and propranolol improved CAR T cell activity. This is based on recent successful studies with losartan and propranolol in dogs with OS and glioma. We found that combination losartan and propranolol decreased the population of mouse CD11b+Ly6Chigh tumor associated macrophages (TAMs) to xenografted canine OS tumors. In vitro assays showed that immune suppressive macrophages enhance B7-H3-CXCR2 CAR T cell function when co-cultured, likely through CAR activation by macrophage B7-H3 expression. Collectively, the results from these studies pave the way for assessing B7-H3-CXCR2 CAR T cells in dogs with metastatic OS. Clinical outcomes in spontaneous OS in dogs will likely give more clinically relevant results serving as a platform for evaluating new CAR T cell therapies and combination therapies with TME modification, radiation, or checkpoint blockade. Success of this work can provide a new adoptive cell immunotherapy treatment option to patients both canine and human with metastatic osteosarcoma. Additionally, B7-H3-CXCR2 CAR T cells can be applied to other B7-H3 positive CXCL8 secreting tumor types.
Open Access
Feline oral squamous cell carcinoma: a comprehensive approach to improve treatment outcome
(Colorado State University. Libraries, 2013) Yoshikawa, Hiroto, author; LaRue, Susan M., advisor; Ehrhart, E. J., committee member; Randall, Elissa, committee member; Thamm, Douglas, committee member
To view the abstract, please see the full text of the document.
Open Access
Identification of therapeutic targets in canine bladder cancer: a translational model for MAPK pathway-targeted and immune-based therapies
(Colorado State University. Libraries, 2021) Cronise, Kathryn Elizabeth, author; Duval, Dawn, advisor; Gustafson, Daniel, advisor; DeLuca, Jennifer, committee member; Page, Rodney, committee member; Thamm, Douglas, committee member
Activating mutations in the proto-oncogene BRAF are drivers of oncogenesis in several human cancers, including melanoma, thyroid and colorectal carcinomas, and hairy-cell leukemia. Small molecule inhibitors targeting oncogenic BRAF demonstrate initial efficacy in approximately 50% of BRAF mutant melanoma patients; however, acquired resistance invariably develops. Other individuals, including the majority of colorectal cancer patients, exhibit intrinsic resistance to BRAF inhibitors. Combined inhibition of BRAF and its downstream target MEK improves the rate and duration of patient response, but resistance remains an issue. Thus, more effective and robust therapies are necessary. Transitional cell carcinoma (TCC) is the most common bladder cancer in dogs and humans. In this study, we provide a molecular characterization of 11 canine TCC (cTCC) tumors and identified BRAF mutations in 8 out of 11 samples. All BRAF mutations were valine-to-glutamic acid missense substitutions at amino acid residue 596 of canine BRAF (V596E), analogous to the V600E driving variant in human cancer. Additionally, 22 out of 32 formalin-fixed paraffin embedded samples expressed mutant BRAF, indicating an overall prevalence of 70%. Further analysis identified four tumors, three being BRAF mutant, that exhibited increased expression of immune gene markers and gene signatures associated with complete clinical response to checkpoint inhibition in human bladder cancer. We also found that all TCC tumors overexpress cell cycle, DNA repair, and immune-related genes. The high prevalence of BRAF mutations in cTCC makes targeting BRAF with small molecule inhibitors an attractive therapeutic option. We explored this possibility in vitro and determined that BRAF mutant cTCC cell lines are insensitive to the BRAF inhibitor vemurafenib but are sensitive to the newer, "paradox-breaking" BRAF inhibitor PLX7904. All tested cTCC cell lines were sensitive to the MEK1/2 inhibitors trametinib and selumetinib. A phenomenon observed with single-agent BRAF or MEK inhibition was the reactivation of ERK1/2 within 24 hours post-treatment, suggesting built-in mechanisms of bypassing BRAF and MEK inhibition. We also observed upregulation of genes encoding the ErbB family receptors, EGFR and ERBB2, and the EGFR ligand, EREG, in cTCC cell lines compared to other canine cancer cell lines. Treatment with the pan-ErbB inhibitor sapitinib synergized with BRAF or MEK inhibition in the BRAF mutant Bliley cell line and in the BRAF wild-type Kinsey cell line. Next, we generated trametinib-resistant clonal derivatives of the BRAF mutant Tyler1 cTCC cell line (Tyler1-TramR). Tyler1-TramR cells exhibited trametinib IC50 values over 500 nM and maintained suppression of ERK1/2 phosphorylation for 24 hours following trametinib treatment. This response, combined with the insensitivity of Tyler1-TramR cell lines to the ERK1/2 inhibitor ravoxertinib, suggests that resistance to trametinib is independent of ERK1/2 reactivation. Further analysis of two Tyler1-TramR clones using RNA-Seq identified a loss of epithelial gene markers, while mesenchymal genes and transcription factors controlling the epithelial-to-mesenchymal transition were upregulated. Analysis of basal cellular metabolism using a Seahorse XF analyzer revealed that one of the Tyler1-TramR clones exhibited altered metabolism compared to the parental Tyler1 characterized by decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and decreased glycolytic reserve. Collectively, these results demonstrate that spontaneous, BRAF mutant cTCC can be utilized as a translational model for investigating novel targeted and immune-based therapies that may improve treatment in both canine and human MAPK-driven cancers.
