|Colleen Hannon||A glycolytic mutant in Drosophila with neurodegeneration and shortened lifespan
Several neurodegenerative diseases, such as Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease have been linked to metabolic disruption in neurons. This suggests that neurons are particularly sensitive problems in energy metabolism. However, a controversy currently exists regarding the role of glycolysis in neurons. One hypothesis, the astrocyte-neuron lactate shuttle (ANLS) model, proposes that neurons do not perform their own glycolysis but depend on lactate provided by glial cells as an energy substrate for ATP production. A neurodegenerative mutation in Drosophila melanogaster has been identified, and mapped it to a small region containing the gene that encodes aldolase. Aldolase is a vital glycolytic enzyme, and a mutation in its coding sequence would likely cause energy deprivation, possibly leading to degeneration of neural tissue and early death. These Drosophila mutants provide a model system to investigate the energy requirements for neuronal viability. To test the ANLS hypothesis, tissue-specific rescue experiments were performed, in which aldolase function was selectively restored to mutants in their neurons and glia individually. The results have shown that restoring aldolase to neurons can rescue lifespan defects in the mutants as effectively as restoring its function to the entire organism, suggesting an essential role for glycolysis in neurons.
|Alexandra Linz||Characterization of hydrogen production from carbon fixation mutants of Rhodobacter sphaeroides
In the search for renewable fuels, hydrogen is a promising option. Producing hydrogen gas using photosynthetic bacterial cultures would potentially allow large scale, clean, renewable energy production. Rhodobacter sphaeroides is being studied for this project because it naturally produces large amounts of hydrogen gas via the nitrogenase enzyme under photosynthetic, anaerobic conditions in the absence of ammonia. To maximize hydrogen yield, mutant strains of R. sphaeroides are being investigated. One of these mutants (CfxA-B-) has its main carbon fixation pathways genetically inactivated and typically cannot grow under the conditions needed to produce hydrogen. This growth defect has been hypothesized to be due to the buildup of excess reducing power in the cell. However, strains of CfxA-B- that can survive under these conditions and produce hydrogen gas have been selected for. These strains produce more hydrogen than the wild type under certain conditions. The gas production of this strain has been characterized and reasons for its survival and increased production hypothesized.
|Elizabeth Chadwick||Characterizing the role of a novel Aspergillus nidulans gene in secondary metabolism
Secondary metabolism is the production of natural, chemical products that enhance an organism’s ability to survive in harsh conditions. In fungi, secondary metabolites play a role in protecting the fungi from the environment and predators. These metabolites are of great interest because many of them have either a beneficial or harmful effect on humans and other organisms. In the model system Aspergillus nidulans, it is known that a heterotrimeric complex of proteins, the velvet complex, is responsible for regulating secondary metabolism. However, there are many unknown interactors with this complex. We have found one particular gene, rsmD, that shows some qualities of a velvet gene suppressor mutant and that also affects secondary metabolism both if overexpressed and knocked out. In this study we use genetic and biochemical assays to determine what metabolites are affected by overexpression and knock out of this gene, and where the RsmD protein is located within the cell.
|Analise Ruegsegger||Development of a Bioluminescence Resonance Energy Transfer Assay to Monitor the Interaction of ERRa1 and GRIP1
Estrogen-related receptor alpha1 (ERRa1) is a human nuclear receptor that has been causally linked to a subset of aggressive breast cancers, called triple-negative breast cancers, that do not respond to any of the targeted therapies currently in use to treat breast cancer. Triple negative breast cancers account for 15% of all breast cancers, indicating a great need the development of drugs to treat these patients. The activity of ERRa1 depends on protein-protein interactions with co-activators and co-repressors. Disruption of the interaction between ERRa1 and co-activators is the basis of drug development for triple-negative breast cancers. The bioluminescence resonance energy transfer (BRET) assay is a new technique that allows the monitoring of protein interactions in live cells. I have developed a BRET assay that monitors the protein-protein interaction between ERRa1 and the co-activator glucocorticoid receptor interacting protein (GRIP1). I am using the BRET assay to determine the ability of several synthetic and plant-derived compounds to disrupt the interaction between ERRa1 and GRIP1. Compounds identified as inhibitors of this interaction would be potential candidates for the development of novel breast cancer therapies.
