Browsing by Author "Argueso, Lucas, committee member"
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Item Open Access Characterization the role of telomeric RNA (TERRA) in telomeric DNA double-strand break (DSB) repair in human ALT (telomerase independent) cells(Colorado State University. Libraries, 2019) Alturki, Taghreed Mohammed, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Wiese, Claudia, committee member; Yao, Tingting, committee memberTelomeres are specialized nucleoprotein complexes that protect natural chromosomal termini from degradation and prevent their detection as of DNA damage. Therefore, telomeres play critical roles in maintaining genomic stability. Telomeres are composed of tandem arrays of conserved repetitive sequences (TTAGGG in vertebrates), bound by a suite of proteins collectively termed "shelterin". Shelterin proteins are essential for telomere length regulation and end-capping structure/function. Due to their repetitive nature and together with telomeres possessing an abundance of heterochromatic marks, telomeres have long been regarded as silenced, non-transcribed features of the genome. The relatively recent discovery of telomeric RNA (TElomere Repeat- containing RNA; TERRA) opened many new avenues of investigation. TERRA is a long, noncoding RNA (lncRNA) that serves a structural role at telomeres, as well as function in regulation of telomere length and telomerase activity, the specialized reverse transcriptase capable of elongating telomeres de novo. Further, TERRA participates in telomeric recombination in tumors that maintain telomere length in a telomerase independent fashion via the Alternative Lengthening of Telomeres (ALT) pathway. Emerging evidence also supports telomeric "DNA- TERRA hybrids" as indispensable for end protection and capping function; e.g., RNA interference mediated depletion of TERRA induced telomeric DNA damage responses (DDRs) and aberrations. Thus, TERRA participates in facilitating telomeric recombination and in preventing inappropriate telomeric DNA damage responses. We hypothesized that TERRA plays a critical role in the repair of telomeric DNA damage. To address the intriguing possibility that TERRA plays a role in the telomeric DNA damage response, we evaluated the colocalization of TERRA and γ-H2AX, a well-accepted marker of double-strand breaks (DSBs), at broken telomeres in human Osteosarcoma U2OS-ALT cells in different phases of the cell cycle. To test our hypothesis, we generated U2OS-ALT cells that stably expressed FUCCI green fluorescent signals to label cells in G2 phase. Telomeric DSBs were then induced in FUCCI-U2OS cells utilizing the ENT endonuclease fused to Telomere Repeat Factor 1 TRF1 (ENT-TRF1), and validated via colocalization of telomeres with γ-H2AX-FLAG. Forty-eight hours following transfection, FUCCI-U2OS cells were also treated with EdU to label cells in S phase. Cells negative for both FUCCI and EdU identified cells in G1 phase. Using this powerful strategy to distinguish cells in G1, S and G2 phases of the cell cycle, we showed that FUCCI-U2OS cells accumulate in G2 phase following transient transfection with ENT-TRF1. We validated that expression of ENT-TRF1 generates telomeric DSBs in U2OS-ALT cells through detection of telomere- γ-H2AX-FLAG colocalization events. Importantly, our data revealed that telomeric DSB induction triggers enrichment of TERRA in G2 phase. Taken together, these observations suggested that TERRA is increased in cells transfected with ENT-TRF1; i.e., in U2OS cells harboring telomere-specific DSBs. TERRA recruitment to telomeric DSB damage sites in G2 was validated by assessing co-localization between TERRA and ENT (FLAG). Similarly, TERRA recruitment to telomeric DSBs in G1/S was also evaluated. Futhermore, non-denaturing Telomere DNA FISH was employed to visualize G-rich and C-rich single-stranded (ss)telomeric DNA. Treatment of U2OS ENT transfected cells with Rnase A and Rnase H to remove TERRA, uncovered elevated levels of resected 5' C-rich (ss)telomeric DNA (complementary TERRA sequence), suggesting a potential role for TERRA in protecting resected telomeric DNA prior to cells entering G2 phase where Homologous Recombination (HR)-mediated elongation/repair would be possible. Consistent with published reports, telomeric DSBs were also significantly induced in cells transfected with TRF1-only (positive control). However, although TERRA co-localized with FLAG/broken telomeres, resected (ss)telomeric DNA was not detected upon removal of TERRA. Therefore, our results further support induction of telomeric DSBs with overexpression of TRF1, and additionally indicate that they are repaired via a different pathway than those induced by ENT, potentially alternative End Joining (alt-EJ), as previously proposed. In conclusion, our work revealed for the first time that the telomeric RNA TERRA, is enriched specifically at telomeric DNA DSB sites in U2OS (ALT) cells.Item Open Access Elucidating the role of iron in