Students in biology

SUMMER RESEARCH PROGRAM

2013 Student Research Abstracts 

SRP 2013

 

Neha Ahuja, Marquette University

Mentor: Dr. Krassimira Hristova

Presence of plasmid-mediated antibiotic resistant E. coli in the Milwaukee urban waterways

 

Ellen Barton, Marquette University

Mentor: Dr. Michael Schlappi

Genotyping of rice for cold tolerance QTL mapping

 

Cody Boese, Marquette University

Mentor: Dr. Martin St. Maurice

The role of two putative proteins in the function of bacterial urea amidolyase

 

Sonia Chris-Ukah, University of Wisconsin-Oshkosh

Mentor: Dr. Pinfen Yang

Creating Chlamydomonas strains expressing tagged flagellar calcium channels

 

Nikala Gigliotti, Milwaukee School of Engineering

Mentor: Dr. Lisa Petrella

Evolution over time of C. elegans fertility at different temperatures

 

Kirstan Gimse, University of Wisconsin-Parkside

Mentor: Dr. Stephen Downs

Investigating the involvement of AMP-activated protein kinase in the mitotic divisions of the early embryonic development of mice

 

Marian Lund, Gustavus Adolphus College

Mentor: Dr. Michael Schlappi

HyGRP stress response genes, qLTG3-1 and Glyma 17g14850, improve seedling germinability under low temperature and salt stress conditions in Arabidopsis thaliana

 

Katherine Maniates, St. Catherine University

Mentor: Dr. Allison Abbott

Characterization of microRNA regulation of ovulation in C. elegans

 

Joseph Rehfus, Marquette University

Mentor: Dr. Robert Fitts

The effects of exercise training on myocardial action potential duration and calcium handling

 

Kara Signorelli, Marquette University

Mentor: Dr. Thomas Eddinger

Different levels of protein expression in coarcted, corrected, and  control aortic tissue

 

Taylor Stevens, University of Tennessee at Knoxville

Mentor: Dr. Martin St. Maurice

Investigating allosteric activation in pyruvate carboxylase using chimeric enzymes

 

Maureen Thompson, Milwaukee School of Engineering

Mentor: Dr. James Maki

Enzyme activiy of cell-free extracts from biofilms of Bacillus JK7 and Stenotrophomonas JK3

 

Emma Weber, Marquette University

Mentor: Dr. Anita Manogaran

Characterization of genes involved in appearance of the [Het-s] prion

 

Amanda Youmans, Milwaukee School of Engineering

Mentor: Dr. Edward Blumenthal

Possible interactions of genes with drop-dead in Drosophila melanogaster

 

  

 

 

Neha Ahuja, Marquette University

Mentor: Dr. Krassimira Hristova

Presence of plasmid-mediated antibiotic resistant E. coli in the Milwaukee urban waterways

There is a growing need for an understanding of anthropogenic factors that can increase antibiotic resistance.   In particular, the waste water treatment process [WWTP] is thought to aid in the selection of mutli-drug resistant bacteria. We have previously phenotypically screened E. coli isolated from the influent waters and effluent sediment of Jones Island Waste Water Treatment facility, as well as the downstream contaminated KK River. The results showed a dramatically higher ABR level of E. coli isolates from the effluent sediment and KK River as compared to the influent waters.  The purpose of this project was to investigate the role of plasmid transfer in engendering ABR bacteria. We extracted plasmids from E. coli isolates from the aforementioned locations and utilized PCA statistical analysis to determine the correlation between the plasmid profile and resistance of the E. coli  strains in which the plasmids were extracted from.  Single plasmids were isolated via transformation of chemically competent Top 10 E. coli cells and were phenotypically screened to determine the resistances that the plasmid carried.  Finally, to verify that the plasmids were being transferred between E. coli strains, mating experiments using K12 strain CV601gfp were performed.  Initial results indicate that plasmids from the effluent sediment are more correlated with antibiotic resistance than influent, and that plasmids containing resistance genes to multiple antibiotics have the ability to be transferred via transconjugation.   This plasmid-mediated ABR could serve as the mechanism behind increased levels of ABR in clinically relevant settings. 

