http://digital.library.wisc.edu/1793/6665 http://minds.wisconsin.edu:80/retrieve/5653 http://digital.library.wisc.edu/1793/6665 http://digital.library.wisc.edu/1793/38590 Pilot screen for second site suppressors of wstd Tontillo, Kate Triosephosphate isomerase (TPI) loss of function (wstd), in flies has been shown to have increases in methylglyoxal and advanced glycation end products (AGEs) which have also been found in increased levels in diseases such as Parkinson?s and Alzheimer?s disease. By showing that an increase in AGEs leads to neurodegeneration in drosophila, then the accumulation of AGEs might also be the cause of neurodegeneration in human diseases. This experiment is a pilot version of a screen looking for second site modifiers that interact with the TPI gene. A TPI rescue construct was performed from a cross with mutated male drosophila with a null allele for TPI with females deficient for TPI. The experiment selected for non-balancer phenotypes and restored viability in the F1 generation of the cross. The cross resulted in a low number of offspring, and no rescue phenotypes were observed. A fertility test showed that one possibility for the low number of offspring produced is that the mutant male drosophila had low fertility. 14 p. http://digital.library.wisc.edu/1793/38588 Going from embryo to adult: How zebrafish zic genes are regulated during development Holman, Nathanael Thomas This project aims to further our understanding of developmental genetics by identifying unknown transcription factor binding sites that play an important role in regulating zic2a, a gene required for proper neural development in vertebrates. To do this, transgenic zebrafish were created that have various versions of the zic2a gene coupled with green fluorescent protein coding sequence. The transgenes differ by a single mutation in one of the several evolutionary conserved sequences in the enhancer region, which are thought to be transcription factor binding sites. By comparing the expression patterns of the green fluorescent protein among the transgenic zebrafish I identify sites that are required for zic2a transcription during development. 12 p. http://digital.library.wisc.edu/1793/37686 Identification of QTLs for voluntary running activity in mice for the CBA/J and DBA/2J mouse strains Trammell, Sam Our goal is to understand the genetic factors related to voluntary running activity. Voluntary running activity in humans and mice is known to be a heritable behavioral trait. The genetic factors influencing voluntary running activity may be associated with human diseases, such as obesity. Therefore, identifying these factors may not only further our understanding of the behavior at the molecular level but also better our understanding of related disorders. We have found that CBA/J and DBA/2J inbred mouse strains have different voluntary running activities. We used quantitative trait locus (QTL) analyses to locate the genetic loci responsible for this difference. As a quantitative trait, we measured voluntary running activities in the parental mouse lines, their F1, and F2 progeny. We also identified genetic markers distinguishing CBA/J and DBA/2J alleles. We genotyped 176 F2 mice. We performed QTL analyses to identify loci associated with the phenotype. We identified significant QTLs on chromosomes 5 and 6 (LOD scores=3.697 and 5.047). We identified interactions between loci on chromosomes 3 and 6 (LOD score-6.737) and between loci on chromosomes 5 and 6 (LOD scores=8.063). In this study, we identified multiple loci influencing voluntary running activity in mice, providing a guideline for future identification of responsible genes. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. http://digital.library.wisc.edu/1793/37683 The role of dynamin and Rho1 during cytokinesis in C. elegans Amodeo, Amanda Cytokinesis is the final phase of cell division in which a single cell is physically separated into two daughter cells. This is accomplished by constricting the acto-myosin contractile ring and inserting new membrane along the furrow. The large GTPase dynamin is essential for cytokinesis. Cynamin is required for endocytosis and also regulates the dynamics of the actin cytoskeleton. We hypothesize that dynamin may regulate the acto-myosin contractile ring. In order to test this hypothesis, we use in vivo microscopy to shed light on the role of DYN-1 in regulating actin dynamics by using GFP::RHO-1 as a marker. These experiments yield insight into the establishment and regulation of the contractile ring during cytokinesis. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. http://digital.library.wisc.edu/1793/37502 Haplotype frequencies at the miR156 and miR172 transcript regions show a significant response to divergent recurrent selection for vegetative phase change in maize Rutkoski, Jessica Sweet corn (Zea mays L). plants that transition from juvenility to the adult phase earlier during development are generally more resistant to common rust (Puccinia sorghi), a disease that causes substantial yield loss in sweet corn. This developmental transition is called vegetative phase change (VPC). VPC is regulated in part by miR156 and miR172. We found that natural variation at the miR156 and miR172 transcript regions are important in determining variation in the timing of VPC. We sequenced the miR156 and miR172 transcript regions in populations originating from the same source population and divergently selected for eight generations for early and late VPC. For both the miR156 and miR172 transcript regions we detected four distinct haplotypes. Overall and individual haplotype frequencies showed significant responses to selection for both early and late VPC. These data contribute to the identification of important loci involved in VPC. Understanding the genetic basis for VPC will facilitate maize breeding aimed at combining early VPC with other desirable traits. 14 p. http://digital.library.wisc.edu/1793/37500 Preneoplastic Lesion Growth in B6.Hcs7C3H 219R8 Congenic Hepatocarcinogen-Sensitive Mus musculus Poli, Elizabeth A region of mouse Chromosome 1, called the Hcs7 locus, is involved in the susceptibility to liver carcinogenesis in the inbred mouse strain, C3H/HeJ (C3H). Linkage analysis of a cross between C3H and a strain that is resistant to carcinogen induced liver cancer, C57BL6/J (B6), has mapped this locus to the distal end of the Chromosome 1. Several genes in this area could play a role in the increased susceptibility in C3H mice. Initially, however, it should be determined whether or not the locus is affecting the initiation of tumors or the promotion of tumor growth in C3H mice. Early experiments have suggested that Hcs7 affects the promotion phases of hepatic tumors after the carcinogen treatment. By observing the number and size of preneoplastic lesions in C3H, B6, and in B6 mice carrying the C3H allele of Hcs7, it was determined that there is a correlation between the locus and an increased growth rate in preneoplastic lesions in the liver. Further understanding the mechanisms of liver cancer in mice can allow one to begin to question and test whether the same processes exist in human liver cancer. 14 p. http://digital.library.wisc.edu/1793/37498 Semantic category learning through explicit and implicit knowledge of complex sequences Li, Annabel Jerry Objects with similar functions are judged to be similar "kinds of things," even when they have few directly-observable properties in common. How do people learn such "functional" similarity? One possibility is that objects with similar functions occur in similar contexts: there is overlap in the sequence of events that precede and follow their use. To test this hypothesis, participants learnt a novel sequence with multiple interchangeable objects at each position and subsequently were taught to name the objects. As expected, participants learnt category structures consistent with the positions in the sequence. However, contrary to hypothesis, this learning did not transfer to naming and participants did not learn names consistent with the category structure more quickly than random names. Interestingly, an interaction between naming condition (consistent and random) and learning (participants who learned the sequence and participants who did not) was found also and warrants further investigation. 8 p. http://digital.library.wisc.edu/1793/37496 The Biochemical Characterization of a Translational Repressor Luo, Xinwei Sarah Iron regulatory proteins (IRP) are crucial post-transcriptional regulators of iron metabolism. They influence the synthesis of proteins involved in iron uptake, storage and use by binding to their mRNAs. IRP1 stands out particularly with its ability to incorporate a [4Fe-4S] cluster to double function as the enzyme, cytosolic aconitase (c-acon). Since RNA binding and c-acon activity are mutually exclusive, the incorporation of this Fe-S cluster serves as a regulatory switch between the two forms that is dependent on iron availability in cells. This project aims to characterize the biochemical properties of IRP1 in order to understand the finely tuned translational regulation it performs. Polysome profiles were used to isolate different forms of IRP1 based on their presence in actively translating or repressed pools of mRNA. Functional assays like aconitase activity assays and RNA-binding assays were used for biochemical characterization of these forms. This study establishes baseline activities for these assays and also demonstrates that IRP1 differentially regulates the mRNAs of ferritin, the iron storage protein, and ferroportin, which exports iron from the cell. My research will allow further understanding of IRP1 as an agent of regulatory control. 