|Lauren Marinaro||Discovery of the toxicity of AB-47 and TS-17 vitamin D analogues to various types of cancer in vitro and in vivo
1,25-Dihydroxyvitamin D, the hormonal form of vitamin D, has been found to induce differentiation of human promyelocytic leukemia HL-60 cells in vitro halting their proliferation. TS-17 and AB-47, analogues of 1,25-Dihydroxyvitamin D (1,25-(OH)2D3), have been found to not only halt proliferation through inducing differentiation, but completely exterminate HL-60 cells at 10-5 M in vitro. We have found that a 4-day treatment of various other cancer cell lines (small cell lung carcinoma (H-82), human leukemic monocyte lymphoma (U937), human T-cell lymphoblast-like cells (Jurkat), human embryonic kidney cells (HEK-293), and human keratinocytes (HaCaT)) with TS-17 and AB-47 at 10-5 M results in complete toxicity (0% cell viability) compared to only induced differentiation with 1,25-(OH)2D3 in vitro. Toxicity of AB-47 was further explored in vivo on 6-week old female athymic (Nu/Nu) mice. 3×106 HL-60 cells were subcutaneously injected in each flank. Mice were initially treated with daily intraperitoneal injections of 100 µg/kg body weight (b.w.) AB47 with dosage being increased to 10 mg/kg b.w. based on ability of mice to tolerate high levels of AB-47 compared to 1,25-(OH)2D3 (mice can only withstand 0.5-1 µg/kg b.w. 1,25-(OH)2D3). The ability to tolerate the compound was based on levels of serum calcium in the blood and body weight loss. Further studies are necessary to ensure results, however there exists a correlation between AB-47 treatment and decreased size and number of tumors compared to the vehicle (ethanol) treatment. The ability to administer high doses of AB-47 to limit tumor growth without being toxic to the animal suggests that AB-47 works through a mechanism different than the typical vitamin D. Based on our findings, AB-47 and TS-17 may be of interest as possible cancer treatments.
|Pan San Chan||Effects of Hypoxia on Human Ovarian Cancer Cell Growth
Most cancer cells reside under hypoxia (= 1% O2) in vivo. However, our current knowledge on cancer cells is largely built on cell models established under 21% O2. To closely mimic in vivo condition, herein, we investigated effect of low O2 on cancer cell growth using human ovarian cancer cell lines (SKOV-3 and OVCAR-3) established under 21% O2. Our results showed 1% O2 culture for 6 days significantly decreased cell numbers by 5.4 and 11.0 fold, respectively for SKOV-3 and OVCAR-3 as compared with 21% O2. These data suggest that 1) these cancer cells have adapted to the higher O2 levels and 2) establishing cancer cell lines closer to their in vivo O2 levels might be necessary for better understanding cancer cell behaviors in vivo.
|Anonymous||Effects on Endoplasmic Reticulum Stress and Lipogenesis from Inhibition of Stearoyl-CoA Desaturase-1 in RAW 264.7 Macrophage Cells
There has been an increasing trend in the world for the past few years of metabolic diseases such as obesity and diabetes. It is believed that the inhibition of Stearoyl-CoA Desaturase-1 can help treat these disorders derived from the metabolism based on results from previous studies. The relationship between ER stress and inhibition of SCD-1 provides a possible outlook for solving this crisis. It would be best to explore this topic from a biochemical perspective using many biochemical techniques such as cell culture and RT PCR. The preliminary results show that gene expression levels increase as the concentrations of the treatments (TG, Tm) increase. The conclusions thus far show that there are no definitive relationships that can be made about ER stress and SCD-1 inhibition and further tests are needed to come to a consensus.
|Ken Cleveland||Evaluating the efficacy of fungicide programs for the control of potato early blight in the central sands of Wisconsin
Potato early blight is a perennial and potentially destructive disease caused by the fungus Alternaria solani. Appropriately-timed, effective fungicides are necessary to limit yield and quality loss. In 2010, we evaluated 38 fungicide programs for early blight control at the University of Wisconsin Hancock Agricultural Research Station on ‘Russet Burbank.’ Programs included an untreated control, conventional and organic grower standard programs, and newer chemistries, all replicated 4X and arranged in a randomized complete block design. Programs were initated on 16 Jun and all other production inputs were commercial standard. Plots were treated every 7 days and evaluated for disease bi-weekly using a modified Horsfall-Barratt scale. Plots were machine-harvested on 22 Sep and tubers were graded for size and yield. No tuber early blight was observed and the specific gravities of tubers from top yielding programs were not significantly different. Programs that had the lowest Area Under the Disease Progress Curve values were the highest yielding. The highest yielding program was the Wisconsin conventional grower standard. Organic treatments were ineffective. Several newer chemistries and modified standard programs were effective. At this time, and in the registration pipeline, there are excellent fungicides for the control of potato early blight that will contribute to good fungicide resistance management practices.