 

 

Ellen Barton, Marquette University

Mentor: Dr. Michael Schlappi

Genotyping of rice for cold tolerance QTL mapping

Cold sensitivity limits rice productivity and viable regions for rice cultivation.  Cold tolerance is believed to be a quantitative, or polygenic, trait in rice.  Based on preliminary data using genome wide associations studies (GWAS), genes for cold tolerance are thought to be located on chromosomes 3, 4, and 12.  The objective of this SRP project was to confirm or reject the presence of these strong effect cold tolerance quantitative trait loci (QTL) using classical genetics.  DNA was isolated from parents with different cold tolerance phenotypes and from over 200 progeny resulting from crosses between those parents.  Three methods using 60 primer pairs covering the 12 rice chromosomes were used to genotype the progeny. DNA was amplified by polymerase chain reaction (PCR) and analyzed using agarose and polyacrylamide gel electrophoresis, or quantified using real-time qPCR. Most primer pairs were determined to be workable for genetic mapping and the vast majority of markers followed a 1:2:1 segregation in the F2 progeny, indicating that there was no bias for one or the other parent in the viable offspring.  In future genetics mapping experiments, the completed F2 genotypes will be correlated with cold tolerance phenotypes to validate strong effect QTLs on chromosomes 3, 4, and 12.

 

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Cody Boese, Marquette University

Mentor: Dr. Martin St. Maurice

The role of two putative proteins in the function of bacterial urea amidolyase

Urea amidolyase (UAL) catalyzes the formation of ammonia and bicarbonate from urea with the concomitant hydrolysis of ATP, and is a means for fungi, algae and bacteria to utilize urea as a source of nitrogen.  Fungal urea amidolyase is a single polypeptide chain with two enzymatic activities, urea carboxylase (UC) and allophanate hydrolase (AH).  In bacteria, UC and AH are on entirely separate polypeptide chains.  Previous research has shown no evidence for a direct physical interaction between the AH and UC enzymes.  In Pseudomonas syringae, two genes with unknown function, PSPTO_4241 and PSPTO_4242, are clustered very closely with UC and AH, suggesting that they may play a role in mediating physical interactions, enzymatic activity, or substrate channeling between UC and AH.  To test this hypothesis, PSPTO_4241 and PSPTO_4242 were cloned from P. syringae genomic DNA into Escherichia coli pET expression vectors. Protein overexpression tests indicated that each of these proteins were insoluble when expressed individually.  In addition, co-expression of PSPTO_4241 and PSPTO_4242 with either UC or AH did not improve solubility.  However, co-expression of PSPTO_4241 with PSPTO_4242 resulted in soluble protein expression for both proteins, indicating that they may interact with each other.

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Sonia Chris-Ukah, University of Wisconsin-Oshkosh

Mentor: Dr. Pinfen Yang

Creating Chlamydomonas strains expressing tagged flagellar calcium channels

In general, flagellar proteins are delivered from the cell body to flagella by the bi-directional intraflagellar transport in a way reminiscent to the railroad system.  Curiously, some molecules have distinct locations. For example, TRP11 and CAV2, which are mechano-sensitive and voltage-dependent calcium channels respectively, are located at the opposite end of Chlamydomonas flagella. In order to elucidate how discrete channels, both supposedly transported by IFT, are localized at different regions, this project focused on CAV2 that is primarily positioned at the tip. Specifically the goal is to create tagged CAV2 strains for purification of tip-determining molecules and for tracking CAV2 in vivo. Notably, CAV2 is a 2062s-aa protein, consisting of four regions of 6 transmembrane regions. The nearly 15 kB GC-rich sequences and positioning of tags posed substantial challenges.  Firstly, a BAC clone containing the CAV2 sequence was identified and the entire CAV2 gene was cloned into two plasmids. The DNA encoding green fluorescent protein (GFP) or 3HA12His was generated by PCR and inserted in-frame into a restriction site downstream to the first ATG but upstream to the first transmembrane region in 5’ clone.  The tagged 5’ and 3’ fragments were then cloned into a binary vector that carries an antibiotic cassette to aide selection in Chlamydomonas. The usage of DH10B bacterial strain; and the control of the growth conditions permitted the cloning and optimal yield of the final 19-kB plasmids. The two types of tagged constructs, confirmed by restriction digest, were transformed into the Chlamydomonas CAV2 mutant.

