2013 Presenters

Emily Fischer Sequencing genes on chromosomes 1 and 17 to find modifiers of hepatocellular carcinoma

Hepatocellular Carcinoma is one of the leading contributors to cancer deaths world-wide. The study of this disease in mice has determined many chromosomal regions that likely contain tumor-inducing genes, two of which are located on chromosomes 1 and 17.1 Locus Hcf1 of chromosome 17 affects the production of tumors, and females of the C57Br/cdJ (BR) strain are much more susceptible than those of the C57BL/6J (B6) strain.2 The genes in this region are being sequenced so that strains can be compared for potentially causative polymorphisms. Many genes have already been sequenced, but it is important to know the allele composition of every gene in the locus, for accurate comparison between strains. The sequencing of the remaining BR genes will be done using PCR and Sanger Sequencing. Once known, the sequence will be compared to C3H/HeJ (C3H), a resistant strain, to determine if the sequence of DNA in BR is unique. It is expected that a polymorphism will be found which is real and can be linked to the production of tumors in the liver.

Kayla McKaveney Saccharomyces cerevisiae chaperones Ssb and Zuo1 influence long-term cell viability and population growth

Molecular chaperones assist in protein folding, transport, and degradation. The action of chaperones prevents the buildup of protein aggregates within the crowded environment of a cell. In Saccharomyces cerevisiae, the J-protein Zuo1 stimulates the ATPase domain of Hsp70 Ssb. ATP hydrolysis by Ssb drives transient binding of nascent polypeptides and protects exposed hydrophobic residues from aggregation. This interaction also influences the total density reached by cells in liquid culture; deletion of any or all of the Zuo1-Ssb complex results in cell overgrowth compared to wild-type cultures. The mechanism behind the overgrowth is not known. We investigated the conditions necessary for this phenotype and determined that both glucose and nitrogen must be restricted in order to confer a growth advantage to cells without the functioning chaperones. Future investigations will focus on whether overgrowth possible nutrient sensing actions are related to Zuo1-Ssb chaperone activity or an unknown function of the proteins. Determination of novel activities of Zuo1-Ssb may enhance understanding of physiological drivers of the population dynamics and longevity phenotypes.

Jennifer N. Molenda Generating Mutant Lines Expressing Auxin-Sensitive Promoters to Investigate the Role of GLRs in Arabidopsis thaliana Root Development

Plant GLUTAMATE RECEPTOR-LIKE (GLR) genes have been shown to play a role in amino acid-gated Ca2+ channels at the plasma membrane. By creating knock-out mutants in the members of the GLR gene family, the resulting loss of function in Ca2+ channels may affect lateral root development. To test this hypothesis, mutant lines glr3.2 and glr3.4 were created with vector recombination in Arabidopsis thaliana. These knockouts were isolated for homozygosity of the mutated allele, and then for homozygosity of the GUS or GFP-tagged DR5 auxin reporter. DNA isolation, PCR, and microscopy were used to confirm the genetic identities of these lines. These genotypes will be used to determine whether GLRs play a role in auxin sensitivity within root phloem.

Michael Voss The Jaw and SI3 Domains Regulate Transcription through Interactions with the Trigger Loop Structure in Escherichia coli RNA Polymerase

The goal of this research is to improve current knowledge of the fundamental mechanisms of transcription by expanding the understanding of RNA polymerase (RNAP) structure. The RNAPs of many bacterial species contain structures called sequence insertions (SIs), which are not present in the RNAPs of archaea or eukaryotes, and whose functions are relatively unknown. This project will focus on defining the functions of two lineage-specific SIs in Escherichia coli RNAP (EcoRNAP) called the jaw and SI3 domains. Studies of amino-acid deletions within these structures will examine their possible regulatory functions by observing changes in the rates of transcriptional elongation and pausing through in vitro biochemical assays. Due to the location of these structures in EcoRNAP, this project will also examine the extent of their interactions with the trigger loop (TL), which is the primary flexible active-site structure. Observing the effects of these deletions on the TL’s mobility will be accomplished by forming disulfide bonds between mutant cysteine pairs only capable of reacting in certain TL conformations. Combining these results will determine if the SI3 and jaw domains have regulatory roles in EcoRNAP and if this function is a result of their altering TL movements during transcription.

Amy Whillock The Role of Tcf19 in the Pancreatic ß-cell and Diabetes

Insulin is necessary for transporting glucose from the blood into the cells and is produced by the ß-cells in the pancreas. In diabetics, reduced numbers of functional ß-cells lead to insufficient insulin production, resulting in high blood glucose. Tcf19 is a new factor and may play a role regulating the growth of the ß-cell. We have demonstrated that knockdown of Tcf19 expression in ß-cells leads to reduced cell growth. We will examine the protein interactions of Tcf19 and its specific role in ß-cell growth. We will pull down Tcf19 using an antibody to identify its binding partners. The results of this experiment will clarify the role of Tcf19 in ß-cell growth, and could help identify a new therapeutic target to help treat or cure diabetes.