23 p. http://digital.library.wisc.edu/1793/37494 Characterization of nuclear RNAi defective mutants in C. elegans Lachowiec, Jennifer Proper gene expression is crucial for all cellular processes, and changes in normal gene expression has been implicated in a number of human diseases, including asthma, heart disease, and cancer (Guajardo 2005, Tan 2002, Peng 2005). One form of gene regulation is RNA interference (RNAi). Using forward genetics, we have identified three genes required for nuclear RNAi. These genes regulate gene expression by targeting premRNA in the nucleus. We have found that three of these proteins likely act downstream of previously characterized RNAi proteins known to regulate mRNA levels. Based on sequence identity, two of these proteins are conserved. Our results strongly suggest the three proteins act in the same pathway. Greater understanding of the basic mechanisms of RNAi will help maximize its use as a therapeutic to treat disease. 16 p. http://digital.library.wisc.edu/1793/8148 The role of a soluble, competitive FGF receptor in embryonic stem cell-derived hematopoiesis Webster, Daniel E. The fibroblast growth factor (FGF) signaling pathway plays a pivotal role in the maintenance of embryonic stem cells (ESCs) as well as their differentiation into the hematopoietic lineage. We observed that supplementing human and rhesus ESCs in embryoid body (EB) culture with the FGF-2 ligand showed a dose-dependent stimulation of hematopoietic differentiation. However, despite phylogenetic similarities, rhesus ESCs needed a 5-fold greater concentration of FGF-2 relative to human ESCs to achieve optimal hematopoiesis. To elucidate the mechanism behind this species-specific differential requirement for the central growth factor FGF-2, we measured and compared endogenous gene expression of FGF ligands and splice variant isoforms of FGF receptor 1 (FGFRI) between human and rhesus ESC lines. The results indicate that rather than an inherent lack of FGF ligand production in rhesus ESCs relative to human ESCs, there are significant differences in their expression of FGFRI isoforms. Our findings suggest that an increasing fraction of the soluble, competitive inhibitor sFGFRI isoform relative to the functional FGFRlα isoform expressed in ESC cultures correlates (r2=0.84) to a decrease in the degree of hematopoietic differentiation achieved by a differentiating ESC population. This finding not only offers an explanation for the observed differences between rhesus and human hematopoiesis, but also implies a mechanism by which developing cells can self-regulate hematopoietic induction by secretion of competitive, inhibitory receptor isoforms. 17 p. http://digital.library.wisc.edu/1793/8146 An investigation on the effects of mutations in the ATG12 autophagy pathway of Arabidopsis thaliana Damodaran, Ashvini Under nutrient-deficient conditions, plants are forced to recycle secondary cellular constituents to conserve energy for the more essential processes (Klionsky, et al. 2000). Plants accomplish this through a process called autophagy, which involves the capture of cytosolic constituents in vesicles called autophagosomes and transportation of these vesicles to lytic vacuoles for degradation. Macroautophagy, the main autophagy pathway, requires an ATGI2-ATG5 protein conjugate for autophagosome formation (Thompson & Vierstra, 2005). Extensive research has been done on the ATG 12 pathway in organisms such as yeast, but research is yet to be done on ATG12 disruption in plants. By studying protein expression and performing phenotypic analysis oft he model plant Arabidopsis thaliana with mutationsinATG12and other members ofthe ATG12 pathway, we can begin to understand the role of the pathway in autophagic recycling during plant growth and development. Since autophagy controls many important aspects of crop physiology (e.g. seed germination, pathogen defense, and growth under nutrient-limiting conditions), this work will likely provide new venues for improving crop production. 