 

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Nikala Gigliotti, Milwaukee School of Engineering

Mentor: Dr. Lisa Petrella

Evolution over time of C. elegans fertility at different temperatures

Fertility defects occur at high temperatures in C. elegans, humans, and agricultural animals.  We want to observe if C. elegans can evolve better fertility rates at high temperature by maintaining them at intermediate temperatures and apply the findings to how global warming could affect ecosystems if the worms cannot evolve.  We used 15 wild-type strains to determine if there are strains that can adapt to high temperature.  Strains were maintained at two temperatures 24°C and 26°C, and the number of generations that the worms stayed fertile was assayed. These temperatures are higher than the optimal temperature, but lower than the temperature at which they become sterile.  Two different worm maintenance techniques were performed – picking, which is biased, and chucking, which causes intervals of starvation.  Worms survived better at 24°C, and chucking was the better maintenance technique.  More generations will be needed to determine which strain will adapt the best at the higher temperatures.  Another set of experiments was performed to see if worms maintained at intermediate temperatures for many generations resulted in adaptation to produce more offspring when up-shifted to the high temperature fertility threshold of 27°C.  There was not an increase in progeny from the worms maintained at the higher temperatures.  This could be due to the stress that they undergo at the higher temperatures.  Maintaining worms at gradual increasing temperatures or at alternating high and low temperatures to mimic day and night conditions then up-shifting are future experiments to be considered.

 

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Kirstan Gimse, University of Wisconsin-Parkside

Mentor: Dr. Stephen Downs

Investigating the involvement of AMP-activated protein kinase in the mitotic divisions of the early embryonic development of mice


AMP-activated protein kinase is an important regulatory enzyme involved in the maintenance of cellular energy homeostasis. Recent studies have uncovered several additional functions of AMPK including action on the hypothalamus to regulate feeding behavior, regulation of circadian rhythms, and cell cycle regulation.

Prior research in this lab has focused on the role of AMPK in the meiotic divisions that characterize oocyte maturation and activation. These studies have shown that AMPK is essential for the resumption of meiosis and the prevention of premature activation; the latter process normally occurring in response to fertilization.

The goal of this study was to investigate the possible involvement of AMPK in the mitotic divisions of the early embryonic development of mice. Exposure of both pronuclear stage embryos and two-cell stage embryos to the AMPK inhibitor Ara A resulted in a dramatic decrease in the frequency of subsequent mitotic cleavage. Immunofluorescence staining of embryos with an antibody specific to active AMPK revealed an association of the active kinase with condensing chromosomes during prophase, spindle poles during metaphase and the spindle apparatus during anaphase. Additional immunofluorescence staining for the upstream kinase LKB1 showed a similar localization pattern. These data suggest that AMPK is involved in the regulation of mitosis during the early stages of embryonic development. It is tempting to speculate that AMPK plays a role in the checkpoint mechanisms that control the embryo’s progression through the stages of mitosis.

