Browsing by Subject "College of Biological Sciences"

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Absence of Twisted Gastrulation (Twsg1) Limits the Population of Cranial Neural Crest Cells
(2009-04-08) Mittelstaedt, Gina
Craniofacial defects are among the most common birth defects with cranial neural crest cells (NCCs) playing a fundamental role in craniofacial development. The cranial NCCs migrate from dorsal neural folds to populate the branchial arches. Then they differentiate into the cells that form facial structures. For example, the mandible forms from the first branchial arch (BA1). Twisted gastrulation (Twsg1) is a gene that has been found to influence craniofacial development. A mutation in Twsg1 in mice produces a spectrum of craniofacial defects ranging from normal appearance to underdevelopment of the mandible, midline facial defects, and forebrain defects resulting in holoprosencephaly. Previous research has shown that Twsg1 acts as a bone morphogenetic protein (BMP) antagonist and thereby limits apoptosis in BA1, a key destination of cranial NCCs. Apoptosis is increased in BA1 in the absence of Twsg1, leading to a loss of BA1 derivatives. The hypothesis of this work is that the absence of Twsg1 also increases apoptosis in the cranial NCCs and thereby limits the population of NCCs prior to their migration to BA1. This study focused on determining whether the NCC population was depleted in Twsg1 knockout mice and whether the midbrain region itself was affected. We found that the midbrain markers were normal, but the markers specific for NCCs were significantly reduced. This reduction is consistent with a depletion of the NCC population. Future research will directly study proliferation, apoptosis, and BMP signaling in NCC populations in the absence of Twsg1.
Adaptive Evolution of a Blood-Clotting Gene in Venom-Resistant Opossums
(2010-04-21) Sosa, Tim
Action of snake venomHemolytic snake venoms—such as those found in rattlesnakes, moccasins, and lanceheads— are complex cocktails of proteases, phospholipases, and phospho-diesterases. One protein found in the venom of lancehead vipers (genus Bothrops) is botrocetin, which causes aggregation of blood platelets wherever von Willebrand Factor (vWF)and Factor VIII are present in the bloodstream (1).The blood plasma protein vWF and Factor VIII circulate freely in the blood vessel lumen during ordinary (laminar) flow. During normal blood clotting, turbulent blood flow—such as when a blood vessel ruptures—induces vWF to disengage from Factor VIII and complex with glycoprotein Ibαand collagen. This complex, by a series of reactions, aggregates platelets and fibrin to form blood clots (2). Thus, by binding vWF, botrocetin promotes inappropriate systemic clotting, reducing the ability of vWF to respond to ruptures caused by proteolytic venom proteins and promoting hemorrhage. Resistance in opossumsSeveral species of opossums in both North and South America are known to be resistant to lancehead venom—in fact, large opossums will even eat poisonous snakes (Figure 1). The mechanism by which they withstand snakebite is not known; our aim was to assess the possibility that mutations on the vWF gene may play a role in resistance.HyothesisBy calculating the rate of synonymous substitutions dSin the gene and the rate of non-synonymous substitutions dN(i.e., mutations that change the amino acid sequence of the resulting protein), we hope to detect posititve selective pressure. The ratio ω = dN/dS is a key statistic: if ω < 1, purifying selection is acting on this gene (i.e., mutations are purged); if ω = 1, no selection can be inferred; if ω> 1, positive, directional selection is acting on this gene. Our expectation is to find greater rates of substitution in the group of opossums highlighted in red in the phylogeny at right, because these are the species known to be resistant to snake venom.