|Massimiliano Lehnus||Functional Assessment of the role of Lysine Acetylation of the ER chaperone BiP|
|Patrick Halbach||Gender Dependent Interplay of Iron and Lead in Newborns at-risk for Iron Deficiency
Gender Dependent Interplay of Iron and Lead in Newborns at-risk for Iron Deficiency
Patrick J. Halbach, Beth A. Fischer, Sharon E. Blohowiak, Elizabeth Goetz, Daphne D.-Q. Pham, Anthony P. Auger, Pamela J. Kling
Background: Iron is required for in utero development of the central nervous system. The fetus receives iron through a complex placental transport mechanism facilitated by membrane proteins, including divalent metal transporter 1 (DMT 1). Interrupting this mechanism may compromise fetal neurodevelopment. Lead, in addition to possessing gender-specific, neurotoxic properties, interferes with iron-DMT 1 binding. Objectives: The present study investigates the gender-specific, gestational interaction between iron and lead using the umbilical cord blood of newborns at-risk for iron deficiency. We hypothesized that cord blood lead levels (BLLs) would correlate to cord blood iron status in a gender-specific manner. Methods: In a prospective study of 259 term newborns at-risk for infantile iron deficiency, cord BLLs and iron measures were studied. Risk factors, which were evaluated using medical records and a postnatal retrospective stress questionnaire, included abnormal fetal growth, maternal diabetes mellitus (DM), psychosocial stress, and iron deficiency anemia (IDA). Cord blood iron status was established by measures of RBC iron (zinc protoporphyrin/heme), storage iron (plasma ferritin), and transport iron (plasma transferrin). Cord BLL was measured by Inductively Coupled Plasma Mass Spectrometry. Results: The cord BLLs were neither elevated nor gender-specific. Maternal DM and high maternal stress did not affect cord BLLs, while maternal IDA lowered cord BLLs, p<0.05. Collectively, cord BLLs correlated to worsening RBC iron, p<0.01, but a gender-specific analysis revealed males dominated this correlation. Maternal DM, stress, and IDA caused a direct correlation between cord BLLs and worsening RBC iron in males, while maternal IDA caused an inverse correlation in females. Cord BLLs were not correlated with measures of transport or storage iron. Conclusion: Our findings show males have a stronger correlation between impaired RBC iron and cord BLLs. Although BLLs were low, our work supports the previously suggested phenomenon that lead neurotoxicity is gender specific, with greater deficit in male neurodevelopment than female, despite comparable BLLs. Moreover, our work suggests an interaction between lead neurotoxicity and RBC iron in males. Maternal IDA, with prenatal iron prescribed, may be protective, causing lower BLLs. Our work supports that the interplay between iron and lead status early in infancy is complex, gender-specific, and requires further physiological study.
|Melissa Breunig||Immunomodulatory properties of mesenchymal stem cells derived from human pancreatic islets
Mesenchymal stem cells (MSCs), originally isolated from bone marrow (BM), are a type of stem cell that now can be derived from many other tissues. MSCs differentiate into various cell types, pertinent for application in cellular replacement therapies. However, they also play a significant role in tissue homeostasis through their unique immunomodulatory properties. Type-1 diabetes mellitus is caused by autoimmune destruction of insulin producing pancreatic islets. The objective of this study is to test the hypothesis that MSCs can be expanded from human pancreatic islets and to examine if they have immunomodulatory properties similar to bone marrow derived MSCs. We aim to characterize pancreatic islet MSCs’ (PI-MSCs) immunomodulatory properties through analysis of interferon-? (IFN-?), and intercellular cytokine expression, relative to BM-derived MSCs. PI-MSCs are culture-expanded from human cadaveric islets and characterized by fluorescence activated cell sorting (FACS). Preliminary results indicate that PI-MSCs analyzed by FACS share characteristics with BM-MSCs. We anticipate that PI-MSCs will have an immunophenotype and immunomodulatory properties comparable to BM-derived MSCs.
