|Sarah Gleason||A comparison of chromic oxide and yttrium oxide as inert markers for determination of digestive efficiency in Lithobates pipiens tadpoles
The uptake of nutrients, or digestive efficiency, is important for determining an animal’s energy budget, especially those that are sensitive to temperature fluctuations that may affect these processes. Although digestive efficiency is a useful tool, the procedure for its determination has not been extensively studied in Lithobates pipiens tadpoles. No study has been conducted to determine whether chromic oxide or yttrium oxide serve as accurate inert markers that do not affect digestive efficiency. This study will focus on answering these questions by using 3 treatment groups; one control, one treatment using 1% chromic oxide spiked food and the last using 1% yttrium oxide spiked food. Feces of 5 individual tadpoles from each treatment will be collected and pooled into each respective treatment to create one replicate. It is predicted that neither marker will affect digestive efficiency, however yttrium oxide may be a more accurate inert markers because smaller fecal masses are needed.
|Joseph Kutschenreuter||Analysis of Genetic Interactions in Gravity Signal Transduction in Arabidopsis thaliana
Gravitropism is the process by which plants direct root and stem growth in response to gravity. The mechanical force of gravity is converted to a chemical signal in a process not fully understood. This investigation is of the genetic relationships between genes known to function in gravitropism (PIN3, ADK1, and MAR2), providing insight into the gravity signal transduction pathway. I created two double homozygous knockout mutant lines (pin3 mar2 & adk1 mar2) and analyzed their respective root phenotypes. The current results support a new genetic pathway model of gravity signal transduction.
|Jessica Kazmierczak||Cellular Gene Regulation by Overexpression of the Epstein-Barr Viral Protein EBNA1
EBNA1, Epstein-Barr Nuclear Antigen 1, encoded by the Epstein-Barr virus (EBV) has been identified as a survival factor for Burkitt’s lymphomas, as it is necessary for B cell proliferation. Using cellular DNA sites to which EBNA1 is bound, along with EBNA1-binding sites in the viral genome, a consensus sequence depicting EBNA1’s DNA binding sequence was recently generated by Dresang et al. (2009). This sequence can now be used to search for additional sites in the human genome to which EBNA1 may bind. This thesis project seeks to identify precisely which cellular genes are regulated by EBNA1’s binding them. EBNA1 is a transcriptional transactivator that causes reduced cell survival when it is overexpressed in B cells. To accomplish this goal, cell lines that inducibly overexpress EBNA1 have been generated. Ultimately, microarray analysis results will be discussed using isolated RNA from these cell lines in order to identify which cellular transcripts are changed in their levels, which of these are predicted to bind EBNA1, and which are thus likely to be regulated by EBNA1’s binding.
|Bingming Chen||Detection of Filamentous Salmonella on Low-aw Food Items and Enumeration method of Filamentous Salmonella
Under certain stresses, Salmonella can form multi-chromosomal filaments, which can septate under favorable conditions. Filaments also form a single colony on a plate, which would lead to underestimating the number of viable cells. Filamentous Salmonella were detected on the surface of dried food items. Therefore, it is important to develop an enumeration method that accounts for each filament septating into multiple viable units. By incubating filamentous Salmonella cells in 0.2x Luria-Bertani broth at room temperature for 8 hours, the percentage of filamentous cells decreased from 70% to 18% of the total number of cells. Concurrently, the concentration of cells increased, indicating septation and division. Therefore, it is important to develop a method for enumerating the number of viable cells of the filamentous Salmonella.