Open Access
In vitro and in vivo characterization of RAD51AP1 in homologous recombination DNA repair
(Colorado State University. Libraries, 2020) Pires, Elena, author; Wiese, Claudia, advisor; Argueso, Lucas, committee member; Thamm, Douglas, committee member; Yao, Tingting, committee member
Cancer embodies a large group of diseases that is responsible for illness and deaths in millions of people annually around the world. Many tumors arise due to accumulated, unrepaired damage and alterations to genes, from endogenous or exogenous sources of DNA damage. Among the DNA lesions associated with cancer, DNA double-strand breaks (DSBs) are considered the most dangerous and require coordinated and conserved machinery to prevent unfavorable consequences, such as apoptosis and cancer-causing mutations. One crucial DNA repair pathway for mending DSBs and maintaining genome integrity is homologous recombination (HR) DNA repair. This relatively error-free mechanism employs the RAD51 recombinase and involves the joining of homologous DNA strands to restore lost DNA sequence information at the damage site. RAD51-Associated Protein 1 (RAD51AP1) is a key protein that interacts with RAD51 and stimulates its activities during HR. Nonetheless, there are knowledge gaps in understanding how this HR player functions mechanistically and in vivo for protection against DNA damage. To test our overarching hypothesis that disrupted RAD51AP1 inhibits cellular and organismal protection against spontaneous or induced DNA damage, we assessed the biochemical and biological functions of RAD51AP1 through three main avenues of study: its role in the context of chromatin, the effects of its post-translational modifications in cells, and the penalties of its loss in an animal system. This dissertation describes findings from these pursuits that have not been previously characterized and offers new insights into RAD51AP1's functions in vitro and in vivo. At the start of this dissertation, our first objective was to define key attributes of RAD51AP1 in the HR reaction by further characterizing the DNA binding properties of recombinant human RAD51AP1. Using the electrophoretic mobility shift assay, we found that RAD51AP1 avidly associates with both naked and chromatinized double-stranded (ds)DNA. Deletional and mutational analyses were used to further define the chromatin-binding region in RAD51AP1, which occurs within its C-terminal DNA binding domain. Two post-translational modification (PTM) sites, which undergo phosphorylation at S277 and S282 (in isoform 2) and lie within its C-terminal DNA binding region, were also evaluated and showed decreased affinity to chromatinized dsDNA. These results unveil a novel RAD51AP1 interaction with chromatin DNA. Next, we further assessed these PTMs in regard to their impacts on RAD51AP1 function and HR capability in cells facing spontaneous or induced DNA damage. Using RAD51AP1 KO cells expressing phosphorylation mimic (S2D) or non-phosphorylatable (S2A) mutants, we found that S2D expressing cells behaved similarly to wild-type expressing cells. Notably, S2A expressing cells were significantly compromised in their growth, cell survival, cell cycle progression, and HR kinetics. The results of these studies provide an important role for PTMs that affect RAD51AP1's functions during HR. To examine the role of RAD51AP1 in providing protection against DNA damage in an animal system, we utilized a recently available mouse knockout model to evaluate the impacts of Rad51ap1 deletion from spontaneous DNA damage. Given the role of RAD51AP1 in meiotic HR and its high expression in murine testes, we specifically monitored fertility ratios, spermatogenesis in testes cross sections, and meiosis via synaptonemal complex formation. We found that Rad51ap1 heterozygous mice do not breed in a Mendelian pattern. Furthermore, while synaptonemal complex formation was not impaired in Rad51ap1 KO mice, advanced stages of spermatogenesis were impacted, suggestive of a biological role for RAD51AP1 in maintaining the fidelity of this process. Collectively, the results of these studies characterizing the in vitro and in vivo roles of RAD51AP1 provide new insights into this important HR player. For the first time, we reveal a new association between RAD51AP1 and chromatinized dsDNA and