the pathogenesis of idiopathic osteoarthritis in the Dunkin-Hartley animal model(Colorado State University. Libraries, 2021) Burton, Lindsey Hammond, author; Tjalkens, Ronald, advisor; Santangelo, Kelly, advisor; Legare, Marie, committee member; Goodrich, Laurie, committee member; Argueso, Lucas, committee memberOsteoarthritis (OA) is the most prevalent musculoskeletal disorder, affecting millions of individuals worldwide. While OA is characterized by the progressive loss of articular cartilage, it is now widely accepted to be a whole joint disorder, with changes such as synovial hyperplasia, subchondral bone remodeling, and osteophyte formation accompanying cartilage degeneration. The knee is one of the joints most affected by OA. Patients with knee OA exhibit painful and/or limited mobility as a consequence of the disorder, resulting in an increased risk of comorbidities such as heart disease, obesity, diabetes, and depression. Unfortunately, the mechanisms driving OA pathogenesis remain poorly understood, and there are no effective therapies available for treating the disorder. Therefore, there is a need to understand factors contributing to OA to identify potential targets for combating the condition. Iron is the most abundant mineral in the human body and is essential for conducting numerous physiologic processes. However, unbound or partially-liganded iron can participate in redox reactions that produce reactive oxygen species and free radicals capable of inciting tissue damage. As such, iron needs to be tightly managed within the body. Mammals do not possess a regulated mechanism for excreting iron, and iron progressively accumulates within tissues throughout the aging process. Primary/idiopathic OA does not have any known, identifiable cause for disease development, but the largest risk factor associated with the disorder is advancing age. To address this gap in knowledge, we designed a series of experiments to elucidate the contributions of iron to the pathogenesis of idiopathic OA. The main animal model used for this work is the Dunkin-Hartley guinea pig, which spontaneously develops age-related OA with a histopathology similar to that observed in humans. In the first study, we quantified tissue iron levels at different ages in OA-prone Dunkin-Hartley guinea pigs relative to an outbred, control strain not used in OA research. While the control strain accumulated iron in the liver with age, but not within cartilage, the Hartleys demonstrated a significant increase in cartilage iron concentration at 7-8 months-of-age. This increase in cartilage iron concentration was more significant in males, though it was also observed in females. As this timepoint corresponds to a moderate stage of disease progression, this finding suggests that iron may play a role in OA development and/or progression in Hartley guinea pigs. This concept was supported by gene expression analysis of iron-related genes. Notably, both male and female Dunkin-Hartley guinea pigs had decreased transcript expression of ferritin heavy chain and ferroportin at 7-8 months, which may contribute to cartilage iron accumulation at this age by inappropriately storing iron in chondrocytes. Because of this intriguing association, we wanted to investigate the gene expression changes occurring with systemic iron manipulation in knee joint tissues. Exogenous iron overload resulted in worsening of OA pathology in the disease-resistant Strain 13 guinea pig. The systemic administration of iron dextran caused iron to accumulate within articular cartilage from a diarthrodial joint environment and was accompanied by gene expression changes within knee tissues. Notably, systemic iron overload altered the expression of several iron-related genes in this control strain, indicating that both the cartilage and a large adipose depot, the infrapatellar fat pad, were able to detect and respond to changes in tissue iron levels in the presence of joint pathology. Conversely, systemic iron deficiency, achieved by supplying an iron deficient diet, decreased cartilage lesions within OA-prone male Hartley guinea pigs. In this proof-of-principle study, the reduction in cartilage iron concentration was accompanied by the altered expression of two iron transport genes, the importer transferrin receptor 1 and the cellular iron exporter ferroportin. As iron deficiency is not a recommended pursuit, we investigated the effects of systemic iron reduction, without clinical iron deficiency or anemia, on OA pathogenesis. The commercially available pharmacologic iron chelator deferoxamine (DFO) was used to reduce total iron levels in the body of male and female Dunkin-Hartley guinea pigs. In males, administration of DFO was successful at reducing tissue iron levels both systemically and in a diarthrodial joint environment, and this was accompanied by a significant decrease in the severity of cartilage lesions. The reduction in joint pathology observed with treatment was largely attributed to a decrease