Adipocyte Secretion in Metabolic Syndrome
(2020) Bell, Marcus; Hertzel, Ann; Bernlohr, David
Metabolic syndrome is characterized by chronic, low grade inflammation of the adipose organ and increased basal white adipocyte lipolysis. These processes may promote insulin resistance and cell senescence. Dr. Ann Hertzel and Dr. David Bernlohr showed that, in response to lipolysis, adipocytes secrete hundreds of proteins unconventionally (UPS). Among the most abundant of these secreted proteins is fatty acid binding protein 4 (FABP4), a leaderless lipid carrier and known agonist of several mechanisms of insulin resistance, hepatic steatosis, and atherosclerosis. The mechanism of secretion is not well understood, and proteins may be secreted freely or in vesicles. Another notable aspect of lipolytic UPS is its dependence on autophagy, which leads to the hypothesis that it may rely on sphingosine-1-phosphate (S1P) pathway. Additionally, the chronic inflammatory state of the adipose organ may lead to cellular senescence. The molecule 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, may promote cellular senescence via the carbonylation of DNA and proteins. In 2015, Hauck and Bernlohr showed vastly increased intracellular levels of 4-HNE in adipocytes treated with inflammatory cytokines. The passive membrane diffusion of 4-HNE is plausible because it is small and amphipathic yet largely nonpolar. If 4-HNE is secreted from adipocytes, it may act in a paracrine manner and cause local cells to senesce. Adipocyte senescence may result in a decrease in triglyceride storage ability. When this occurs subcutaneously, visceral fat depots become preferred for triglyceride storage – a hallmark progression of obesity and its comorbities. 4-HNE secretion from adipocytes has not been shown in biochemistry literature but is hypothesized to be under lipolytic or inflammatory control due to its high abundance in obese adipocyte cytoplasm. Characterizing the mediating factors of UPS and 4-HNE secretion may show that adipocyte secretions can exacerbate metabolic syndrome and promote cell senescence. Furthermore, showing that adipocytes secrete 4-HNE may introduce an area of study for researchers of aging processes, especially as they relate to obesity and metabolic syndrome.
Analysis of a Genetic Adaptation for Glycerol Utilization: Implications for Microbial Fuel Cells
(2010-04-21) Torchia, Mike
Biodiesel promises a renewable source of energy yet is unable to be an economically viable alternative to petroleum. One way to solve this is to convert glycerol, a by-product of the biodiesel production process, to higher value commodities. Shewanella oneidensis can respire insoluble extracellular substrates such as electrodes. Furthermore, when the pGUT2PET plasmid is transformed into wild type S. oneidensis, the non-redox balanced conversion of glycerol to ethanol is permitted. This engineered bacterium permits the generation of two higher value products (ethanol and electricity) from the original glycerol feedstock. Since any future industrial application of this microbe will necessitate optimization of all its parameters, we were interested in studying how S. oneidensis grows faster on glycerol.
The Analysis of Pyridyloxobutyl RNA Adducts in Rats Treated With 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
(2009-04-08) Muzic, John
Tobacco-specific nitrosamines are potent carcinogens formed from parent compounds such as nicotine during the curing process of tobacco. The cytochrome P450 enzyme family metabolically activates these nitrosamines, which then attach pyridyloxobutyl (POB) groups to DNA bases. The formation of POB-DNA adducts in tissues of rats treated with tobacco-specific nitrosamines have been demonstrated in previous studies. These adducts can potentially lead to mutations in DNA which promote the formation of tumors. However there exists no data on the formation of POB-RNA adducts. RNA adducts could be important in carcinogenesis, and could potentially be a more reliable biomarker than DNA adducts for tobacco-specific nitrosamine exposure. The objective of our project was to chemically characterize and quantify POB-RNA adducts in the tissue of rats treated with a nitrosamine compound, specifically 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). RNA was isolated from rat liver treated with NNK over a course of 20 weeks and from untreated liver. Combined liquid chromatography-mass spectrometry and high performance liquid chromatography were used to identify RNA adducts. The results indicate the presence of POB-RNA adducts in the treated livers. Further work will involve synthesizing standards by reacting NNK with nucleosides and confirming the structure of adducts with NMR. The results of this study will confirm the presence of RNA adducts due to nitrosamine exposure and provide insight into the utility of RNA adducts as a biomarker used for chemopreventative strategies.