propose a model integrating this interaction within the HR reaction, when homology search and hetero-duplex formation after presynaptic filament formation occurs. Additionally, previously uncharacterized PTMs were assessed functionally in cells, and we unveil that the lack of these PTMs negatively impacts cells against spontaneous and induced DNA damage. Lastly, our studies on the biological effects of Rad51ap1 loss in a recently available Rad51ap1 KO mouse describe a novel role of Rad51ap1/RAD51AP1 during late spermatogenesis in an animal system for the first time. Ultimately, by understanding the mechanisms and biology of this important HR protein, this knowledge can guide the optimization of treatments for cancers that exploit DNA repair factors as well as help us comprehend how this factor protects against DNA damage in mammals.
Open Access
Investigation of clinical gastrointestinal toxicity and underlying normal tissue damage associated with concurrent abdominal radiation therapy and tyrosine kinase inhibition
(Colorado State University. Libraries, 2023) Prebble, Amber R., author; Boss, Mary-Keara, advisor; LaRue, Susan, committee member; Leary, Del, committee member; Thamm, Douglas, committee member
Tyrosine kinase inhibitors (TKIs) may be combined with radiation therapy (RT) to enhance tumor control due to their anticancer and antiangiogenic effects; however, clinical evidence has emerged which suggests the treatment combination of RT and TKI may result in higher incidence of normal tissue side effects, dependent on the organs at risk in the radiation treatment field, than would be expected for either modality alone. We evaluated the incidence of gastrointestinal (GI) toxicity in canine cancer patients receiving concurrent hypofractionated abdominal RT and the TKI toceranib and compared to those receiving abdominal RT alone, toceranib alone, or concurrent non-abdominal RT and toceranib. Medical records of canine cancer patients were retrospectively reviewed and identified dogs were included in the following treatment categories: dogs which received RT to a portion of the abdomen and concurrent TOC (n = 19), abdominal RT alone (n = 29), TOC alone (n = 20), or non-abdominal RT plus TOC (n = 9). Toxicities were graded using the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events criteria and compared to published data on TOC-associated GI toxicity. Patients receiving TOC while undergoing abdominal RT had significantly increased rates of any grade of diarrhea (p = 0.002), hyporexia (p = 0.0045), and vomiting (p = 0.003), as well as severe hyporexia (p = 0.003) when compared across the treatment groups. This retrospective study revealed significantly increased incidences of GI toxicity when abdominal RT was combined with TOC in canine patients. Following these findings, we investigated the morbidity and underlying histological changes associated with combined abdominal RT and the TKI sunitinib in a mouse model. Prior to the experimental study, we identified a dose of abdominal RT in CD1 outbred mice which would induce mild GI toxicity according to weight loss and histologic changes in GI tissues harvested 7 days after irradiation; 12 gray (Gy) was selected as the optimal dose for the subsequent experiment. Twenty-five mice were then assigned to control (n = 5), sunitinib alone (n = 7), RT alone (n = 6), or RT + sunitinib (n = 7) groups and were weighed daily. All mice received daily oral gavage of vehicle or sunitinib (40 mg/kg) in vehicle for the entire study. On day 7 mice received 12 Gy abdominal RT or sham irradiation. On day 14 mice were euthanized and their entire GI tract was harvested for histopathologic evaluation, semiquantitative scoring of inflammation, and immunohistochemical quantification of cells positive for CD31 (vascularity) and Ki67 (proliferation). Major findings of this study included that mice in the combined therapy group, RT + sunitinib, lost significantly more weight than sunitinib alone (p < 0.0001) or RT alone (p = 0.0258). Mice in the RT alone group had a significant increase in GI vascular density, as determined by CD31, when compared to the SUN group (p = 0.0252). The mice in the RT + sunitinib group did not mount the same GI vascular response as the RT treated mice. The RT + sunitinib group had more crypt abscessation when