Abstracts

Mannans are hemicellulosic polysaccharides within plant cell walls with cellular functions including structure, storage, and signaling. Understanding the patterning of mannan synthesis will enhance our understanding of the roles of these polysaccharides in plants. Numerous members of the CSLA gene family encode mannan synthase enzymes involved in the synthesis of the backbones of mannans. This project aims to create an expression atlas that documents the expression pattern for each of the nine CSLA genes in the model plant Arabidopsis thaliana by using GUS-reporter gene fusions and microscopy throughout much of the plant life cycle. This will be used to identify the contribution of each CSLA throughout development.

While there are animal models of kidney disease, the mechanisms by which they act are not well understood. Here, we analyze an injectable compound, previously used as a cancer adjuvant, to investigate its role in causing reversible proteinuria. Intra-peritoneal injections of 40 mg/kg body weight/day resulted in massive proteinuria by day 2, which cleared after cessation of treatment. Tubular walls of the cortex appeared porous and diffuse when stained with H&E or Mason's Trichrome. SEM of visceral epithelial cells of the glomerular filtration barrier, known as podocytes, showed marked retraction and flattening, or effacement, following treatment for 3 days. Immunofluorescence demonstrated no discernable variation in the localization of slit-diaphragm proteins nephrin, neph1, and podocin in glomeruli of treated mice. Proteinuria is likely due to loss of adequate filtering at the podocyte junctions but may also indicate leakage from the tubules. It is unclear if the proteinuria and kidney damage observed is a primary result of acute treatment or secondary to animal sickness caused by the compound. We have not ruled out use of this compound as an inducer of proteinuria, but the mechanism of action must be better understood before it will prove useful as a model of disease.

Plant cell walls are composed of a matrix consisting primarily of cellulose and hemicelluloses. The unique characteristics of this matrix are exploited in many useful ways. Because of their abundance, plant cell walls also hold great promise as a feedstock for biofuels. Much remains to be learned about the synthesis of hemicelluloses, including the identities of genes encoding glycan synthases and glycosyltransferases that biosynthesize these polymers. It has been previously demonstrated that some CELLULOSE SYNTHASE LIKE (CSL) genes encode glycan synthases involved in cell wall polysaccharide synthesis. While it is hypothesized that these genes encode glycan synthases, no members of the CSLG family have been functionally characterized. Here, the three members of the CSLG family from Arabidopsis thaliana are studied using heterologous expression in a moss (Physcomitrella patens) and a yeast (Pichia pastoris). Preliminary results of these efforts are presented.

P190RhoGAP, also known as p190A, is an important regulator of the monomeric GTPase Rho, which in turn regulates the cytoskeleton. We know little about the expression of p190RhoGAP in Xenopus laevis embryos. The purpose of this project was to visualize the expression patterns of p190RhoGAP in developing embryos. Using an antibody for p190, expression was visualized in whole embryos. Sections of the antibody-stained embryos were taken using a microtome to observe the protein expression within the embryos. During the early stages of development, the protein was localized to the cortical cytoplasm in the animal hemisphere of the embryo. P190RhoGAP appeared to be expressed within specific structures including the neural folds of neurulation. The expression of p190RhoGAP in particular regions during embryogenesis suggests an important function for this protein in the developmental of Xenopus laevis. Knockout studies are being performed to further determine the developmental function of p190RhoGAP.

Microbial communities differ within varying aquatic environments, with land usage impacting the natural makeup of these communities. There is a general trend for lower diversity found at areas of heavy human use than those at sites that are considered more natural. Water, sediment, and biofilm samples were obtained from three different sites (natural, agricultural, and urban) within the Huron River Watershed. Bacterial 16S rDNA was cloned and PCR amplified, and representative bacterial samples were sequenced at the University of Michigan DNA Sequencing Core and identified using the Ribosomal Database Project (http://rdp.cme.msu.edu). The 16S rDNA bacterial sequences from the water, sediment, and biofilm samples were analyzed for diversity and the role of possible human impact. Identification of the different bacterial samples suggested the role of land use in the formation of microbial communities within stream habitats.