Analyzing Coronary Heart Disease Risk Factors and Proper Clinical Prescription of Statins
(2012-04-18) Thorne, Peter
A sample of adults participating in the first 7 months of visit 5 of the Atherosclerosis Risk in Communities (ARIC) study were analyzed to determine if they were receiving statin treatment, when indicated, based on their risk factors for coronary heart disease. This sample consisted of male and female adults ages 67 through 89. Data collected from the ARIC study along with the Framingham risk score were used to calculate participants ten year risk. Next, the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults report was used to determine if the 10 year risk was great enough to warrant statin use. It was found that 163 of 831 males and 213 of 1145 females were not on statins even though their risk factors suggested they should be.
Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer
(2012-04-18) Hanson, Kevin
This year in the United States, cancer is projected to cause one out of every four deaths. Prostate cancer alone is estimated to take the life of over 30,000 men. One area of intense research as a potential therapy against cancerous growth is in tumor suppressing genes, which function in cell cycle checkpoint responses, detection and repair of damaged DNA, protein ubiquitination and degradation, mitogenic signaling, cell specification, differentiation and migration, and tumor angiogenesis. One particular gene, the mammary serine protease inhibitor (maspin), is critically important in both breast and prostate cancer, which in the latter the gene is repressed entirely. However, the exact mechanism that leads to the repression of the maspin gene is not entirely understood. In my thesis work, I first showed that the maspin tumor suppressor gene was under direct regulation by the androgen receptor protein. By analyzing the quality and quantity of the mRNA produced under various growth conditions, we confirmed that the activation of the androgen receptor was critical for the repression of maspin. To do this, cell cultures were selectively grown with or without androgens and the mRNA was extracted, converted to cDNA, and RT-PCR was performed to analyze relative levels of maspin expression. I next attempted to verify that the androgen receptor was physically binding the promoter region of maspin by performing chromatin immunoprecipitation (ChIP) assays. After immunoprecipitating any chromatin fragments with the androgen receptor bound, a PCR to amplify the maspin promoter was completed. Obtaining a PCR product confirmed that the AR binds the androgen response element (ARE), repressing its expression.
ATP Rapid Testing in Health Care Settings
(2012-04-18) Elias, Aaron
Infection Prevention in hospitals is an integral part of what makes hospitals effective at total patient care. Environmental surfaces in patient rooms in hospitals include counters, tray tables, nursing equipment, and many other high touch surfaces, all of which can carry bacteria and harmful substances, and can cause secondary infections if they are not cleaned properly. This study was conducted to test how well these high-touch environmental surfaces are cleaned using a rapid test, as opposed to standard microbiological methods. This rapid test measured the amount of ATP found on a surface (given in RLUs), in order to estimate the amount of contamination. In general, most environmental surfaces in different departments across Fairview Health Services met cleanliness standards. A standard must be made for this type of testing in order to know what ATP levels are acceptable. It was also determined that using this ATP rapid test is a viable way to check how effective patient rooms are being cleaned.
Bark Beetle: Fungus Interactions in Declining Hickory Trees
(2010-11-29) Zenner, Bobbi
The objective of this study is to determine whether C. smalleyi is commonly carried by hickory bark beetles when they emerge from beetle infested, declining bitternut hickory in late spring. The findings will help answer the question of whether the beetle is an important vector of C. smalleyi. These results will be added to those of additional assays of beetles from the same site as well as from a second Wisconsin location.
Biological Function of Nectin 4 in Ovarian Cancer
(2012-04-18) Meyer, Adam
Ovarian Cancer Overview: 5th leading cause of cancer death for women in the U.S.; Most patients diagnosed after metastasis, when survival rates are low; Early diagnosis yields 93% five year survival rate. Biomarkers and Nectin 4: Biomarkers have different properties: early detection, clues to therapy effectiveness, and possible prognostic applications; Nectin 4 has been identified by the Skubitz lab as a possible biomarker. Nectins belong to a family of immunoglobulin-like proteins important in cell to cell adhesion; A study of 500 ovarian cancer tumors showed 48.6% stained for Nectin 4 in the tumor while 53% of ovarian cancer blood samples tested positive. Nectin 4 also expressed in lung cancer. Our hypothesis is that knocking down the expression of Nectin 4 will inhibit ovarian cancer cell function and metastasis.