21 p. http://digital.library.wisc.edu/1793/8142 Starvation responses of invasive and non-invasive Eurytemora affinis populations Chau, Fong Ching Within the past century, the copepod Eurytemora affinis have invaded freshwater from saline sources throughout the Northern Hemisphere. Two genetically divergent clades (Atlantic and North Atlantic) of E. affinis coexist in the St. Lawrence estuary, but only the Atlantic clade was able to invade into freshwater ecosystem. A common-garden reaction norm experiment was performed on populations from these two clades to determine their differences in survival and development time across three food levels (high, medium and low) in fresh water. The populations were fed with the freshwater alga Rhodomonas minuta. The developmental progress and survival of the populations were monitored throughout the experiment. The results indicated that the population from the invasive Atlantic clade is more starvation resistant and has faster development time to adulthood. The differences between the populations might be due to their different osmoregulatory mechanisms and energy usages. These different physiological responses to starvation are likely to have functional consequences for the populations' ability to expand their ranges into novel environments. 24 p. http://digital.library.wisc.edu/1793/7987 To determine the functional relevance of circadian Fabp7 expression in memory consolidation Lai, Tsz Chung Fatty-acid binding protein 7 (Fabp7) is closely related to sleep/wake cycle and regulated in a circadian pattern. To investigate whether the expression affects learning and memory behavior, we used the Drosophila model. Transgenic lines of FABP7 were conditionally expressed in order to test the hypotheses that FABP7 altered the formation of long-term memory (LTM). Unlike the circadian rhythm, much less has been known for the behavioral state and the homeostatic regulation of sleep control at the molecular and genetic levels. Previous results show that all synapses are affected in a similar manner, and the molecules that have a diurnal pattern of expression at synapses throughout the brain after sleep/wake cycle would be excellent candidates for cellular correlates responsible for the homeostatic arm of sleep regulation, and they are also targets of therapeutic approaches to neurological diseases. Therefore, identifying and investigating the targeted synaptic molecules involved in the sleep/wake rhythm is important for a better understanding of mental illness. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. http://digital.library.wisc.edu/1793/7979 Transgenic zebrafish that carry inducible zic2a fusion proteins Matson, Daniel R. Zic genes encode proteins that contain zinc fingers used to bind DNA and modify transcription of target genes. Several zic genes have been identified that are critical in regulating nervous system development in a wide variety of organisms. We have chosen to study zic2a because its transcription activity has not been well defined. The goal of this study was to begin determining the regulatory nature of zic2a in the zebrafish (Danio rerio) central nervous system. To do this, three constructs were assembled that, when induced, over expressed constitutively activating, constitutively repressing, or wild type zic2a function. These constructs were further modified and then used to develop three stable transgenic zebrafish lines, which allow conditional over-expression of zic2a in zebrafish embryos. These transgenic, zic2a-expressing zebrafish will be used in further studies to characterize the function of zic2a in the developing nervous system. 11 p. http://digital.library.wisc.edu/1793/7973 A genetic approach to understanding co-regulated MAPK genes in Arabidopsis thaliana Ho, Bonnie Yiu Mitogen-activated protein kinase (MAPK) signaling cascades connect cellular signal and gene transcription in cells. The pathway can regulate cell division, cell growth, and other pathways in eukaryotes. We used the Comprehensive Systems Biology Project database to find out that MPK1, 2, and 6 have a similar co-response level, as do MPK8, 19 and 20. We hypothesized that the genes in each group serve similar functions and would respond to stress similarly in Arabidopsis. We created a homozygous knockout mutant in MPK1, 2 and 6, and another one in MPK8, 19 and 20. Under normal growth conditions, the triple mutant seedlings appeared identical to wildtype seedlings. We compared the mutant and wildtype seedlings in different stress conditions (0.3 uM kinetin, 2% sucrose, 5% sorbitol, ethylene and 14°C chilling). We measured root growth of light-grown seedlings in the first three and chill treatments and hypocotyls length for sucrose-treated and ethylene-treated dark-grown seedlings. Results showed that there was no significant difference in the root or hypocotyls length growth between wildtype and mutant seedlings in all the experimental conditions. Thus, MPK1, 2, and 6 are not required in responding to stress based on the MPK1, 2, 6 triple mutant; MPK8, 19 and 20 are not required in the stress response cased on the MPK8, 19, 20 triple mutant. Future experiments have to be done to give information of their functions. This project shows a reverse genetic approach to study gene function. 16 p.