The Eph family of receptor tyrosine kinases is involved in mediating interactions between cells during embryonic development. Activation of the EphA4 receptor in early Xenopus laevis embryos leads to perturbation of cell adhesion, which causes exterior lesions and disruption of blastocoel development. The downstream effectors of this signaling pathway are not well understood, although previous studies have shown RhoA GTPase to be inhibited by EphA4 catalytic activity. We have investigated the involvement of rho-associated protein kinase (ROCK), which includes a RhoA binding domain, in the signaling downstream of EphA4 by ectopically expressing mutant forms via RNA microinjections into Xenopus embryos. Our results showed a significant rescue of the EphA4 phenotype when expressing a constitutively active mutant isoform ROCK1, with normal blastocoel formation increased from 32% (n=50) to 60% (n=42) (twosample t-test, p<0.01). Thus, we provide evidence supporting the down-regulation of ROCK1 during EphA4 signaling.

Salmonella typhimurium is a leading cause of infectious gastroenteritis and results in systemic disease when it escapes into the bloodstream. One of the most important components of human serum in fighting bacterial infections is the complement system. A strategy S. typhimurium uses for survival outside of cells is resistance to killing by complement (serum-resistance), a trait conferred by several known virulence genes. Our goal is to investigate a complement-resistance gene located on the chromosome of S. typhimurium. A chromosomal TnphoA insertion of S. typhimurium strain EM876 results in reduced serum-resistance. Inverse PCR with DNA sequence analysis revealed the insertion to be located in the gene glpQ. To determine the gene's role in serum-resistance, we will use complementation assays, and the resultant isolates will be tested to determine whether the reduction in serum-resistance has been repaired. We hypothesize that glpQ is responsible for the decreased serum-resistance phenotype seen in EM876.

Common terns (Sterna hirundo) in the lower Great Lakes have declined in number since the 1960s and nest mostly on artificial structures adjacent to water. These colonies are often spatially fixed and prone to excessive annual predation, especially in the urbanized Detroit River, where nest predators are common. This study investigates the spatial and temporal patterns of habitat use of one colony underneath a busy bridge in the Detroit River with high variability of habitat. I compared substrate and vegetation variables of nest sites with random points throughout the colony. I also characterized the vegetation at three areas with different substrate and explored differences in early nests and late nests and successful and unsuccessful nests. I employed a technique for monitoring general patterns of nocturnal nest desertion through the use of temperature sensors placed within incubated nests.

AOT is an anionic surfactant and a common ingredient in consumer products, including stool softeners. This study was aimed at finding the role of surfactant AOT as an antibacterial against wide range of micro-organisms from pathogenic probiotics to yeast. It involved culture and identification of organisms and MIC (Minimum Inhibitory Concentration), followed by determination of MBC (Minimum Bactericidal Concentration) of AOT. In this study, AOT was found to be an effective antibacterial substance against a wide range of micro-organisms, including pathogenic organisms. Its antibacterial effect is limited to the Gram-positive organisms tested, including several Staphylococci. It is interesting to note that one strain of Lactobacillus was found to be resistant to AOT. Further studies will be directed toward elucidating the mode of action of AOT against Gram-positive bacteria and yeast, using strains sensitive to AOT.

This research focuses on the detection and quantification of long interspersed DNA element (LINE), short interspersed DNA element (SINE), and Mys retrotransposon expression in the deer mouse (Peromyscus maniculatus). These 'mobile elements' increase in genomic copy number via a process referred to as retrotransposition. This study is designed to determine if germ-line expression of these elements is the limiting factor associated with their relative successes in generating newly inherited copies. This will be analyzed by contrasting the RNA levels in gonadal tissues with the number of copies of the elements found within the Peromyscus genome. Thus far, expression of LINE and Mys retrotransposons were detected in deer mouse brain, liver, testes, and ovary tissues, using the RT- PCR technique. For SINE analysis expression, a novel use of columns was incorporated as a size-fractionation tool to validate isolation of true RNA pol III transcripts. SINE expression was detected in gonadal tissue for all the analyzed elements. Currently I am developing the appropriate conditions for real-time PCR for RNA quantification of the elements within the gonadal tissue. In addition to addressing our hypothesis, this will enable us to determine whether the male or female is more responsible for transferring these elements to the next generation.