in chondrocyte cell death; this finding was supported by the decreased expression of several proapoptotic genes within knee articular cartilage. Conversely, tissue iron levels were not altered by administration of the same dose of DFO in females, suggesting the presence of sex differences in systemic iron homeostasis. There was a relative reduction in histologic OA score in treated female animals, which may be due to the beneficial mobilization of iron by DFO that was also noted in males. The modest reduction in female joint pathology with treatment was largely driven by decreased tidemark advancement. Tidemark replication is associated with articular cartilage mineralization and was almost completely absent in all males evaluated, implying there may also be differences in OA pathogenesis between male and female Dunkin-Hartley guinea pigs.Item Open Access Functional redundancy between the RAD51 accessory proteins RAD51AP1 and RAD54 in homologous recombination DNA repair(Colorado State University. Libraries, 2021) Selemenakis, Platon, author; Wiese, Claudia, advisor; Argueso, Lucas, committee member; Kato, Takamitsu, committee member; Kim, Seonil, committee memberCancer is responsible for the death of millions of people annually. Factors that increase the risk of tumorigenesis are endogenous challenges and exogenous compounds. These insults are responsible for the generation of DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different DNA repair pathways, among which homologous recombination (HR) is the least error prone. In HR, DNA strand exchange is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While the pre-synaptic steps of HR in human cells have been studied extensively, there are still extensive knowledge gaps with respect to the molecular mechanisms of synapsis and post-synapsis and the roles of RAD51AP1 and RAD54 in these later steps of HR. Here, I hypothesized that RAD51AP1 and RAD54 may exhibit functional redundancy in human cells. Also, I speculated that Rad51ap1 disruption in mice would be associated with an increased susceptibility of these mice to radiation carcinogenesis. Finally, I hypothesized that post-translational modification and, more specifically, phosphorylation may regulate the activity of human RAD51AP1. To test for functional redundancy between RAD51AP1 and RAD54, we investigated the impact of simultaneous RAD51AP1 and RAD54 disruption in human cancer cell lines and in response to DNA-damaging agents in cell survival and DNA replication assays. We found that cells lacking both RAD51AP1 and RAD54 (i.e., double KO cells) are more sensitive to the cytotoxic effects of mitomycin C (MMC) or olaparib exposure than cells lacking either RAD51AP1 or RAD54. Accordingly, double KO cells exhibit a more pronounced G2/M arrest, higher levels of chromosomal aberrations and increased sensitivity to DNA replication stress as determined by DNA combing experiments. These results show that RAD51AP1 and RAD54 can compensate for each other in human cancer cell lines. To investigate the consequences of RAD51AP1 loss in mice we utilized a novel mouse model that lacks Rad51ap1 and determined the susceptibility of these mice to radiation carcinogenesis. We found that compared to wild type mice, loss of Rad51ap1 does not affect the survival of mice after whole body IR. We speculate that functional redundancy between RAD51AP1 and RAD54 may also exist in mice, and that Rad51ap1-/- Rad54-/- double KO mice may exhibit pronounced susceptibility to radiation carcinogenesis. Finally, we sought to characterize the regulation of RAD51AP1 activity by post-translational modification. To achieve this objective, we identified two critical residues in RAD51AP1 that appear to be regulated by phosphorylation, S277 and S282. We found that mutation of these residues to the non-phosphorylatable S277A and S282A compromises RAD51AP1 function as measured by DNA replication and cell survival assays. These results suggest that phosphorylation of S277 and/or S282 is crucial for RAD51AP1 function. Collectively, our studies clarify one aspect of functional redundancy within the HR pathway, and the role of post-translational modification of RAD51AP1. Our results provide new insights on the mild phenotypes associated with RAD51AP1 or RAD54 deficiency in human cells and mice. Our findings highlight the importance of development of personalized approaches for cancer treatment.Item 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 memberCancer 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.Item Embargo Of microbes and mothers: evaluating the complex maternal-neonatal interaction and microbiome-immunity development with novel Lactobacillus vaccination(Colorado State University. Libraries, 2024) Ecton, Kayl E., author; Abdo, Zaid, advisor; Dean, Gregg, advisor; Wrighton, Kelly, committee member; Vilander, Allison, committee member; Argueso, Lucas, committee memberThe task of identifying an optimal vaccination strategy for neonates has