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Marian Lund, Gustavus Adolphus College

Mentor: Dr. Michael Schlappi

HyGRP stress response genes, qLTG3-1 and Glyma 17g14850, improve seedling germinability under low temperature and salt stress conditions in Arabidopsis thaliana

The ability of plant seeds to germinate (germinability) under low temperature and osmotic stress conditions is an important trait for agronomic purposes. Previous experiments have shown that hybrid proline-rich protein (HyPRP) or hybrid glycine-rich protein (HyGRP) genes such as EARLI1 in Arabidopsis thaliana or qLTG3-1 in rice, respectively, improve germinability under stress conditions. The objective of this SRP study was to determine whether the two HyGRP genes qLTG3-1 and Glyma17g14850 from rice and soybean, respectively, would improve seed germinability under low temperature and salt stress conditions in transgenic Arabidopsis thaliana lines when expressed from a strong constitutive promoter. Transgenic line and wild-type control seeds were germinated on agar plates containing half strength Murashige Skoog (MS) medium under room temperature (22°C), cold stress (10°C), and salt stress conditions (half strength MS medium supplemented with 50 mM NaCl at 22°C). HyGRP gene expression was analyzed under all germination conditions using RT-PCR and real-time qPCR to quantify gene expression. qLTG3-1 lines with high transgene expression had significantly better germinability rates under stress conditions than wild-type controls or lines with relatively low transgene expression. A similar result was obtained for transgenic lines with relatively high expression of Glyma17g14850. These results indicate that qLTG3-1 and Glyma17g14850 have similar stress protective functions and suggest that Glyma17g14850 may help soybean seeds to germinate under adverse stress conditions.

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Katherine Maniates, St. Catherine University

Mentor: Dr. Allison Abbott

Characterization of microRNA regulation of ovulation in C. elegans

microRNAs are essential regulators of animal development and physiology. They are small, non coding ~22 nucleotide RNAs that function post-transcriptionally to regulate gene expression. Mutations in the microRNA biogenesis pathway result in lethality in worms, flies, fish and mice. One process in worms that requires microRNAs is the process of ovulation. Although microRNAs are required for ovulation, the steps that require microRNAs remain unexplored. Using a conditional allele of the gene, pash-1, which is required for microRNA biogenesis, we are able to study the effects of reduced microRNA activity on the process of ovulation. Worms with the mj100 temperature sensitive pash-1 allele are unable to ovulate at the restrictive temperature of 25°C at which few, if any, mature microRNAs are generated. In this study, I examined ovulation in worms grown at the permissive temperature of 15°C and an intermediate temperatures of 17.5°C. pash-1 (ts) mutants have a reduced number of progeny as well as a reduced oocyte maturation rate. Using video microscopy, we find spermatheca entry defects in which part of the proximal oocyte gets pinched off as it enters the spermatheca. These data indicate that the process of ovulation is sensitive to reduced microRNAs. Specifically, spermatheca valve dilation and gonadal sheath contractions require microRNA activity. Future work will include performing calcium imaging to determine if pash-1(ts) worms have defects in calcium signaling during ovulation.

 

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Joseph Rehfus, Marquette University

Mentor: Dr. Robert Fitts

The effects of exercise training on myocardial action potential duration and calcium handling

Cardiovascular disease (CVD) is the leading cause of death for both men and women and health care costs exceed $500 billion annually in the US. Frequently, CVD involves ischemic heart disease (IHD), characterized by reduced blood flow to the myocardium. Exercise-training has been shown to reduce the incidence and severity of IHD. Thus, understanding its role in protecting against and treating ischemic injury is important. This study evaluated how voluntary wheel running in rats altered the electrical activity of the heart and Ca2+ handling within isolated cardiomyocytes. Using a Langendorff apparatus, hearts from trained (TRN) and sedentary (SED) rats were subjected to regional ischemia (RI) and reperfusion. Action potential durations (APD) were recorded at multiple stimulation frequencies from the base and apex and compared before, during, and after RI. APDs (in ms) were shorter in TRN than SED rats at rest during RI (121+/-20 vs. 129+/-9 at base, 111+/-11 vs. 130+/- 8 at apex), and at high heart rates after 60 minutes of reperfusion (81+/-16 vs. 99 at base, 83+/-15 vs. 99 at apex). Cardiomyocytes from SED rats were incubated with Fluo-4AM and Ca2+ transients measured at various stimulation frequencies. The Ca2+ content (fluorescent intensity of 242.4+/-43 vs. 172.1+/-30), influx (4.6+/-0.8 vs. 3.3+/-0.8) and removal (-0.7+/-0.1 vs. -0.26+/-0.08) rates were higher in cardiomyocytes from the base compared to the apex. In conclusion, training shortened APD at rest during RI and following 60 minutes recovery from ischemic injury, and base compared to apex myocytes showed higher Ca2+ transients and faster kinetics.