Bloated Rhizobia: The effect of PHB storage on Bradyrhizobium japonicum mortality during desiccation
(2011-04-13) Underbakke, Kyra
Soil bacteria known as rhizobia infect the roots of legumes, forming nodules, where they benefit the host by fixing atmospheric nitrogen into a usable form for the plant. In return, these rhizobia use plant-derived energy to reproduce, and many species accumulate large quantities (>50% cell dry weight) of the storage lipid poly-3-hydroxybutyrate (PHB). Since high PHB stores give rhizobia up to a threefold reproductive advantage over cells with low PHB during starvation conditions, genotypes of rhizobia that are able to escape from nodules with more PHB would be expected to increase in frequency through time. However, not all strains of rhizobia synthesize large amounts of PHB. This research investigates a potential disadvantage to storing too much PHB: rhizobia that accumulate excessive amounts of PHB may be more likely to die in a drought environment. Using soybean (Glycine max) as host plants, I extracted Bradyrhizobium japonicum from nodules, desiccated the rhizobia, and measured PHB and percent of cells killed by this treatment using flow cytrometry. The results showed a positive correlation between PHB storage and percent killed by desiccation, indicating that high PHB storage may not always be selected for in rhizobial populations, despite its reproductive advantages.
Can we increase the nitrogen fixation efficiency of Sinorhizobium meliloti?
(2011-04-13) Bower, Justin
Rhizobia are soil bacteria that can grow and reproduce in nodular swellings on legume plant roots. In return for carbon resources, rhizobia provide their host plant with fixed nitrogen (N2 → NH3) for proteins, etc. In alfalfa nodules, rhizobia that fix atmospheric nitrogen lose their ability to reproduce. These non-reproductive rhizobia may divert resources from nitrogen fixation to rhizopine, which can be used as an additional carbon source for still- eproductive rhizobia in the same nodule. Consequentially, rhizopine producing rhizobia would benefit at the host’s expense and could harm agriculture. From June 2010 to March 2011, I have been measuring nitrogen-fixing efficiency to determine if rhizopine production actually reduces the quantity of nitrogen fixation in several genetically different strains of rhizobia. I measured how much carbon dioxide (from respiration) and atmospheric hydrogen (a byproduct of nitrogen fixation) were given off by nodules as a function of oxygen concentration. Of the four strains of rhizobia tested by this method, two strains synthesized rhizopines and two strains did not. The respiration and fixation data for each strain fit a linear regression with the inverse slope = nitrogen-fixation efficiency and the y-intercept = carbon cost in the absence of nitrogen fixation. My results show that one rhizopine producing strain had lower nitrogen fixation efficiency. However, this did not occur in a commonly used rhizopine producing laboratory strain. Therefore, I was able to conclude that rhizopine production may not be the most important factor controlling nitrogen-fixation efficiency.
The Cancer Vaccine: Identifying Barriers to HPV Vaccination in Ugandan Adolescent Girls
(2017) Gravagna, Katie
Candida albicans Mutagenesis: Response to Stress
(2009-04-08) Bruck, David Joachim
Candida albicans is a model eukaryotic yeast and an opportunist human pathogen. It generates novel drug resistance through mutation and mitotic recombination. The rate loss of heterozygosity, or rate LoH, is a measure of this mutation and recombination. Previous research has shown that some stresses like the anti-fungal drug fluconazole cause the yeast cells to increase their rate LoH while other stresses do not. On the assumption that fluconazole does not directly affect DNA replication, it is possible that the increased rate LoH is due to a stress response pathway. My UROP project aimed to create a list of computationally curated gene targets using microarray expression data analysis which would then be tested for mutant phenotype rate LoH. The majority of the time allocated to the project was spent doing ortholog searches of gene lists curated from another yeast, Saccharomyces cerevisiae, as well as studying any previous papers where any of the orthologs in question were directly examined.