been challenging scientists and physicians alike. Multiple factors contribute to the difficulty in establishing an optimal platform including the complexity of the maternal-fetal dyad, a neonatal Th2 skewed profile and the role of the parallel development of the immune system and the gut microbiome (8). Disease remains a main cause of infant morbidity and mortality, encouraging the discovery of novel infant vaccinations to be delivered during the first 28 days of life to provide protection (41). Passive protection from the maternal transfer of transplacental IgG and both IgG and IgA in breastmilk has a limited window of operation, leaving the maturing neonate at risk (128). Although exact mechanisms remain to be elucidated, here we examine the complex crosstalk between mother-fetus and maternal-neonate dyads, neonatal microbiome-immunity development, and optimal delivery strategies for neonatal vaccine development. In this dissertation we investigated the role of maternal infection prior to gestation, neonatal challenge after vaccination, and vaccine effectiveness after exposure to virus. We evaluated the use of a novel vaccine platform developed previously in the lab as an orally delivered mucosal targeting subunit vaccine in Lactobacillus acidophilus. We investigated the effectiveness of the recombinant vaccine with and without adjuvants in a neonatal experimental design model and discovered increased virus specific responses in neonates vaccinated with adjuvants when challenged with rotavirus. We show a significant impact of maternal influence on neonatal outcomes. Beyond the immunogenic strength of the novel Lactobacillus acidophilus vaccine platform in neonates, we identified induced shifts to the gut microbial communities that occurred with vaccination or infection. We saw a shift in the gut microbiome over the course of a 7-day rotavirus challenge in neonates that did not return to baseline during the observation period, even after no virus shedding was detected in fecal samples. We also evaluated the impact of different doses, 1x106 CFU/dose and 1x109 CFU/dose, on the immune response and the gut microbiome. We confirmed the role of fecal microbiome transplants in breeding does to normalize for the maternal microbiome prior to gestation. Our results indicate that there are modifications to the gut microbiome and changes in immune antibodies during vaccination and infection. While we did not pursue a specific mechanism crosslinking the maternal-neonatal interaction and the gut-immunity relationship, we do consider the presence of such a connection.Item Open Access SARS-CoV-2 viral RNA biology and its impact on the infected cell(Colorado State University. Libraries, 2023) Altina, Noelia H., author; Wilusz, Jeffrey, advisor; Argueso, Lucas, committee member; Geiss, Brian, committee member; Perera, Rushika, committee member; Yao, Tingting, committee memberThe fine-tuning of the replication and transcription of RNA viruses often requires the interaction of viral RNAs with cellular RNA binding proteins. This project addresses fundamental knowledge gaps on the molecular mechanisms that underlie SARS-CoV-2 gene expression, regulation, and viral RNA-host interactions. After infection, SARS-CoV-2 generates a large set of sub-genomic mRNAs, each containing an identical ~70 base 'leader' region in their 5'UTR (from position 14 to position 75 in RefSeq NC_045512) and a 229 base region at the 3'UTR (from position 29,675 to position 29,903 in RefSeq NC_045512) generated by discontinuous transcription. The accumulation of a considerable amount of these leader/3'UTR regions during the infection represents a possible sink for cellular RNA binding proteins. We demonstrated that PTBP1, a cellular protein involved in the regulation of alternative splicing, binds to the SARS-CoV-2 leader region. SARS-CoV-2 infection critically impacted the splicing of several cellular pre-mRNAs that are normally regulated by PTBP1. Mechanistically, we suggest that SARS-CoV-2 sequesters and influences the re-localization of shuttling splicing factors like PTBP1 from the nucleus to the cytoplasm resulting in significant effects on the host cell splicing machinery leading to changes in cellular mRNA splicing patterns during SARS-CoV-2 infection. Given the current extensive interest in epigenetic methylations of both cellular and viral RNAs, our study also explored the role of post-transcriptional RNA modifications on viral mRNAs. We demonstrated that SARS-CoV-2 can usurp the cellular enzyme, namely PCIF1, to place the m6Am modification at the cap proximal position in its mRNAs. This double methylation is usually found on all host mRNAs that initiate with an adenosine residue, and thus SARS-CoV-2 likely installs this modification on its mRNAs to avoid host immune recognition. Interestingly, we also discovered that capping and m6Am modification are tightly regulated throughout the infection. The highest levels of these 5' end RNA modifications were observed at 12 