 

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Kara Signorelli, Marquette University

Mentor: Dr. Thomas Eddinger

Different levels of protein expression in coarcted, corrected, and  control aortic tissue

Aortic coarctation is a narrowing of the aorta, and can have life threatening implications due to the hypertensive conditions it creates above the point of constriction and hypotensive conditions below. It is believed that stressful conditions can induce changes within smooth muscle cells, which may contribute to additional adverse pathologies associated with aortic coarctation. One way to assess the changes occurring in smooth muscle cells is to evaluate the varying levels of protein expression. This project was designed to investigate the levels of different proteins being expressed under coarcted, corrected, and control conditions in a rabbit model. In order to execute this investigation, the aortic tissues of these rabbits were harvested and subjected to immunohistochemical staining to determine the intensity, as well as the location of protein expression within the tissue. Methodological issues such as failure of tissues to adhere to slides and limited antigen availability were encountered. For this reason, protocols needed to be optimized before experimentation could proceed. It was found that the ideal protocol for insuring tissue adherence was to first coat the slides in albumen and allow them to dry prior to picking up the tissue samples. The slides then needed to be incubated overnight just below the melting point of the paraffin wax they were preserved in. The ideal procedure for antigen retrieval consisted of heating the tissue coated slides in a buffer just below its boiling point. These procedural changes have helped enhance immunostaining effectiveness, and will allow experimentation to proceed from this point. 

 

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Taylor Stevens, University of Tennessee at Knoxville

Mentor: Dr. Martin St. Maurice

Investigating allosteric activation in pyruvate carboxylase using chimeric enzymes

 

Pyruvate carboxylase (PC) is a multidomain enzyme important for several metabolic processes, including pyruvate cycling in humans, a process thought to be tied to glucose stimulated insulin release. Acetyl coA, an allosteric activator of PC, has been shown to bind to the enzyme at a site located on the central allosteric domain, but the actual mechanism of activation is unknown. In this study, we investigated the role played by different domains in the allosteric activation of PC. Chimeras were constructed from domains in Aspergillus nidulans PC, one of the few forms of PC not activated by acetyl coA, and Rhizobium etli PC, a form that is fully activated by acetyl coA. Overall, our kinetic data reveals that the mechanism behind activation is complex and involves interactions between multiple domains. More specifically, these results suggest that the major determinant for allosteric activation resides in the biotin carboxylase domain of PC, and that the affinity for the activator is primarily dependant on the allosteric domain.

 

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Maureen Thompson, Milwaukee School of Engineering

Mentor: Dr. James Maki

Enzyme activiy of cell-free extracts from biofilms of Bacillus JK7 and Stenotrophomonas JK3

 

Biofilms of the bacteria Bacillus JK7 and Stenotrophomonas JK3 inhibit adult zebra mussel attachment when developed on polystyrene, but not when developed on glass. Data has shown that the inhibitory factor is extracellular, but its nature is unknown. Experiments were conducted to better understand the inhibitory factor and biofilms of these bacteria in general. First, it was hypothesized that exopolymers from biofilms of B. JK7 and S. JK3 may contain toxins. In order to test this, a bioassay using nematode embryos was developed. Data showed no significant difference in nematode survival between the control and the treatments (p>0.05, ANOVA). This may be due to the collagen structure of the embryo egg shell; results may be different if tested with adult nematodes.  Second, biofilms of bacteria may disassemble due to secretion of D-amino acids, reducing biofilm biomass. Biofilms of B. JK7 and S. JK3 were treated separately with D- and L-tyrosine (10µM) and the effect on the biomass of attached cells was measured. Addition of D-tyrosine did not reduce the biofilm biomass compared to control and L-tyrosine treatments (p>0.05, ANOVA), but other D-amino acids might be more effective; these need to be tested.  Finally, degenerate PCR primers from the literature designed for amplification of protease gene profiles from Bacillus sp. were used to examine DNA extracted from B. JK7. No pattern of amplified products was evident on the gel. These primers may not be universal enough to amplify protease genes from B. JK7. Different primers may have to be designed.