Plant cell walls provide an abundant renewable resource used for the production of biofuels, paper, textiles, and many other useful products. These composites consist mainly of carbohydrates, including cellulose and non-cellulosic polysaccharides. Previous studies of cell wall polysaccharides have revealed their complex structures but have not identified all the enzymes needed for their synthesis. Numerous members of the Cellulose Synthase-Like A (CSLA) family have been demonstrated to encode glucomannan synthases, enzymes that produce mannan polysaccharides present in plant cell walls. The genome of Arabidopsis thaliana contains nine CSLA gene family members; however, mannans are not accumulated in abundance in this plant. This raises questions about the functional significance of some members of the CSLA family. We are using promoter:GUS fusions to study the expression patterns of CSLA genes in Arabidopsis. Progress of these efforts, with special emphasis on the CSLA1 and CSLA14 genes, is presented.

SINEs (short interspersed DNA elements) are families of non-coding regions of DNA that amplify within genomes via an RNA intermediate and are referred to as retrotransposons. These elements mobilize using machinery from other retrotransposons and therefore are non-autonomous. It has been demonstrated that both nucleotide sequence and the 3' A-tail are important contributors for successful amplification. We propose that the level of germ-line transcription of SINE 'master genes' is a primary factor in their successful mobility and vertical transmission. By RT-PCR, the GPIDL SINE family appears to be active in the guinea pig germ-line. Since copy numbers of different SINE families are known to be highly variable in genomes of various rodent species, a relationship in germ-line SINE expression levels, determined by real-time PCR, would support our hypothesis. Additionally, this analysis may provide insights as to whether inherited SINE integrations are more likely to occur in oogenesis or spermatogenesis.

Filamentous cyanobacteria have caused ecological problems in the southern United States. One species of particular concern is the filamentous Lyngbya wollei, which has recently been observed in Lake Erie. Some problems arising from invasion by Lyngbya include alteration of natural communities and skin lesions in mammals. Unless an effective management strategy can be developed, there may be consequences for Lake Erie. Therefore, the reason Lyngbya wollei has taken hold in Lake Erie must be ascertained. The purpose of this study was to determine if Lyngbya is nutrient-limited in Lake Erie. Nutrient-diffusing substrata were used to establish whether Lyngbya is nutrient-limited. We hypothesized that Lyngbya wollei has been nutrient-limited in Lake Erie and that historical data will show a shift towards conditions conducive to the growth of Lyngbya. Preliminary results suggest that Lyngbya wollei is nitrogen-limited in Lake Erie.

Nerve transection injuries may necessitate surgical intervention. We previously established the use of scaffold-less engineered neural conduits fabricated from primary cells in sciatic nerve repair surgeries. We proposed that engineered neural conduits fabricated from induced stem cells would successfully recover a similar nerve transection. The ability to utilize stem cells would eliminate the limitation of conduit material and have minimal host donor site morbidity. We established protocols for isolating stem cells from inguinal adipose tissues, differentiation into fibroblast and neural lineages, and co-culturing methods. Both cell types were integrated into a 3-dimensional scaffold-less conduit. In vitro morphology and histology of these conduits showed similarity to primary cell conduits. These conduits can now be evaluated in vivo as a bridge for regenerating nerve in transection injuries.