compared to groups not receiving RT (vs. Control, p = 0.0076; vs. sunitinib alone, p = 0.0023). And, while it did not reach statistical significance when compared to the RT alone group, the RT + sunitinib group had more abscessation than RT alone (p = 0.0862) which could indicate a trend of higher levels of crypt abscessation with this combined treatment modality. The results from our canine retrospective clinical study and the preclinical mouse model experiment suggest that abdominal RT + TKI increases morbidity and GI toxicity at the RT and TKI doses investigated. Continued investigation of the underlying normal tissue effects associated with concurrent TKI and abdominal RT are recommended in order to determine whether combining these therapies could be optimized for safety and efficacy, such that GI toxicity is minimalized while achieving optimal tumor control.
Open Access
Markers and mechanisms of resistance to Toceranib phosphate (Palladia®) in canine cutaneous mast cell tumor
(Colorado State University. Libraries, 2014) Halsey, Charles H. C., author; Gustafson, Daniel, advisor; Thamm, Douglas, committee member; Ehrhart, EJ, committee member; Worley, Deanna, committee member
To view the abstract, please see the full text of the document.
Open Access
Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length and mRNA export
(Colorado State University. Libraries, 2011) Sagawa, Fumihiko, author; Wilusz, Jeffrey, advisor; Wilusz, Carol J., advisor; Reddy, Anireddy S. N., committee member; Thamm, Douglas, committee member
During polyadenylation the multi-functional protein nucleophosmin is deposited onto all cellular mRNAs analyzed. Premature termination of poly(A) tail synthesis using cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for nucleophosmin binding. Nucleophosmin appears to be a bona fide member of the complex involved in 3' end processing as it is directly associated with the AAUAAA-binding CPSF-160 protein and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of nucleophosmin results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knock down of nucleophosmin results in retention of poly(A)+ RNAs in the cell nucleus, indicating that nucleophosmin binding influences mRNA export. Collectively these data suggest that nucleophosmin plays an important role in poly(A) tail length determination and helps network 3' end processing with other aspects of nuclear mRNA maturation.
Open Access
Pharmacological characterization of losartan as a CCR2 antagonist and pre-clinical and pharmacodynamic assessment as a potential anti-metastatic therapy
(Colorado State University. Libraries, 2017) Regan, Daniel P., author; Dow, Steven, advisor; Schenkel, Alan, committee member; Thamm, Douglas, committee member; Slansky, Jill, committee member; Basaraba, Randall, committee member
To view the abstract, please see the full text of the document.
Open Access
Progress towards the enhanced synthesis of FK228 and analogs; and the total synthesis of Largazole-Azumamide hybrid
(Colorado State University. Libraries, 2009) Troutman, Ann E., author; Williams, Robert M., advisor; Wood, John, committee member; Thamm, Douglas, committee member
Progress towards an improved synthesis of HDAC inhibitor FK228 and work towards the completion of FK228 and Largazole Azumamide hybrids are presented. Through investigation of previous syntheses of FK228, a synthesis aimed at improving the overall yield and efficiency was planned out. After overcoming initial synthetic hurtles, a synthesis was attempted with moderate success. Further optimization would be needed for completion. Interest in creating Class specific HDAC inhibitors has gained popularity due to results of recent studies. The potent biological activity of FK228 and Largazole made them ideal candidates for modification. We were interested in developing these analogs by attaching side chains of other known HDAC inhibitors onto each macrocyle core, and in doing so we hoped to increase the reactivity of each molecule. Synthesis of the FK228-Azumamide hybrid began well, but hit several synthetic obstacles that have yet to be overcome. In contrast, the synthesis of Largazole- Azumamide hybrid proceeded smoothly, and was completed in thirteen steps and 11% yield. We are currently waiting for results of the biological activity studies.