Carbon soil dynamics in secondary tropical dry forests in Northwestern Costa Rica regenerating from grazing
(2012-04-18) Nowicki, Jesse
The goal of my research project is to understand how ecosystem processes like carbon sequestration in soils changes as young forests regenerate on lands that were previously used for grazing. The objective is of my project is to re-visit the same area that Dr. Powers studied 5 years ago and determine if the chronosequence and longitudinal studies reveal the same soil carbon dynamics as previously predicted, as well as to see if soil carbon sequestration increases with forest age, as many conceptual models predict. From the data analysis, the carbon concentration and carbon isotope followed our predicted assumptions for the concentration to increase slightly and the carbon isotope would become more negative. We predicted the bulk density would decrease over time as the soil became less dense but our data showed mixed results on whether it increase or decreased.
Characterization of Parkinsonian Neuropathophysiology and its Modulation by Deep Brain Stimulation in the Behaving, Nonhuman Primate Model
(2016) Weinstock, Zachary;
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by debilitating motor disturbances. It is believed that the signs and symptoms of PD are caused by idiopathic cell death in the basal ganglia (BG), a network of subcortical nuclei with a well-established connection to motor control. Although there is no cure for PD, deep brain stimulation (DBS) of the internal Globus pallidus, a constituent nucleus of the BG, offers hope for patients who don’t respond well to conventional medications. However, the mechanisms underlying the therapeutic effects of DBS remains unclear, a fact largely attributed to a poorly characterized pathophysiology. Here we identify characteristic, electrophysiolgical biomarkers of PD that preempt the emergence of its behavioral signs and show that DBS works to shift cortical activity back towards a more “normal” state. Using a behaving non-human primate model of PD, we observed a disruption in the normal firing patterns and frequencies of both single motor units and neuronal populations in the primary motor cortex (M1) and supplementary motor areas (SMA) following the induction of parkinsonism. During DBS, we observed an increase in task-related neuromodulation. Taken together, our results hint at a therapeutic mechanism for DBS whereby signaling in M1 and SMA is made more salient and shifted towards “normal” activity. We anticipate that our findings will serve to guide future research and instruct the development of more effective, adaptive DBS technologies.
Characterizing the Catalytic Activity and Specificity of RNF168 using Targeted Mass Spectrometry and Ubiquitination Stoichiometry-Based Quantitative Analysis
(2021) Wei, Lai
The Chen lab developed a new technique that uses quantitative proteomics approach for site-specific ubiquitination stoichiometry analysis, named the Isotopically Balanced Quantification of Ubiquitination (IBAQ-Ub), which is a method that quantifies the absolute abundance of ubiquitination. IBAQ-Ub and in vitro ubiquitination enzymatic assays were conducted to characterize the site-specific ubiquitination dynamics and linkage specificity of RNF168 in DDR(DNA-damage-response).
Chloroplast capture between Clarkia xantiana xantiana and Clarkia xantiana parviflora
(2022) Suri, Amal M.
Introgression occurs when genetic material from one gene pool gets incorporated into another gene pool. Introgression of chloroplast genomes is when one species inherits the chloroplast genome of another species, termed chloroplast capture. Understanding introgression can provide invaluable insight into reproductive barriers and speciation. Clarkia xantiana xantiana and Clarkia xantiana parviflora are subspecies that diverged about 65,000 years ago. Subspecies xantiana is an outcrossing species, while ssp. parviflora is a selfing species. We hypothesized that chloroplast capture is likely occurring between the two subspecies in sympatric areas, where they co-occur. In this study, we performed a phylogenetic analysis with about 200 complete chloroplast genomes of the subspecies from 6 different hybrid zones and 16 allopatric sites to determine whether chloroplast capture occurred. Additionally, we sampled more vigorously from 4 sympatric sites, where we then performed Polymerase Chain Reaction on those individuals to determine chloroplast genotype. In accordance with current theory, we found that chloroplast capture was asymmetric such that, chloroplasts flowed mainly from the selfing species, ssp. parviflora, into the outcrossing species, ssp. xantiana. There was, however, notable variation in asymmetry among sympatric populations.
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.
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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