Abigail Gadbois Spatial scales of gene flow and genetic structure in Maianthemum canadense based on next generation sequencing

My project is to quantify the spatial scales of gene flow and genetic structure of populations of the wildflower Maianthemum canadense, in conjunction with a larger project studying changes in other understory species. Using tissue samples collected and preserved over silica gel the previous summer, I will be isolating DNA from which I will prepare a double digest restriction size associated DNA sequencing (ddRADseq) library for Illumina HiSeq2000 sequencing. Sequence data will be compiled into a genetic library for the species, and using the programs SPAGeDI and Structure, we will assess the spatial scale of gene flow and analyze the variance within and between populations of this herb to quantify the genetic diversity of Maianthemum canadense.

Thomas Alderson Structural and kinetic studies on the isolated nucleotide-binding domain of Escherichia coli protein heat shock cognate A
Madeline Ford Examining Field Cancerization in the Pirc Rat Colon Cancer Model
Alexandra Linz Overexpression of the Rnf complex to increase hydrogen production in Rhodobacter sphaeroides

Photosynthetic bacteria have the potential to produce biofuels and other useful compounds using only sunlight and waste carbon. The purple non-sulfur bacterium Rhodobacter sphaeroides produces hydrogen gas under photoheterotrophic conditions via the nitrogenase enzyme. To make microbial production of hydrogen gas viable, the rate of hydrogen production must be increased. This can be accomplished by making more reductant available for nitrogenase to use. The Rnf complex is part of the cell’s electron transport chain that reduces ferredoxin, which then passes its electrons to nitrogenase. In this research, we attempt to overexpress the Rnf complex by creating strains in which the rnf operon is controlled by different promoters. A subset of these strains were found to have higher hydrogen yield than the corresponding strains in which the rnf operon is controlled by its native promoter. This result could be used in combination with other factors to create a R. sphaeroides strain with extremely high hydrogen yield that is suitable for use in industry.

Kaitlin Wollak Specificity of the Attine-microbe relationship: Analysis of optimal inhibition of the parasitic fungi Escovopsis by Actinobacteria
Samuel Ekstein Postmenopausal Hormone Replacement Therapy Controversy at the Biochemical Level
Hannah Grossberg Atmospheric Simulation Chamber for the Measurement of Volatile Organic Compound Oxidation

Atmospheric pollutants such as ozone and secondary organic aerosol (SOA) have a significant effect on human health and climate change. The photo-oxidation of volatile organic compounds (VOCs) contributes to the formation of SOA and ozone. We studied the oxidation of two VOCs using a Teflon chamber, a proton-transfer-reaction mass spectrometer (PTR-MS), and a laser-induced fluorescence formaldehyde instrument. The oxidation of 2-methyl-3-buten-2-ol (MBO) was used to validate our experimental methods. We are now studying the oxidation of a hydroxyl-hydroperoxide (ISOPOOH), which is hypothesized to contribute to SOA formation. The determination of VOC reaction pathways as a function of anthropogenic influence will provide insight into what emission control policies should be implemented to reduce such pollution.

Kellie Kolb RNA hairpin stem enhances pausing of transcriptional elongation complexes

RNA polymerase (RNAP) transcribes DNA to synthesize RNA. During this process it can temporally halt nucleotide addition by pausing. Transcriptional pausing regulates gene expression and in many instances serves to synchronize transcription and translation in bacteria. Pausing at the his site, which occurs at the histidine biosynthetic operon in bacteria, especially E. coli, is stabilized by a hairpin structure in the nascent RNA. The hairpin increases pause half-life; this effect can be mimicked by annealing a complementary RNA oligonucleotide to the nascent RNA. To investigate the contribution of the length of the RNA hairpin stem on hairpin stabilized transcriptional pausing, oligonucleotides of different sizes were added to preformed elongation complexes (ECs; RNAP on a nucleic acid scaffold) and pause half-lives of these EC’s were measured. Interestingly, a duplex of at least 8 RNA bp of 11 DNA:RNA bp was required for maximum effect, and RNA oligonucleotides enhanced pausing more strongly than DNA oligonucleotides at every base-pair length. The transcription elongation factor, NusA, interacts with the elongation complex and hairpin and stimulates pausing. Although NusA recognizes both RNA-RNA and DNA-RNA duplexes and further enhances pausing, its effect on pause enhancement was more prominent in ECs containing duplex RNA and saturated at 10 bp.