Open Access
Repeated sequences encoding Cys2His2 zinc finger motifs influence mRNA polyadenylation and localization
(Colorado State University. Libraries, 2017) Jalkanen, Aimee L., author; Wilusz, Carol, advisor; Wilusz, Jeffrey, advisor; Bailey, Susan, committee member; Bouma, Gerrit, committee member; Thamm, Douglas, committee member
The Cysteine2 Histidine2 zinc finger (C2H2-ZNF) proteins are a vast family with over 700 members in primates, many of which are transcription factors with important roles in development, differentiation, cell cycle progression, and tumor suppression. Due to the sheer number of C2H2-ZNF proteins and their roles in modulating expression of other genes, any mechanism for coordinating their expression could have wide-ranging impacts on cell function and phenotype. Previously, a large subset of C2H2-ZNF transcripts were determined to have significant populations with short poly(A) tails. Here, we show that multiple C2H2-ZNF mRNAs accumulate with very short or undetectable poly(A) tails, even when newly transcribed. Furthermore, these C2H2-ZNF mRNAs are restricted to the nucleus. Reporter mRNAs with sequences from the ZNF12 open reading frame (ORF) and/or the 3' untranslated region (3' UTR) have short poly(A) tails and are retained in the nucleus. Deletion analysis suggests that repeated sequence elements in the ZNF12 mRNA that code for zinc finger protein motifs are important in controlling both poly(A) tail length and nuclear localization. Remnants of C2H2-ZNF motif sequences found in the ZNF12 3' UTR are also able to confer short poly(A) tails and nuclear retention. Finally, we use RNA-fluorescence in situ hybridization (RNA-FISH) to reveal that ZNF12 reporter transcripts are found in foci within the nucleus that could represent sites for storage or processing. Overall, our findings suggest repeated sequence elements encoding C2H2-ZNF protein motifs play a dual role as regulatory elements that may coordinate expression of the C2H2-ZNF protein family by controlling post-transcriptional events.
Open Access
Telomere length, telomerase activity, and structural variants as biomarkers of extreme environments
(Colorado State University. Libraries, 2017) McKenna, Miles J., author; Bailey, Susan M., advisor; Dow, Steven, committee member; Kato, Takamitsu, committee member; Simon, Steven, committee member; Thamm, Douglas, committee member
Mammals, and in particular humans, are masterful at overcoming and adapting to extreme environments. Whether astronauts in low earth orbit aboard the International Space Station (ISS) or U.S. military veterans exposed to nuclear fallout from atomic weapons testing, humans can persist through a wide range of physical, psychological, and environmental stressors. The overall goal of the studies presented here was to evaluate the biological influences of extreme environments not commonly experienced by the general population. Whether spaceflight or exposure to nuclear fallout, results improve our understanding of short- and long-term effects of low gravity environments, exposure to ionizing radiation (IR) of mixed qualities, as well as low dose and low dose effects of IR. We explored these scenarios by evaluating biomarkers of stress, specifically telomere length dynamics, and biomarkers of DNA damage, specifically transmissible structural rearrangements. Telomeres are not only regarded as valuable biomarkers of aging and age-related degenerative pathologies like cardiovascular disease and cancer, and so are reflective of overall health status, they also serve as "hallmarks" of radiosensitivity. Stable chromosomal structural rearrangements (translocations and inversions) persist with time and so provide informative signatures of IR exposure as well. During the 1950's United States military personnel and weathermen, collectively known as the atomic veterans, were unintentionally exposed to nuclear fallout during atomic bomb testing following WWII. Here, directional Genomic Hybridization (dGH) for high-resolution detection of IR-induced chromosomal inversions and translocations was assessed as a more sensitive, quantitative retrospective biodosimetry approach. The influence of IR exposure on telomere length dynamics was also evaluated to determine the long-term influence of such exposures. Our results illustrate that