|Joel Prince||Determination of ubiquitin dimerization kinetics via UV spectrophotometry
The Ubiquitin–Proteasome System (UPS) is integral to the regulation of cellular protein homeostasis. The affixation of a polyubiquitin tag to a substrate is a common signal for 26S proteasomal degradation, and results in the lysis of the substrate into constituent amino acids. The basic unit of this signal is the ubiquitin monomer. These units are iteratively linked through an amide-forming reaction between the side chain of one of the seven lysine residues of one ubiquitin moiety and the carboxy-terminus of the next ubiquitin, eventually generating a polyubiquitin tag. The most thoroughly characterized polyubiquitin tags are homopolymers of the Lys48 and Lys63 linkages. We have designed a kinetic assay to compare the initial dimerization reaction of all seven linkages. This was accomplished via measurement of the release of the UV-active 2-nitro-5-thiobenzoate. Ultimately, these results will aim to explain why certain linkages are more commonly observed in vivo. Since it has previously been shown that human ubiquitin possesses 12 dehydrons, it is also of interest to determine whether the distribution of linkages observed in vivo is due to the specificity of numerous ubiquitin ligases or the favorable association of dehydrons.
|Kia Ludwig||Dietary supplements, conjugated linoleic acid (CLA) and ginger extract, as preventive and therapeutic agents for gynecological cancer
Ovarian Cancer and related carcinomas affects many women annually and often, treatments are limited to halt or suppress the cancer’s progression. In an effort to identify new prevention and treatment strategies for gynecologic cancers, we are investigating the potential of natural dietary substituents. We tested the potential of Conjugated Linoleic Acid (CLA), found in rudiment products, dairy, and other meats along with essential oils isolated by steam distillation of ginger rhizomes on the proliferation of ovarian and endometrial cancer cell lines. The trans10:cis12 isomer of CLA significantly inhibited the growth of the ovarian cancer cell lines A2780, SKOV-3, and CaOV-3 at an IC50 of 7 µM. Treatment with t10:c12 CLA did not result in cell death. Instead, this agent caused cell cycle arrest and autophagy in the ovarian cancer cell lines. The ginger extract had limited effect on the growth of ovarian cancer cell lines, however, it was highly potent in blocking the proliferation of two endometrial cancer cell lines, Ishikawa and ECC-1 at 300 pg/ml concentrations. The ginger extract induced apoptosis in the endometrial cancer cell lines. Our research indicates that supplementation of these natural dietary products, will be effective in preventing and treating patient with ovarian and endometrial malignancies. Our on-going research will investigate the combined use of chemo- or radiation therapy along with CLA or ginger extract as a treatment modality for gynecologic cancer.
|Kelsey Kyle||Differences in Pragmatic and Cognitive Disorders in Individuals with Traumatic Brain Injury|
|Yamini Karandikar||Effect of Climate Change on Tannin Levels in Aspen and Birch
To test the effects of climate warming on tree defensive chemistry, we measured condensed tannin levels in aspen and birch plant leaves. Tannins are used as a defense mechanism by plants to protect from herbivory. The Growth Differentiation Balance Hypothesis states that as plants gain the ability to grow faster, less energy is allocated towards defense. Under warmer growing conditions, such as those experienced under predicted future temperatures, we expect plants to grow faster and produce less condensed tannins. To test this, leaf samples from aspen and birch trees grown under control, +1.8, and +3.6 C temperature-controlled forest plots were analyzed for tannin content. We found that birch and aspen grown under higher temperatures have lower levels of condensed tannin.
|Cristina Vaughan||Gender Determination of Two-toed Sloths (Choloepus hoffmanni) with the SRY Gene
It is difficult to identify the sex of juvenile two-toed sloths (Choloepus hoffmanni) due to genitalia that are not fully developed. To aid field biologists in collecting demographic information on this species of conservation concern, we developed a modified polymerase chain reaction (PCR) amplification of a Y chromosome-linked gene, the sex-determining region Y (SRY), to determine the sex of individual sloths. Mature sloths of known sexes were used to test a previously developed SRY amplification routine and optimize the technique. When electrophoresed on an agarose gel, the amplified SRY region was visualized at 466bp when the sloth was male. If the sloth was female, no band was detected. Testing was performed on 47 adult sloths of known sex (22 male and 25 female), which resulted in a 6.9% error rate. Of the 35 unknown juveniles tested, 20 were male and 15 were female. The ability to identify sex of juvenile sloths will aid researchers by providing them with more detailed information about the two-toed sloths which can in turn aid in answering questions of population ecology that require an understanding of sex ratios.