Eric Polich AMPA receptor, GluA1, Regulates the Maturation of New Adult Hippocampal Neurons

The maturation of young neurons is the final step of neurogenesis and abnormalities in this process are frequently found in neurodevelopmental disorders. We recently found that deficits in RNA binding protein FXR2, leads to impaired hippocampal neuronal maturation which is likely due to reduced mRNA stability of GluA1. However, the function of GluA1 in maturation of adult-born new neurons has not been investigated. Here, we show that knockdown of GluA1 leads to impaired dendritic development which phenocopies the neuronal maturation deficit in FXR2 knockout mice. Reintroduction of GluA1 expression rescues such deficits in these mice. Taken together, the results unveil a novel regulating mechanism of GluA1 by FXR2, which may play an important role in neuronal development and pathogenesis of neurodevelopment disorders.

Ismat Bhuiyan The role of Fragile X Mental Retardation Protein (FMRP) in differentiation of human fragile X patient-derived neural stem cells (FXS-NSC)

Fragile X syndrome (FXS) is an inherited learning disability caused by the lack of the fragile X mental retardation protein (FMRP). Despite extensive studies, how FMRP deficiency affects human neuronal development is unclear. We hypothesize that FMRP plays a crucial role in regulating neuronal differentiation of human neural progenitors (NSCs). We will use FXS patient-derived NSCs (FXS-NSCs). In this study, FXS-NSCs and control NSCs will be infected with lentivirus expressing red (mCherry) and green (GFP) fluorescence proteins, respectively, mixed, and then transplanted into immune-deficient (SCID) mice. We will then use histological analyses to determine whether FMRP-deficiency impairs the survival and neuronal differentiation of transplanted cells. The results of this study will unveil the functional of FMRP in human neuronal development.

Karli Hochstatter Estradiol, and not increased calorie intake, determines sexual behavior in female marmoset monkeys.

The objective of this study was to determine the effects of estradiol and high calorie diet on female sexual behavior. We used common marmosets (Callithrix jacchus), a well established nonhuman primate model for studying female sexual behavior. Fifteen female marmosets, pair housed with a single adult male as part of a larger study, were ovariectomized to remove the influence of the ovarian cycle on sexual behavior and implanted with silastic capsules that were either empty (control, C) or filled with estradiol (E). Four treatment groups were designed: E-capsule and normal calorie diet, E-capsule and high calorie diet, C-capsule and normal calorie diet and C-capsule and high calorie diet. Two observation sessions were performed after 2 and 5 months on treatment during which sexual and affiliative behaviors were recorded. Findings were most pronounced at 5 months, when females receiving E, regardless of diet, showed higher (p<3×10-6) frequency of acceptance of male mounts (receptivity) compared to C females. In females receiving E but not C, and regardless of diet, there was a positive correlation (p<0.007) between total mounts by males and female receptivity. In contrast, in C but not E females, total mounts by males correlated positively (p<1.8×10-5) with female rejection of mounts. In all females combined, female receptivity correlated positively (p<0.036) with uterine weight (a biomarker for E) harvested at necropsy 1 month after observations. The supportive effects of estradiol on female sexual behavior and reproductive tract appear unaltered by high calorie diet.

Tamayanthi Rajakumar Role of SCD4 in Murine Lipid Metabolism
Bridget Mais Regulation of Gene Expression using Hypoxia Driven Viral Vectors

Parkinson’s disease is a degenerative disorder of the central nervous system characterized by death of the dopaminergic neurons in the substantia nigra and reduced levels of dopamine in the striatum. Current treatment options for Parkinson’s disease do not restore endogenous dopamine levels, cannot regenerate neurons, and are unable to attain site-specific delivery. Therefore, alternative mechanisms for treatment, such as gene therapy, are required. Genes such as Glial-Derived Neurotropic Factor (GDNF), promote the survival of dopaminergic neuron but cannot cross the blood brain barrier. This can be solved by using Adeno-Associated Virus (AAV), a gene transfer vector, to deliver GDNF directly into the brain. However, chronic over-expression of GDNF can cause cellular abnormalities. A proper regulator, such as a hypoxia regulator, is required to control the basal expression. The goal of this experiment was to develop a hypoxia regulated AAV transfer vector that exploits hypoxic conditions as a trigger for dosing GDNF in the brain to combat the effects of Parkinson’s disease. By regulating GDNF in vivo, the negative side effects associated with taking external medications will not be present as the protein is synthesized directly in the brain.