|James Oldeschulte||Mitochondrial ATP-sensitive potassium channels protect against myocardial ischemia
Sulfonylurea receptors-2A (SUR2A) are the regulatory subunits of the ATP-sensitive K-channels. Mitochondrial membrane potential (mmp) differences in transgenic mice expressing different levels of SUR2A-55 (a splice variant of SUR2A) may explain how the SUR2A and its gene-products affect cardioprotection. Preliminary studies using florescence spectrophotometry and isolated cardiac mitochondria have shown that transgenic mice expressing 3 times as much SUR2A-55 (tg+3) have decreased mmp (7.258±0.672% afu) compared to wt (10.35±0.7523% afu). Also, tg+3 mitochondria are resistant to Ca2+ loading (40uM Ca2+ to achieve 40% depolarization; 30uM Ca2+ in wt). This decreased mmp and tolerance to Ca2+ are indicative of a cardioprotective phenotype.
|Trever Greene||Mutagenic analysis of a potential self-ligand in the voltage gated potassium ion channel mEAG
The Ether a go-go (EAG) family of channels are voltage gated potassium channels important in many physiological processes and implicated in the progression of certain cancers. The EAG family shares a region of homology with the cyclic nucleotide gated (CNG) family of channels. Interestingly, while the cyclic nucleotide binding domain (CNBD) is highly conserved in EAG channels, they have been shown to be unaffected by cyclic nucleotides. Based on homology modeling we hypothesize that the side chains of two of the CNBDs residues fill the binding pocket where the cyclic nucleotide would normally sit, substituting for the cyclic nucleotide and allowing the channel to undergo self-gating. This project used site directed mutagenesis in combination with two electrode voltage clamp to analyze the function of these residues in mEAG channels. Modifications of these residues resulted in phenotypes ranging from channels with complete abrogation of current, to channels with currents identical to wild-type. Overall our studies indicate this region of mEAG’s CNBD is important in the development of functional channels, although the mechanism has yet to be elucidated.
|Kayla McKaveney||Roles of Interleukin-4 Receptor a-Chain on Glioma-Infiltrating Monocytes
Gliomas account for nearly 80% of primary malignant brain tumors. Several epidemiological studies have indicated association of single nucleotide polymorphisms and haplotypes in the IL-4Ra gene with altered glioma risk and prognosis. IL-4Ra is expressed on immunosuppressive cells of monocyte lineage and mediates their production of transforming growth factor (TGF)-ß in response to IL-4 or IL-13. Thus, it was hypothesized that IL-4Ra expression on monocytes would play a significant role in glioma development. We induced de novo gliomas in BALB/c-background mice by intracerebroventricular transfection of oncogenes using the Sleeping Beauty transposon system. IL-4Ra-deficient (Il4ra-/-) mice exhibited significantly prolonged symptom-free survival compared with WT mice; median survival was 89 and 55.5 days, respectively. Consistently, gliomas induced in the WT mice were infiltrated with significantly higher numbers of CD11b+Gr1+ immunosuppressive monocytes than those in the Il4ra-/-mice. We subsequently isolated glioma-infiltrating CD11b+Gr1+monocytes to address their functions. RT-PCR and ELISA revealed that the monocytes derived from WT mice express significantly higher levels of TGF-ß. As it is possible that type-1 skewed T cells in Il4ra-/- animal contributed to the observed better survival compared to WT mice, we next depleted CD4+ and CD8+ T cells using antibodies. While T-cell depletion shortened the overall survival of both WT and Il4ra-/- mice, T-cell depleted Il4ra-/- mice still exhibited enhanced survival over T-cell depleted WT mice. Taken together, these data suggest that IL-4Ra expression on glioma-infiltrating monocytes may promote the immunosuppressive microenvironment of gliomas through a variety of mechanisms including TGF-ß production and T cell inhibition, thereby facilitating glioma development.
|Victoria Ziebert||Sharing rates of gargle repertoires of Black-capped Chickadees (Parus atricapillus) from two locations in southern Wisconsin
Black-capped Chickadees (Parus atricapillus) are locally resident throughout the year. Therefore, it is assumed they have very little contact with populations other than their own. This species learns their vocalizations as young from their parents. The gargle is a specific, defensive vocalization produced by this species. Past studies have shown a difference in gargle repertoires among populations that are relatively near each other. This study tests this conclusion with recent equipment. Black-capped Chickadee subjects were captured from two populations located near Madison, Wisconsin. Gargles were collected using recording equipment on the University of Wisconsin – Madison campus using birds’ reflections as a dominance challenge. Gargles were recorded and analyzed from captured specimen using computer software. The gargles were tested for patterns and differences between the populations. There was a definite pattern of shared gargles within a population, and a lack of a pattern when comparing the two populations. This evidence supports the theory of varying repertoires between populations. T tests showed significance in two scenarios and a lack of significance in another; however, this is probably due to a lack of sample size.