|Corie Borchert||Inactivity-induced phrenic motor facilitation is associated with decreased phrenic burst-to-burst variability
Reduced respiratory neural activity in ventilated rats elicits rebound increases in phrenic discharge once neural activity has been restored, a form of plasticity called inactivity-induced phrenic motor facilitation (iPMF). We hypothesized that iPMF is associated with reduced phrenic burst-to-burst variability. Phrenic discharge was measured in anesthetized, vagotomized and ventilated Harlan (H) and Charles River (CR) Sprague Dawley rats exposed to a 30 min neural apnea or an equivalent duration of baseline conditions (time control). As expected, H rats expressed iPMF at 5 and 60 min (p<0.05) post-apnea, whereas CR rats expressed iPMF only at 5 (p0.05) min. Inspiratory time (Ti), expiratory time (Te) and peak area variability was assessed using Poincare plot analyses for burst n vs. burst n+1 before, and 5 and 60 min post-apnea (400 consecutive bursts). In H rats exposed to a neural apnea, standard deviations of the Poincare plot (SD1 and SD2) for Ti and Te were significantly decreased at 5 and 60 min post-apnea (p< 0.01). In CR rats exposed to a neural apnea, significant decreases in SD1 and SD2 for Ti and Te were observed at 5 (p0.05) min post-apnea. No changes in SD1 or SD2 for Ti or Te were observed in time controls (p>0.05), and no changes in SD1 or SD2 for peak area were observed in any rat group (p>0.05). These data suggest that iPMF is associated with decreased variability in Ti and Te. UW-Madison Regent Scholars Fund.
|Joe Schmidtt||Inhibiting Androgen Receptor and JunD protein interaction to prevent prostate cancer progression
Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS plays a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa.
A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as AR-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 µM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented.
|Kayla McKaveney||Interactions of Ssz1 and Zuo1 with transcription factor Pdr1 in S. cerevisiae
The phenotype of pleiotropic drug resistance is characterized by the action of ATP-binding cassette transporters, which allows S. cerevisiae to pump toxins beyond the confines of its outer membrane. Transcription factor Pdr1 regulates pleiotropic drug resistance through the transcriptional activation of PDR5 and other ABC transporter genes. The Hsp70 Ssz1 and J-protein Zuo1 function as molecular chaperones in a trimer with Ssb1/2; however, altered forms of Ssz1 and Zuo1 independently induce pleiotropic drug resistance in a PDR1-dependent manner. The involvement of Ssz1 and Zuo1 in pleiotropic drug resistance suggests that the two proteins are each capable of binding to Pdr1 or acting in a pathway leading to conformational changes in Pdr1. Previous study using a yeast two-hybrid system provides evidence of interaction of Zuo1 variant ZuoC* with Pdr1. GST- and TAP-tag pulldowns will evaluate interactions between Ssz1*/ZuoC* with domains of Pdr1 based on their ability to be co-purified in vitro.
|Grant Barber||Methods for Identifying Substrates of Atypical Mitochondrial Kinases
Defects in Coenzyme Q (Q) biosynthesis cause human disease and may have a significant impact on aging. Particularly, patients with mutations in an atypical human mitochondrial kinase develop Q deficiency and progressive cerebellar ataxia. However, the mechanisms by which this kinase affects Q biosynthesis and ataxia are unknown. Identifying the endogenous substrates of this atypical kinase would represent a key advance in our understanding of its function. Toward this end, we performed multiple in vitro experiments. Radiolabeling of mouse mitochondrial lysates with 32P-ATP and the atypical kinase revealed lipid phosphorylation. We are now testing specific lipids as substrates using a high-throughput in vitro kinase assay (ADP-Glo) we developed for measuring this kinase’s activity. We have also created analog-sensitive mutants of this kinase as tools to selectively manipulate its activity and are testing their activity. Our work toward discovering substrates of this atypical kinase represents important progress in characterizing its biological role in coenzyme Q biosynthesis and cellular aging.