hours post infection, correlating with the full establishment of viral gene expression in infected cells. These findings indicate that 5' end modification of SARS-CoV-2 transcripts is not simply a default process but rather undergoes unanticipated regulation throughout the infection. Collectively, the data presented provide not only new insights into the complex interactions that SARS-CoV-2 has with the RNA biology of the cell during infection, but also identify attractive potential targets for developing novel anti-coronavirus drugs to treat future emerging coronavirus diseases.Item Open Access Site-specific function of Endonuclease G and CPS6 to enable vertebrate function in an invertebrate model(Colorado State University. Libraries, 2021) Czarny, Ryan Scott, author; Ho, P. Shing, advisor; Stargell, Laurie, committee member; Hansen, Jeffrey, committee member; Argueso, Lucas, committee memberThe role of mitochondrial localized Endonuclease G (EndoG) remains relatively elusive. Studies have shown that EndoG has implications in mitochondrial DNA copy number, nuclear DNA cleavage during apoptosis, and oncogenesis; however, the mechanisms and pathways have yet to be determined. Our initial work investigates the nuclease activity of EndoG as well as its binding preference for duplex DNA and Holliday Junctions. It appears that EndoG and its C. elegans homolog, cps6, have slightly different functions in their in vivo systems, which has led us to query the structural modifications between the proteins. EndoG has been shown to have a preference for the 5-hydroxymethylcytosine (5hmC) epigenetic marker, an interesting feature due to the fact that invertebrate systems do not contain 5hmC in their epigenome. A key difference in the homologs arises in their DNA binding domain. The invertebrate model (cps6) contains two additional amino acids within this region that potentially allow for an alpha helix not seen in the vertebrate model to form. This alpha helix repositions a conserved cysteine in a way that it is pointed away from the active site in cps6, which could have consequences with regards to function. Our work investigates the addition/removal of this helix from the vertebrate and inveterate system to elucidate its role. In conjunction with the primary DNA binding site, there is a second site next to and orthogonal to the first. The vertebrate system contains multiple positively charged residues positioned to interact with the DNA backbone while the invertebrate contains two prolines that seem to be responsible for repositioning charges away from the site. We investigate the role of this secondary binding site as well as the importance of the invertebrate prolines. Overall, we propose a model to determine the role of EndoG in vivo utilizing the suite of protein mutations characterized herewithin.Item Open Access T cell independent mechanisms for protection against Mycobacterium tuberculosis infection(Colorado State University. Libraries, 2019) Bickett, Thomas, author; Izzo, Angelo, advisor; Dow, Steven, committee member; McLean, Jennifer, committee member; Bowen, Richard, committee member; Argueso, Lucas, committee memberThe live attenuated Mycobacterium bovis strain Bacille Calmette Guérin (BCG) is a potent innate immune stimulator. Innate Immunity provides the host with the ability to immediately respond to invasion by pathogens and can be utilized through the use of molecular adjuvants to trigger specific innate mechanisms leading to adaptive immunity. In the C57BL/6 mouse model of tuberculosis, BCG stimulated immunity causes a significant reduction of M. tuberculosis burden after pulmonary infection. Our studies indicate that BCG induced protection against pulmonary M. tuberculosis through early monocyte recruitment is present as early as 7 days after vaccination. This protection showed longevity, as it did not wane when mice were infected 30 days post vaccination. As BCG induced mycobacterial killing after 7 days, we sought to identify the contribution of different innate immune components to better understand mechanisms required for mycobacterial killing. When BCG was administered through subcutaneous inoculation, we found that there was significant monocyte recruitment in the lungs within 7 days after vaccination. Further studies revealed that killing of mycobacterium is dependent on BCG being viable and is monocyte derived, independent of trained innate immunity, highlighting a novel mechanism for killing M. tuberculosis. With the rise of drug resistant strains of Mycobacterium tuberculosis, new vaccine development is paramount. A better understanding of the BCG vaccine will hopefully lead to the development of a more effective alternative.Item Open Access Telomeric double strand breaks undergo resection - but not repair - in G1 human cells(Colorado State University. Libraries, 2017) Nelson, Christopher Boulanger, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Kato, Takamitsu, committee member; Wiese, Claudia, committee