Invasive species can substantially alter vital ecosystem functions, such as carbon and nutrient cycling. An invasive aquatic plant, the common reed (Phragmites australis) may strongly affect wetland processes. Reeds form monoculture stands, exhibit high primary productivity, and contain higher proportions of recalcitrant biomass than native macrophytes. These traits enhance Phragmites' ability to dominate large areas. This study compares organic matter cycling in invaded wetlands to cycling in native wetlands. I hypothesized that Phragmites' invasion reduces rates of organic matter turnover and greenhouse gas emission in freshwater wetlands. Sediment incubation experiments were conducted to determine the rate of CO2 and CH4 emission. Oxygen concentration and H2O saturation were manipulated to mimic natural variation. Preliminary results suggest that less CO2 and CH4 is released from Phragmites invaded wetlands. This indicates that Phragmites is impacting wetland function and may be acting as a temporary sink for carbon.

Microbial diversity and content within an aquatic environment plays a significant role in shaping and regulating the rest of the community. Information about the effects of seasonality and location within a river ecosystem remains insufficient from past studies. This study attempts to provide information regarding the microbial composition found in a natural site, agricultural site, and an urban site around the Huron River in Michigan. Molecular techniques were used to analyze microbial composition in bacterial periphyton communities at three sites along the Huron River watershed. Results showed similarities among sites, but there were unique characters within each site that reflected environmental conditions around each individual locale. Phylogenetic analysis of these data will be presented.

Phragmites australis is an invasive plant that has strong negative impacts on wetland ecosystems. To control Phragmites, the broad-spectrum herbicide glyphosate is often applied. While its toxicity effect on macro-organisms and specific model microorganisms has been well researched, effects on microbial community structure have not. Because microbes are important regulators of nutrient cycling in wetlands and changes in their composition can alter this function, the purpose of this study was to determine whether differences in microbial community structure occur after glyphosate treatment. We collected a series of soil samples from a local wetland prior to and following glyphosate application. Using T-RFLP analysis, a bacterial community profile was constructed through time. We hypothesized that an overall shift in the microbial community composition would be seen and some microbial groups would be enriched based on their ability to use glyphosate as a nutrient source. Here we present the results from T-RFLP analysis.

In animals, specific dynamic action (SDA) is the metabolic response following the intake of a meal caused by processes involved in ingestion, digestion, and absorption. Currently, there are only two published studies for SDA response in arachnids, and studies on the effects of dietary composition on SDA response are limited mainly to fish, with very few invertebrate studies. In this study, tarantulas (Poecilotheria regalis) were presented various prey types to determine if there were quantifiable differences in their SDA response; in addition, during prey-capture, prey-handling behaviors were recorded to examine differences in behaviors among three types of prey (crickets, cockroaches, and waxworms). This study provides insight into energetic costs associated with feeding and may lead to inferences on prey preference in a tarantula species.

Adult common terns (Sterna hirundo) temporarily abandon their nests at night when there is nocturnal predation of adults and chicks. We monitored 55 nests in one colony using cameras and temperature sensors to determine the frequency, duration, and synchrony of nocturnal nest-desertion in response to black-crowned night heron predation. Field cameras revealed predation by one heron on 69% of nights from 26 May (9 days after first tern hatched) until 30 June when there were still 43 active nests, resulting in 0.35 (? 0.75) chicks per pair in 2010. There was a mean of 80 (? 94) minutes between the first and last heron detections. Adult terns abandoned nests on 85% of nights for a mean of 5:58 (? 2:41) hours. Terns temporarily abandoned their nests less synchronously than when they arrived to continue incubating.

Human common fragile sites are loci that form gaps and breaks in metaphase chromosomes under replication stress conditions. These sites are often located in tumor suppressor genes. The chromosomal aberrations occurring in tumor cells could be due to instability of the common fragile sites. The exact location at which fragile site breaks occur still remains unknown. Since most of these sites contain AT-rich regions, the formation of secondary structure by the regions could contribute to the instability in these sites. We are using a yeast artificial chromosome (YAC) carrying FRA3B sequence to identify and analyze the breakpoint locations. We determined that the YAC contains most of the FRA3B sequence by using multiplex PCR. By using genetically modified yeast, we induced breaks in the FRA3B under replication stress conditions. The location of the FRA3B breaks will be determined by using multiplex PCR, and the sequence near the breaks will be analyzed.