|Nicholas Sanchez||Polyubiquitin Formation via Thiol-ene Chemistry
Ubiquitin is a post translational modification protein that is used by all eukaryotic cells for numerous cell functions. It functions through polymers of its c-terminus and one of seven lysine residues. We developed a method to synthesize polyubiquitin chains for laboratory use via thiol-ene chemistry. Our method involved the mutation of one lysine residue to a cysteine residue and a radical reaction with allylamine-ubiquitin to form a specifically linked ubiquitin dimer. All seven cysteine mutants successfully reacted with allylamine-ubiquitin to form a dimer. The formation of dimer is the first step to developing a reliable method to synthesize polyubiquitin chains of specific chain length and linkage and is key to better understand ubiquitin’s function in eukaryotic cell processes.
|Kelsey Kyle||Pragmatic and cognitive disorders in individuals with traumatic brain injuries (TBI)
A traumatic brain injury (TBI) can result from a sudden bump, jolt, or blow to the head. Individuals with TBI may acquire disorders in communication and cognition, including memory loss, comprehension difficulties and loss of basic social skills. Conscious memory for facts and events, explicit long-term memory, is often impaired while the memory of habits and procedures, implicit long-term memory, is typically intact. The role of implicit memory in communication after TBI is not known. We predict that individuals with TBI will have normal performance on aspects of communication that are related to implicit memory. We will present experiments that are designed to identify the memory functions that imply a role for implicit memory in TBI patients.The results of our research will help us develop better strategies for rehabilitation of social and communication skills in adults with TBI.
|Roger Diehl||Proline’s Preferential Interactions with Biopolymer Surface in Aqueous Solution
Protein folding is a very active area of study in modern biochemistry, as the three-dimensional structures of proteins are vital to their proper function but are only marginally stable, and the thermodynamics of folding can be changed by small molecule solutes through preferential noncovalent interactions biopolymer surfaces relative to interactions with bulk water. As noncovalent interactions are short-range, they are assumed to be roughly proportional to the accessible surface area (ASA), and the chemical nature of the surface, and are quantified by the derivative of free energy with respect to solute concentration, or µ23, with a positive µ23 indicating an unfavorable interaction. Once the strength of these preferential interactions is known, it will be possible to predict the effects of a solute on a process with known surface area change, or alternately to use solutes as probes to determine the amount and type of surface area buried in a given biopolymer process. This study examines the effects of proline, a free amino acid accumulated by E. coli in response to osmotic stress.
Interactions were determined by measuring the effect of proline concentration on equilibrium processes involving small molecules with several surface types similar to those found in proteins. For most model compounds, vapor pressure osmometry was used to determine the change in activity resulting from proline-model compound interactions. Direct measurements of solubility and micelle formation equilibria were used to determine proline’s interactions with marginally soluble model compounds and SDS tails. Proline shows a large and statistically significant unfavorable interaction with aliphatic surface, and a statistically significant favorable interaction with aromatic surface. Proline also appears to interact unfavorably with hydroxyl and carboxylate surface and favorably with cationic N and amide N surfaces, but these values are not statistically significant due to large uncertainty values. Interactions with amide O surface appear to be nearly neutral. As most protein folding processes primarily bury aliphatic surface, this would suggest that proline stabilizes the folded state of most proteins.