|Steven Marmer||Stability of Zinc Protoporphyrin/Heme Ratios on Filter Paper as a Newborn Screen for Tissues Iron Deficiency
Background: ID, a common nutrient deficiency, impairs brain development. With earlier identification of at-risk children, long-term neurocognitive deficits could be prevented. ZnPP/H is an available, cost effective, and sensitive biomarker of iron status. ZnPP/H, a candidate for newborn screening of iron deficiency, is measurable on washed umbilical cord blood and potentially on filter paper blood spots. Bilirubin interferes with ZnPP/H readings, but interference is reduced with Bilirubin Oxidase (BO). Objective: To examine whether ZnPP/H from filter paper specimens is comparable to washed blood specimens. Methods: De-identified cord blood samples were collected at birth in EDTA and whole blood and washed ZnPP/H ratios were measured by hematofluorometry. Blood was spotted onto Whatman 903 filter paper. Specimens were dried and eluted from the paper using different reagents; PBS elution (control), superoxide dismutase (SOD) and catalase to prevent oxidation of heme from lysed RBCs, and BO to prevent heme breakdown to bilirubin and other autofluorescent molecules. All treatments were repeated at 1, 2, and 7 days to measure the ratio’s stability over time. Results: Filter paper ZnPP/H ratios were higher when eluted, compared to rinsed blood. With = 24 hrs on filter paper, elution with PBS was highly correlated with rinsed blood R2=.74, p<0.01, with slight improvement from SOD/Catalase, R2=.81, p<0.01, as well as BO, R2=.85, p<0.01. With increased time on filter paper, variability of the readings increased and the correlations were weakened. Compared to control PBS, SOD/Catalase and BO yielded stronger correlations which persisted over time. Conclusion: With increasing time on filter paper ZnPP/H readings deteriorated. The relationship between filter and washed ZnPP/H deteriorated less rapidly when eluted with either SOD/Catalase or BO. Although it is unclear what biochemical mechanisms are responsible for this deterioration, our treatments prevented both heme oxidation and heme breakdown. Further studies are needed to determine the biochemical changes in ZnPP and heme on filter paper, but using filter ZnPP/H on dried blood spots may still be feasible.
|Nick Jikomes||The evolution of novel enhancer elements
Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements (“enhancers”) that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve.
|Hannah Lincoln||The Synthesis of Phosphatidic Acid by Diacylglycerol Kinase Zeta is Sufficient to Induce Skeletal Muscle Hypertrophy
Previous studies have shown that activation of the mTOR pathway is sufficient to induce skeletal muscle growth (Glass, 2003). Signaling through mTOR has also been shown to be necessary for skeletal muscle growth in response to stimuli such as mechanical loads. It has been proposed that mechanical loads activate mTOR through a mechanism involving phosphatidic acid (PA). However, it is not known if an elevation in PA is sufficient to induce skeletal muscle growth. There are numerous mechanisms for regulating the level of PA, including the conversion of diacylglycerol (DAG) to PA by diacylglycerol kinase (DGK). In fact, in HEK(293) cells, over expression of DGK has been shown to promote mTOR-dependent signaling to substrates such as p70S6K (Avila-Flores et al., 2005). Therefore, to determine if an elevation in PA is sufficient to induce skeletal muscle growth we over-expressed DGK zeta in skeletal muscle fibers of the mouse tibialis anterior muscle. The results of this study indicate that over-expression of DGK is indeed sufficient to induce a hypertrophic response, and DGK kinase activity is necessary for this event. Thus, we conclude that elevated levels of PA are sufficient to induce skeletal muscle hypertrophy.
|Andrew Kornaus||Verification of Optical Structure Aberrations in Two Oligodendroglioma Tumors
Many large- and small-scale changes occur in the genomes of tumor cells. The optical mapping technique was used previously to determine where large changes in the genome of tumors have occurred, as well as smaller optical structure aberrations (OSAs). OSAs include single basepair changes, which result in missing or extra restriction enzyme sites in the genome, or small insertions and deletions, which can change the local size of restriction fragments at a locus. OSAs can be verified by polymerase chain reaction (PCR) of the regions that differ from reference human sequence. This study looks at two oligodendrogliomas – tumors that have arisen from oligodendrocytes, the cells that insulate axons within the brain.