Lauren Hoffer Differences in Lipocalin-2 Expression and Susceptibility to Mammary Cancer in ACI and BN Rat Strains Treated with Estradiol

Lipocalin-2 (LCN-2) is a secreted extracellular matrix (ECM) protein that forms heterodimers with ECM remodeling proteins called MMP’s. In previous studies, LCN-2 was displayed to be a biomarker for human breast cancer and was shown to promote breast cancer progression. This project studies LCN-2 expression in the ACI rat model of 17ß-estradiol (E2)-induced mammary cancer. In this model, the ACI rat mammary gland that is highly susceptible to the development of breast cancer when treated with E2 shows little to no expression of LCN-2. In contrast, the resistant BN rat mammary gland shows higher expression of LCN-2 relative to the ACI rat. This interesting observation of differential expression of LCN-2 was further explored through western blot analysis of the first generation (F1) progeny of ACIxBN and BNxACI to determine if the observed phenotypes are heritable. It is hypothesized, based on mRNA data, that the F1 rats will demonstrate a co-dominant inheritance pattern for LCN-2 expression. ACI mammary tumor tissues were also analyzed and showed no expression of LCN-2, therefore we can conclude that LCN-2 is not a biomarker for breast cancer in this rat model of E2 induced mammary cancer. Further studies will analyze which cells in the mammary gland of the BN rat express LCN-2 and will also look at congenic lines to see if LCN-2 expression is regulated by any of the estrogen induced mammary cancer (Emca) loci that have already been mapped and linked to reduced susceptibility to E2 induced breast cancer.

Amrit Kanwar The Association Between Anxiety Disorders and Suicide: A Systematic Review and Meta-analysis
Tyler Hansen Investigating the Nuclear Import Factor Regulation of Gamete Cell Fate
Tucker Matthews Transcriptional regulation of Leucine-responsive regulatory protein (Lrp) in Xenorhabdus nematophila

Leucine-responsive regulatory protein (Lrp) is a global transcriptional regulatory protein that is necessary for both the colonization and pathogenesis segments of Xenorhabdus nematophila’s symbiotic lifecycle with the nematode, Steinernema carpocapsae. This study was done to characterize the regulatory mechanisms behind the expression of Lrp, in X. nematophila. Promoter constructs containing varying lengths of the intergenic region of DNA upstream from the Lrp promoter were made and fused to a lac-Z reporter gene, to determine the effects of the different sections of DNA on Lrp promoter activity. This was done in both a wild type and Lrp mutant background, to determine whether or not the regulation is Lrp-dependent. Our results show areas of both Lrp-dependent and Lrp-independent regulation—both positive and negative—in this regulatory region.

Manav Khanna Activated Eosinophils Interacting with Periostin, a Protein Found in Asthma
Cole McBee Examining the role of the SUMO class of genes on the development of Physcomitrella Patens

he study of the SUMO family of proteins (a subclass of the Ubiquitin family) is an important class to study to their presence in almost all eukaryotes. Much is known about the function of SUMO in yeasts and mammals, but relatively little is known about its function in plants, and almost nothing outside of the study of Arabidopsis thaliana. The study of SUMO1 and SUMO2, the two genes which code for SUMO tags in Physcomitrella patens will help give insights into the role SUMO plays in plant development and the homology of the role across different species. This will be done by causing the targeted degradation of SUMO RNA transcripts in Physcomitrella patens and observing the phenotype associated with gene knockouts of just SUMO1, SUMO2, and then both genes simultaneously. It is expected that the function of SUMO1 and SUMO2 are redundant, and that only a knockout of both genes will produce a distinct phenotype, which will result in severe disability or death of the plant after several days without new SUMO transcription.

Scott Lee Characterization of the Phenotypic Development of Cholinergic Neurons in the Mouse Enteric Nervous System

There are many different types of neurotransmitters that are involved in signaling throughout the enteric nervous system (ENS). One of the main excitatory neurotransmitters involved in gut motility is acetylcholine. The goal of this study was the track the progression of cholinergic neurons in the developing gut of the mouse from the phenotypic birthdate all the way through adulthood. This was done through the use of a genetically encoded florescent protein which is expressed when the choline acetyltransferase (ChAT) gene is activated. We used this method to find out what proportion of neurons in four different regions of the gut (jejunum, distal ileum, proximal colon, distal colon) are expressing this gene throughout various stages of development. What we found is that cholinergic neurons have a peak birthdate at approximately embryonic day 12 and reach a maximal expression at approximately postnatal day 30. There also appears to be a wavefront progression of the gene throughout the gut, with more rostral parts of the gut expressing the ChAT gene much earlier than the more caudal regions. The results of this study give us further information about what areas to focus on when determining the exact molecular mechanisms of the phenotypic differentiation of cholinergic neurons, and to identify how this progression of differentiation is affected in congenital diseases affecting the ENS, such as Hirschprung’s disease.