|Angela Schnieder||Protein Expression in the African Trypanosomes
The VSG surface coat of African trypanosomes has been found to be not linked to differences in virulence levels of the strains Trypanosoma brucei rhodesiense LouTat 1 and LouTat 1A. Protein extraction from the cells of trypanosomes of both LouTat 1 (low virulence) and LouTat 1A (high virulence) were analyzed using 2 dimensional analysis to determine whether there is differential protein expression associated with virulence levels of these strains. The first dimension of the gels used IPGphor to separate the proteins by pH whereas the second dimension used gel electrophoresis to separate the proteins by mass. The gels from LouTat 1 and LouTat 1A were then analyzed by computer to find differences in protein expression. The existence of certain unique protein spots on LouTat 1 gels in comparison to LouTat 1A gels as well as large differences in expression of certain proteins of one strain compared to the other was observed. This implies that there may be a correlation between protein expression and virulence in these strains of African Trpanosomes.
|Amy Davis||Ribonucleotide pro-moieties for time-dependent drug release
Time-release drug strategies are of critical importance in pharmaceutical drug design, where ability to control plasma concentrations of a drug yields more effective dosing strategies. Time-release, or extended-release strategies often involve the encapsulation of bioactive materials within an insoluble matrix to prevent instant metabolism and uptake. Encapsulation strategies range from use of phospholipid-derived micelles to use of indigestible, matrix-bound oligosaccharides of cellulose or lactulose. This project investigates a different method of time-dependent release: the inactivation of antibiotic drugs through phosphodiester linkage with a ribonucleotide, and release via endogenous ribonuclease. Human plasma contains a vast number of endogenous ribonucleases that can catalyze the cleavage of RNA molecules into their ribonucleotide subunits. More specifically, the enzyme RNAse 1 cleaves the internal phosphodiester bonds of ribonucleotides on the 3’ end of thymidine bases. In this project, we coordinated the antibiotic metronidazole to adenine-, cytosine-, and thymine-3’-monophosphate through ester linkage. Due to the ubiquitous nature of ribonucleases, the active drug is cleaved in vivo from the ribonucleotide in a time-dependent manner. In this study, RNAse 1 is used to measure the time-release kinetics of the antibiotic metronidazole, and anaerobic bacteria culture is used to measure the mean inhibitory concentration (MIC).
|Mackenzie Egan||Role of Aging and DNA Promoter Methylation in Rat Colonic Tumor Formation
Colon cancer is responsible for tens of thousands of deaths in the United States every year. Following surgical removal of tumors, new tumors often form. This phenomenon could be explained by a field effect, where the colonic epithelium DNA is mutated or epigenetically altered to predispose the surrounding tissue to tumor formation. Aging appears to play an important role on epigenetic changes such as DNA methylation. The association between aging and DNA methylation may contribute to epigenetic silencing of tumor suppressor genes through promoter methylation, but this relationship to colonic tumor formation and the field effect remains unclear. Our laboratory uses a robust animal model of human colon cancer called the Pirc rat. DNA excised from colonic tissue of young and aged Pirc rats provide the material for methylation analysis by bisulfite pyrosequencing. Specifically, the assays measure global DNA methylation of Long Interspersed Elements (LINE) as well as two genes known to be silenced in human colon cancer, O6-methylguanine-DNA methyltransferase (MGMT) and MutL Homolog 1 (MLH1). Finding a correlation between DNA methylation and age in normal colonic tissue will help elucidate any association between aging and promoter methylation, illuminating the field effect hypothesis in colon cancer.
|Tyler Wied||Separation of chemical interactions and volume effects of solute size on DNA triple helix stability
Biopolymer processes such as protein folding or DNA helix formation are affected by solutes due to favorable or unfavorable chemical interactions of the solute with functional groups within the biopolymer. Large solutes also affect biopolymer processes because they exclude volume and create steric crowding effects. Previously, we developed a model to separate chemical effects and volume effects of a solute on a DNA duplex and hairpin. Here, we expand upon that model with a DNA triplex. For this study, we used 12-mer strands of DNA that associate via intermolecular forces into a DNA triplex. The triplex consists of two strands bound by Watson-Crick base pairing. The third strand is bound in the major groove of the other two, called Hoogsteen base-pairing. We studied the effect of a full range of polyethylene glycols (PEG), from the monomer ethylene glycol to PEG 20,000. From preliminary results, we find that the association of the Hoogsteen bound strand is stabilized by large PEGs at constant monomer concentration whereas the association of the Watson-Crick strand is destabilized by small solutes. These observations are consistent with our model that volume exclusion effects increase and chemical interactions diminish as PEG size increases.