member; Miller, Benjamin, committee member; Chicco, Adam, committee memberTelomeres are specialized G-rich repetitive regions at the ends of eukaryotic chromosomes (TTAGGGn in mammalian cells). Telomeres function to prevent double strand break (DSB) repair activities at chromosome ends, in order to avoid fusion events which result in lethal dicentric chromosomes. Telomeric repeats make up an appreciable amount of genomic DNA (1-15kb per chromosome end). Therefore, an interesting question becomes, how is the inevitable DSB occurring within a telomere dealt with by the cell? It has been suggested that DSBs within telomeric DNA may not be repaired at all, as DSB DNA damage response (DDR) foci at telomeres do not resolve following large amounts of global DNA damage (e.g. ionizing radiation). Such studies also suggest that telomere repair may be inhibited specifically in G1, as the majority of surviving cells with unresolved telomere damage responses were senescent (a G1 phenotype). On the other hand, studies on the fragmentation of telomeric DNA following cutting with a telomere-targeted endonucleases indicate that repair of telomere-specific DSBs involves Homologous Recombination (HR) and Break-Induced Replication (BIR). However, a marker of telomeric DSB DDRs was only observed in cells with BrdU incorporation, in support of the view that repair of telomeric DSBs is an S/G2-related process, which does not occur in G1. To follow up on these studies, we investigated telomeric DDRs and DSB repair in individual G1 cells using ionizing radiation (IR) and a targeted telomere-cutting endonuclease. IR exposure could potentially induce loss of telomere function, such that persistent DDRs may not represent actual DSBs. To rule out this possibility, we evaluated whether persistent telomeric DDRs following IR occurred at telomeres that were critically short or lacking TRF2. We found that persistent telomeric DDRs occurred at telomeres of normal length and TRF2 status, in support of the conclusion that G1 telomeric DSBs are irreparable. Additionally, using the telomere-targeted endonuclease we observed that telomeric DSBs in G1 cells elicited a relatively conventional DSB DDR – with one important exception – G1 telomeric DDRs failed to recruit 53BP1, an event implicated in the completion of DSB repair by most pathways, but especially, canonical non-homologous end joining (cNHEJ). Further, shRNA knockdown and kinase inhibition of the cNHEJ factor DNA-PKcs, provided evidence that cNHEJ is not responsible for repair of telomeric DSBs, and that DNA-PKcs does not influence recruitment of 53BP1 to telomeric DSBs in G1. Partial deprotection of telomeres, achieved by siRNA depletion of TRF2, also failed to alleviate inhibition of 53BP1 recruitment to G1 telomeric DSBs, suggesting that 53BP1 recruitment to telomeric DSBs may require full deprotection of telomeres. However, as 53BP1 recruitment occurs at de-protected telomeres, this idea would be difficult to test. Most likely related to the lack of 53BP1 recruitment, an abundance of bidirectionally occurring single-stranded DNA was observed at G1 telomeric DSBs, a characteristic of long-range repair-associated resection. In support of long-range resection, RPA70 and phospho-RPA32 were observed at G1 telomeric DSBs. Additionally, conventional DSB repair-associated resection machinery, including MRE11 and EXO1, but not the telomere processing exonuclease Apollo, promoted resection at telomeric DSBs. We then investigated whether long-range resection-dependent repair was occurring at G1 telomeric DSBs via RAD51 or RAD52 foci, and DNA synthesis (S/G2 related processes). Despite activity resembling long-range repair-associated resection at G1 telomeric DSBs, no evidence for repair by these pathways was found. Taken together, the results presented here provide strong evidence in support of the view that telomeric DSBs in G1 are unrepairable. Therefore, the extensive resection observed at telomeric DSBs must be reflective of an alternative, non-repair related function, perhaps related to structural end-protection. We speculate that resection at G1 telomeric DSBs may serve to prevent 53BP1 recruitment, thereby circumventing a full DDR and activation of cNHEJ, a scenario that would create a serious threat to genome stability.Item Open Access The impact of insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in human and canine osteosarcoma(Colorado State University. Libraries, 2019) Alyami, Nouf Mahdi, author; Duval, Dawn, advisor; Wilusz, Jeffrey, committee member; Argueso, Lucas, committee member; Stargell, Laurie, committee memberOsteosarcoma (OS) is a malignant bone tumor that afflicts over 10,000 dogs. Most dogs and approximately 30-40% of children with OS succumb to metastatic disease. We identified elevated insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as one of the biomarkers of poor prognosis in canine OS. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability, and translation. IGF2BP1 controls the expression of oncogene targets and correlates with poor outcome in a variety of human cancers. Using microarray analysis, we identified elevated insulin-like growth factor II mRNA binding protein 1 (IGF2BP1) expression as a biomarker of poor prognosis in canine osteosarcoma. Also, our preliminary data show that IGF2BP1 knockdown (shRNA) in a human OS cell line increased sensitivity to doxorubicin by ≥ tenfold compared to control. Significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). The current research explores mechanisms for increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines. Gene amplification, hypomethylation, increased transcription, and alterations in microRNA (miRNA) regulation directly or through 3'UTR shortening have all been hypothesized by many studies as mechanisms to increase IGF2BP1 expression in cancer. We evaluated the expression and alternative polyadenylation of IGF2BP1 using RT-qPCR and western blot analysis in human and dog osteosarcoma cell lines. We assessed transcriptional activation of IGF2BP1 using luciferase reporters containing promoter sequences from the human and canine IGF2BP1 genes. To detect genomic amplification and methylation, we used qPCR to assess gene copy numbers and treatment with the DNA methylase inhibitor, 5-Azacytidine, to explore activation of gene expression through hypomethylation. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p = 0.0006, Pearson r = 0.88). We observed no genomic amplification in human cell lines. Significant loss of 3'UTR regulatory sequences was found in 20% of canine cell lines (p < 0.05). The promoter analysis showed that most regulatory elements were located within ~580bp from the translational start site in both species. Using pathway-focused luciferase reporter assays, we identified activation of the following factors: MYC, NF-Kappa B, AP-1, and TCF4: β-catenin. Thus, our data show that multiple mechanisms can contribute to elevated IGF2BP1 expression, and these results can be used to develop new treatment strategies that target elevated IGF2BP1 or regulatory mechanisms. Using the McKinley canine OS cell line, we generated and validated stable overexpression of IGF2BP1 (IGF2BP1-pLVX-Puro, Clontech). The stable OS cell line pool and individual clones with a corresponding empty vector control were analyzed and tested for migration, invasion, proliferation, and resistance to standard chemotherapeutic agents. We analyzed migration and invasion using a scratch wound assay and measured cellular proliferation as a surface confluence for 90 hours on an IncuCyte Zoom. We also assessed the clones' sensitivity to doxorubicin over 48 hours using a bioreductive resazurin-based fluorometric assay. We assessed changes in transcript expression in response to IGF2BP1 from isolated total RNA analyzed on Affymetrix Canine 1.0ST microarrays (University of Colorado Cancer Center Genomic and Microarray Shared Resource). The overexpressing IGF2BP1 clones had increased resistance to doxorubicin compared to the control, and the IC50 levels correlated with IGF2BP1 mRNA levels (p < 0.05, r2 = 0.89). For cellular proliferation, we found that only the IGF2BP1-expressing pool, that represents random insertion of the plasmid without selecting isolated clones, exhibited a significantly higher rate of proliferation relative to the empty vector control (p < 0.05). However, one of the highest expressing IGF2BP1 isolated clones had significantly greater cellular mobility and invasion than this pool, and both the pool and isolated clone had significantly higher rates of migration and invasion that cells transfected with the empty plasmid (p < 0.05). Microarray analysis of control and over¬expressing cells was used to detect global changes in gene expression and to identify potential targets of IGF2BP1. Differentially expressed genes were cross ¬referenced to the RNA¬ Binding Protein Immunoprecipitation database, published by Conway et al. (2016) using human stem cells, to identify direct mRNA targets bound by IGF2BP1. We identified 162 genes that were differentially expressed between control and overexpressing cells (FC ≥2, FDR< 0.05), and 13 of those genes have been previously reported to bind IGF2BP1 directly. Pathway analysis of these 13 genes identified enrichment for genes involved in the regulation of cell adhesion, migration, and the extracellular matrix. Altered expression and IGF2BP1 binding of a subset of these transcripts were confirmed using RNA immunoprecipitation and RT-qPCR. Our data suggest that IGF2BP1 plays a significant role in human and canine osteosarcoma. This study revealed the functional relevance of IGF2BP1 and identified it as a biomarker for aggressiveness in osteosarcoma. With this knowledge, new treatment strategies can be developed that target IGF2BP1 or it is signaling pathways for osteosarcoma, or any cancer that expresses high levels of IGF2BP1. This treatment may have a high impact on the cell's ability to metastasize.