even for nuclear events that occurred six decades in the past, evidence of exposure is still present. We find that although translocations and inversions are reliable biodosimeters independently, a combined approach provides a more sensitive measurement of past radiation exposure. We also report, for the first time, the influence of age and smoking on background inversion frequencies. Furthermore, telomere length was inversely related to IR dose, suggesting that a single acute exposure to nuclear fallout may lead to persistent long-term effects on overall health. Telomere length dynamics and structural rearrangements were also monitored longitudinally in monozygotic twin and unrelated astronauts. NASA astronauts are a unique group of individuals who experience an extreme environment that the human body is not adapted for. Little is known about the biological health effects of a low gravity environment with increased IR exposure including galactic cosmic rays (GCRs), solar particle events, and secondary neutrons. Not only do astronauts have shorter telomeres than age-/gender-matched controls at baseline (pre-flight), but a transient increase in telomere length during space flight was also observed. Results suggest this unexpected finding may be due to an upregulation of telomerase, the enzyme responsible for maintaining telomere length. Moreover, telomerase activity also increased post-flight in both twins, Scott and Mark Kelly. Although not spaceflight specific, this is the first report of telomerase upregulation in humans due to a psychologically traumatic event. A dose dependent increase in inversions, and to a lesser extent, translocations, as a consequence of IR exposure on the ISS was also seen in Scott Kelly. Collectively, the studies presented here demonstrate a profound influence of extreme environments, particularly those involving low dose IR, on human biological responses. Telomere length dynamics and chromosome aberration frequencies (e.g. translocations and inversions) provide insight into the long-term health effects and implications of spaceflight and exposure to nuclear events.
Open Access
Two model systems for studying the effects of acute radiation exposure on gene deletions and amplifications
(Colorado State University. Libraries, 2014) Sharif, Rabab S., author; Weil, Michael, advisor; Argueso, Juan Lucas, advisor; Thamm, Douglas, committee member
Ionizing radiation (IR) poses a severe threat to genome integrity, and is an important source of environmental damage, arising from naturally occurring sources (e.g. radon and cosmic radiation) and medical imaging and therapy. Radiation exposure can lead to somatic changes in chromosomal structure such as copy number alterations (CNAs) resulting in gain or loss in copies of sections of DNA. To study copy number alterations in the human genome resulting from gamma radiation, early passage cultures of normal human fibroblasts were exposed to a single acute 4 Gy dose of radiation. Irradiated cells were kept for 48 h to allow repair of initial DNA damage. Single cell cloning was done by serial dilution in 96 well plates. Standard PCR was performed using seven sequence tagged site (STS) markers (SY 83, SY86, SY88, SY1190, SY1191, SY1201, and SY1206) of the azoospermia (AZF) region in the Y chromosome to test for microdeletions, in irradiated and non-irradiated cells. The comprehensive analysis of the molecular mechanism of copy number changes, requires a more elaborate experimental system in a model organism. Hence, we also investigated copy number alterations in diploid budding yeast cells after exposing them to two acute gamma radiation doses and detecting CNAs via a unique selection system, that involves events at two chromosomes. The copy number selective system used in our yeast samples allowed us to select for copy number alterations (duplications and deletions) in all samples after exposure to radiation, which lead to nonreciprocal translocation events formed by nonallelic homologous recombination (NAHR) mechanism. These results lead us to conclude that acute exposures to gamma radiation, induced deletions and amplifications as shown in both models. The experiments described in the thesis provide a platform for future work aimed at investigating the role low dose ionizing radiation on genome stability.