|Kelsey Phillips||Spatial and temporal variation of bacterial communities in a north temperate bog lake
Humic bog lakes are ideal systems to investigate microbial communities due to easily identified spatial boundaries and well characterized seasonal ecosystem dynamics. Permanently stratified lakes such as Mary Lake in northern Wisconsin provide well-defined thermal layers with different physical and chemical conditions, each thought to harbor distinct microbial communities. Multiple biotic and abiotic factors are thought to influence these communities, and our goal in this study was to analyze the spatial and temporal factors causing the variations between populations within the lake. Molecular techniques and statistical analyses were performed to gain insight into these differences and determine which caused the greatest variability. The bacterial communities in the bog were analyzed based on conserved 16S ribosomal RNA sequences (ARISA) as well as on short base pair sequence reads obtained from a sample time series in 2009. Variations between lake depth, thermal layer, and through time were calculated and assessed in order to determine whether the bacterial community was more variable in space or in time. We found that while the upper and lower thermal layers of the bog were composed of similar microbial communities, these communities had different temporal patterns throughout the time series.
|Amrit Kanwar||Suicide and Anxiety Disorders: A Meta-Analysis
According to the National Institute of Health, in the United States suicide is the tenth leading cause of death. However, the predictive factors of suicide are not well defined and have not been well-studied. Even though depression is common in people who commit suicide, the predictive value of anxiety in suicidal patients is not well known and very little research has been conducted in this field.
|Linda Zhou||Testing a possible metabolic role of plastids and how it may be involved in the gravitropism signal transduction pathway
Gravitropism is a process in which plant roots grow downward due to a gravitational vector. One of the dynamic components involved in gravitropism is the multi-functional organelle known as a plastid. Turning the plant on its side causes the starch-filled plastids in the root tips to fall downward, which results in roots elongating down. However, plastids also have metabolic functions that may be involved in gravitropism.
I researched six candidate genes that have metabolic functions in plastids. I have obtained seeds carrying mutations in these genes and we will cross these plants with known mutants that have been shown to be involved in gravitropism. The objective of this experiment is to not only further elucidate the molecular mechanism of gravity sensing by identifying new genes but also to consider the involvement of metabolic functions of plastids in the gravitropism signal transduction pathway.
|Patricia Gail LaVoie||Treating estrogen receptor alpha (ERa) and estrogen related receptor alpha (ERRa) positive breast cancer cells with an ERRa specific inhibitor to reestablish tamoxifen (TAM) sensitivity
Seventy-percent of breast cancers are ERa positive cancer, and the drug treatment prescribed is TAM. TAM inhibits growth of ERa-positive tumors, but 50% of tumors
inhibited develop TAM resistance (TAM-R) from loss of ERa expression. ERRa may substitute for ERa to continue cancer growth. The objectives of our study include treating TAM-R, ERa-positive, and ERRa-positive cells with XCT790 and determining whether TAM sensitivity is reestablished. We performed cell culture, whole cell protein extraction, and western blotting analysis to determine MCF-7, HER2-18, SKBR3, and BT474 lines expression of ERa and ERRa. Our results showed that MCF-7, HER2-18, and BT474 are positive for ERa and MCF-7, SKBR3, and BT474 are positive for ERRa. This could mean that TAM-R, as indicated in the literature, is not dependent on ERRa within the HER2-18 cells. Due to our novel data on HER2-18, it is necessary to broaden our scope of cell research and create a panel of cells that are TAM-R, ERa-positive and ERRa-positive. The goal of future research will be treating with XCT790 to revert cell lines to the ERa pathway providing treatment to cancer patients in the form of long-lasting TAM.