Browsing by Subject "Department of Genetics, Cell Biology, and Development"

Now showing 1 - 7 of 7
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    Clinical Risk Factors for Infection and Antibiotic Resistance in BMT Patients
    (2012-04-18) Bock, Allison
    Bacterial infections continue to be a leading cause of mortality and morbidity in blood or bone marrow transplant (BMT) patients. The relative importance of different clinical features (donor type, graft cell source, and conditioning regimen) on the incidence and timing of post-transplant bacterial infections is uncertain, but a detailed analysis could better guide prevention and therapy. We retrospectively analyzed the incidence and risk factors for bacterial infection, as well as patterns of antibiotic resistance, in 834 BMT patients at the University of Minnesota from 2005-2010. We found that donor type has the greatest impact on the incidence of infection in BMT patients out to 100 days post-transplant. Full intensity, myeloablative conditioning, compared to reduced intensity conditioning is also associated with a greater risk of bacteremia, as is later development of acute GVHD. Additionally, BMT patients, compared to the contemporaneous hospital population, develop infections with resistance to many antibiotics used for both prophylaxis and treatment against commonly isolated bacterial organisms. These findings have important clinical implications regarding the use and selection of both prophylaxis and empiric antibiotic regimens.
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    Co-Targeting the mTOR and MAPK Pathways is Effective in a Novel Mouse Model of Malignant Peripheral Nerve Sheath Tumors
    (2012-04-18) Anderson, Leah
    Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are soft tissue sarcomas with low 5-year survival rates and no targeted therapies available. Data suggest that the mTOR and MAPK pathways may be involved in the formation and progression of MPNSTs, and both of these pathways can be inhibited with drugs that are currently in use for other tumor types. In vitro, RAD001 and PD-901, inhibitors of the mTOR and MAPK pathways, respectively, are effective at inhibiting proliferation of human MPNST cells, while having little effect on normal human Schwann cells. To better study their therapeutic potential, we tested these drugs in a mouse model of MPNSTs. This model closely resembles genetic changes (Pten loss, EGFR overexpression) and histological feature of human MPNSTs. RAD001 or PD-901 treatment moderately reduced tumor burden and size, and extended lifespan in this model. However, when one pathway is inhibited, there is an increase in signaling through the other pathway, suggesting that these pathways feedback on one another, and that targeting both pathways in combination may be more effective. We found synergistic effects on reducing tumor burden and size, and a significant increase in lifespan when RAD001 and PD-901 are given in combination. The synergy seen is due to the combination therapy allowing for persistent and prolonged reduction in signaling through both pathways, without a subsequent increase in signaling through one pathway, as seen in single agent treatments. These data suggest that co-targeting the mTOR and MAPK pathways could potentially be an effective treatment for patients with MPNSTs.
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    Identification of a Novel p53 Regulated Endogenous Retrovirus MMERGLN
    (2012-04-18) Singhal, Surbhi
    With roughly half of all tumors with mutations in the TP53 gene, TP53 undoubtedly is a tumor suppressor gene that plays a critical role in the prevention of cancer. TP53 responds to cellular stress in cells, either metabolic disorder or genetic damage for example, and becomes transcriptionally activated to produce the p53 protein. Several genes that are directly targeted by p53 have been studied extensively and their role in cellular apoptosis and senescence are well documented. However, whether p53 can also regulate expression of repetitive elements that comprise a significant part of the genome, and their possible consequence, is not known. The mouse genome, in addition to known genes, also has several endogenous retroviruses, short DNA sequences derived from ancient viral infections that are now part of the genome. In my thesis work, I characterize one specific endogenous retrovirus, MMERGLN, and explore MMERGLN’s relationship with the p53 protein. Not only does MMERGLN show p53 regulation according to RNA Sequence data, MMERGLN also possesses p53-binding sites. Here we have identified MMERGLN transcripts that are present in a p53-dependent manner and MMERGLN is expressed body-wide in mouse. Furthermore, we demonstrate that multiple copies of MMERGLN contain all the components necessary for retrotranspostion or infection suggesting this genomic element may still be active. In order to determine a potential function for this genomic element, we investigated the ability of MMERGLN to act as an enhancer by identifying genes with differential transcription near MMERGLN insertion sites in p53 wild type and p53 null mice. Finally, with the purpose of understanding how MMERGLN is prevented from spreading throughout the genome, we investigated the mechanism by which MMERGLN is restricted, and see evidence for cytosine deamination mediated by the Apobec proteins. Our results demonstrate a paradigm shift in how transposable elements are regulated by p53 and suggest a new role for MMERGLN in tumor suppression. We anticipate these studies to shed light on the potential role of transposable elements in preventing disease.