Open Access
Use of liposomal bisphosphonates to deplete macrophages for cancer immunotherapy
(Colorado State University. Libraries, 2011) Hafeman, Scott D., author; Dow, Steven, advisor; Gustafson, Daniel, committee member; Thamm, Douglas, committee member; Zabel, Mark, committee member
In order for continued growth, metastasis and evasion from immune surveillance, tumor cells are dependent on a complex matrix of supportive cells and tissues. These cells make up a significant percentage of the tumor mass and contribute to the hallmarks of malignancy. Of these, the tumor associated macrophage (TAM) has perhaps the most diverse role. In the majority of tumor types studied, increased percentages of these cells in the tumor correspond to a poorer prognosis for the patient. Macrophages are critical in wound healing, and as such provide a wide variety of factors that may be co-opted by the tumor to support its continued growth and metastasis. Macrophages are capable of producing a wide variety of growth factors that directly promote tumor cell growth. These factors can also be used to induce tumor cell migration and invasion, which are critical steps in metastasis. They also produce matrix metalloproteinases that actively degrade basement membranes, further aiding in invasion and metastasis. Macrophages also produce many factors that help induce angiogenesis, providing vital blood supply to the developing tumor. Through both direct and indirect mechanisms they are vital to providing new tumor blood vessels. In addition to these direct tumor aiding effects, macrophages also play a critical role aiding in both local and global immunosuppresion in tumor patients, which allows the established tumor to continue to evade the immune system. Therefore, the targeting and killing of TAMs could potentially be a promising new adjunct to traditional cancer therapies, and may increase the efficacy of traditional therapeutics. One potential drug for this purpose is liposomal clodronate. This drug is produced by encapsulating the bisphosphonate drug clodronate in a liposome. As a free drug, clodronate is very effective at inducing apoptosis of osteoclasts, a close relative to the macrophage. Encapsulation in a lipid bilayer prevents the dissemination of the drug to the bone matrix and instead allows for systemic distribution. However, only cells that phagocytize and degrade the lipsome are susceptible to killing by the enclosed clodronate. Liposomal clodronate has been used extensively to deplete macrophages in studies of autoimmune disease and more recently in tumor models. However the systemic depletion of tumor associated macrophages using liposomal clodronate (LC) has not been previously evaluated in clinical trials, and the effects of systemic LC administration on tumor growth have not been fully elucidated. Studies presented here sought to further determine the role of tumor associated macrophages in tumor growth by studying the effects of their depletion. Specifically, in vitro studies were used to determine an optimal formulation of liposome to more effectively deliver the bisphosphonate drug to macrophages. Using multiple murine macrophage cell lines and proliferation assays the most effective depleting liposome was determined. This formulation consisted of a net neutral charged phosphatidylcholine head group combined with an incorporated mannose group. These liposomes were then evaluated in vivo for their ability to deplete macrophages systemically. Once again, the modified liposome formulation was most effective. The drug was then evaluated for its ability to decrease tumor growth in a mouse fibrosarcoma model, using MCA 205 tumors subcutaneously implanted into C57BL/6 mice. The drug's ability to deplete tumor associated macrophages was also evaluated. Tumor growth rates and tumor associated macrophage numbers were significantly decreased in mice treated with liposomal clodronate as compared to untreated mice or those treated with liposomal PBS. Additional studies were undertaken to determine if liposomal clodronate could be used as an effective cancer therapeutic in a spontaneous tumor model. The tumor evaluated was malignant histiocytosis (MH). This tumor was chosen as it is a tumor derived from macrophages or dendritic cells, and LC could potentially have both primary anti-tumor effects as well as efficacy due to depletion of TAMs. In vitro studies were undertaken which showed that LC was capable of effectively killing MH cells. Based on these results, a clinical trial was conducted for dogs with MH. Dogs were treated with 0.5 mL/kg of liposomal clodronate IV every other week for six treatments. A total of 12 dogs were treated in the study. Treated dogs were evaluated for tumor response, changes in circulating blood cells, and changes in circulating cytokines. We were able to observe a 40% biologic response rate (BRR). The development of a fever was positively correlated with response. Responding dogs also had an increase in neutrophils and a decrease in monocytes while non-responding dogs did not. A significant reduction in serum Il-8 levels occurred post LC treatment. As the clinical availability of LC is currently limited to experimental use additional studies were conducted to determine if combining free bisposphonates, which are readily available, with traditional chemotherapeutics could cause synergistic killing of MH cells in vitro. The combination of clodronate with vincristine or zoledronate with doxorubicin demonstrated synergistic killing in vitro. Further evaluation of these combinations will be necessary to determine if they have a similar effect in vivo.