 

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Emma Weber, Marquette University

Mentor: Dr. Anita Manogaran

Characterization of genes involved in appearance of the [Het-s] prion

 

Prion diseases are brain diseases that are caused by a misfolded protein called a prion.  Misfolding of the non-glutamine rich prion protein, Prp, leads to Mad Cow disease in cattle and Creutzfeldt-Jacob disease in Humans.  These misfolded infectious proteins have the ability to convert normally folded copies of themselves into misfolded forms, thus causing disease.  In order to gain insight into the mammalian prion system, we are studying prions in yeast because they provide an easy means to see how prions appear and progress in this simple system, which is something we wish to gain more knowledge about.

Previous observations of prion appearance have shown that a fluorescently tagged glutamine rich prion,  [PSI+], forms fluorescent rings in yeast cells.  Ring containing cells pass infectious fragments of the rings called aggregates to their daughter cells, which then can be propagated to future generations of cells.   Two classes of gene deletions were found to reduce prion formation. One class, “Class E,” inhibits prion formation in the early stage by inhibiting ring formation. The other class of strains, “Class L,” forms rings but prevents the late transmission of aggregates to daughter cells.   While this glutamine rich prion is controlled by two-classes of genes, we were interested in whether non-glutamine rich prions similar to Prp, are also affected by these two classes.  Therefore, we tested the fungal non-glutamine rich prion, [Het-s].

To test this hypothesis, a plasmid carrying the Het-s gene fused to GFP was transformed into four deletion strains that reduce prion formation: las17, sac6, bem1 and bug1.   We asked whether rings and aggregates were formed with the over expression of Het-s-GFP in all deletion strains.  Our results indicate that only las17 reduces Het-s ring and aggregate formation, suggesting that glutamine and non-glutamine rich prions use different cellular mechanisms during prion appearance. 

 

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Amanda Youmans, Milwaukee School of Engineering

Mentor: Dr. Edward Blumenthal

Possible interactions of genes with drop-dead in Drosophila melanogaster

 

Drop-dead (drd) is a gene in Drosophila melanogaster that when mutated causes shortened lifespan, infertility, and gut defects.  The goal of this project was to figure out the biochemical function of drd, since little is known about this gene.  In order to accomplish this goal, further investigation was required to determine whether a functional relationship truly existed between three gene families: GstE, Jheh, and Cyp450, which were identified by a microarray screen as having significant changes in expression when drd was knocked down in ovarian follicle cells.  Two experimental approaches were utilized.  First, drd was knocked down in the follicle cells and gene expression was measured through qPCR for the 3 gene families identified on the microarray.  Seven of the eleven genes tested produced a significant change in expression as compared to the control.  The second experimental approach was to knock down the genes identified by the microarray in the follicle cells to see if the drd infertility phenotype was seen.  All knockdown progeny were fertile suggesting that the gene may not have a relationship with drd or that the RNAi was not effective.  RT-qPCR ran on the ovarian cDNA of GstE1 and Jheh2 knockdown flies showed that there was no change in expression, so no conclusions about a functional relationship could be drawn from this method.  While no functional relationships were able to be confirmed, many genes from the microarray were verified and therefore provide stronger evidence of a genetic interaction with drd.  

 

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Biology Summer Research group

 


 

 


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