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    Post-Transcriptionally Modified tRNAs for Enhanced Translation
    (2024-04) Prasad, Angelin; Kalb, Evan; Adamala, Kate
    Synthesizing peptides from non-natural amino acids presents challenges such as maintaining a correct reading frame and proper tRNA charging. Flexizyme aids tRNA charging with non-natural amino acids, often using in-vitro transcribed tRNAs. In-vivo transcribed tRNAs, with post-transcriptional modifications, hypothetically offer better stability. Stability for tRNAfMET was assessed via Tm values, indicating higher stability in in-vivo transcribed tRNAs. However, Hibit luminescence assays showed no significant translation efficiency difference between in-vitro and in-vivo transcribed tRNAs. In conclusion, post-transcriptional modifications may not significantly enhance flexizyme reactions. Future steps involve identifying limiting factors in the system or tRNAs.
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    Regulation of Human HRAS1 Minisatellite Stability During Stationary Phase
    (2009-10-07) Brosnan, Laura
    Minisatellites are repetitive tracts of DNA with repeat units ranging from 16-100 base pairs in length. They are stable during mitosis but display changes in repeat number and order after meiosis. Rare alleles of minisatellite tracts thought to arise from repeat instability are associated with human diseases, including cancer, diabetes, and epilepsy. The stability of minisatellites in non-proliferating stationary phase cells is not well understood. Previous work has shown that the zinc transporter ZRT1, the checkpoint gene RAD53, the DNA repair gene RAD27, the endocytosis gene END3, and the protein kinase PKC1 regulate the stability of minisatellites in S. cerevisiae during stationary phase. We inserted the human minisatellite associated with HRAS1 into the ADE2 gene to determine how its stability is regulated during stationary phase. Loss of ZRT1, RAD27, or RAD53 destabilized the minisatellite; loss of PKC1 or END3 had no effect. This work contributes significantly to our understanding of repeat stability and genome stability during stationary phase; this has important implications for human genome stability, since most human somatic cells are non-proliferating.
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    Searching for New Proteins Involved in Mitotic Chromosome Condensation
    (2012-04-18) Grenfell, Andrew
    The segregation of genetic material between newly formed daughter cells during cell division is a highly dynamic, intricately orchestrated cellular process. The sheer size of eukaryotic genomes poses a fundamental problem that must be overcome to ensure proper transmission of genetic material to daughter cells. After DNA replication, a typical human skin cell contains more than twelve feet of DNA, housed within a space measuring less than a millimeter across. Beyond simply containing this mass of DNA, cells must achieve a dramatic reorganization of their chromosomes during mitosis in order for them to be evenly divided between daughter cells. Mitotic chromosome condensation, the process by which chromosomes resolve into discrete bodies prior to nuclear division, must be faithfully carried out to ensure that each pair of sister-chromatids, the identical copies of each chromosome formed during DNA replication, disjoins during nuclear division. If chromosomes fail to condense during early mitosis, tangles between sister-chromatids can lead to breaks in the DNA or an outright failed segregation of sister-chromatids. Previous research has shown that topoisomerase II and the condensin complexes (condensin I and condensin II) are essential for mitotic chromosome segregation. However, a significant body of recent work suggests that there are more components to the chromosome condensation machinery than previously thought. In this work, I identify several novel proteins that could play a role in mitotic chromosome condensation. The identification of these proteins opens up new avenues of study, potentially providing the missing link between the physical phenomenon of mitotic chromosome condensation and our incomplete picture of the process on the protein level. With the future verification, of these proteins as essential players in mitotic chromosome condensation, we will hopefully be poised to take further steps to understand this fundamental cellular process.
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    Suppressor Analysis of a Ubiquitin Ligase Required for Genomic Stability
    (2010-12-20) Bock, Allison
    Analysis of a suppressor of the Ubiquitin Ligase, dia2 mrc1S was identified as a suppressor of dia2 by Chi’s suppressor screen (Koepp Lab 2009) Testing phenotypes mrc1S suppresses to understand the functional relationship between Dia2 and Mrc1 Specifically, assess three phenotypes of both mrc1S and dia2∆