2007 Research Fair - Biology Abstracts
This study was undertaken in an effort to minimize the discrepancy among the science community concerning phylogenetic classification of birds. Through refinement of DNA extraction and sequencing techniques, the fundamental goal of this research was to ultimately produce new and supplementary avian sequence information. DNA extraction was carried out in accordance with Qiagen tissue extraction/purification protocols, and frozen tissue samples from a Mourning Dove (Zenaida macroura) specimen were utilized for an avian DNA source. Additional purification/concentration of extracted DNA was carried out using ethanol precipitation. Gel electrophoresis was employed to confirm extraction/purification success. Resultant data from DNA confirmation led to modifications in the extraction protocols including increased mass of sample tissue. PCR was carried out using primers for the 12S mitochondrial rRNA region based on success in recent studies of phylogenetic relationships. The results of this amplification and resultant sequence information will be discussed at a later date.
Effects of a Urine Environment on Esp and Ace Expression
Enterococcus faecalis is responsible for a variety of infections in the body, including many of the urinary tract. Our focus has been on two genes, esp and ace, identified in previous research to be involved in attachment and adherence of the bacteria to human cells. The clinically obtained E. faecalis strain W34372 was grown in tryptic soy broth containing10 percent urine. We predict levels of esp expression to be higher in urine containing media than levels of ace; though previously published experimentation has been inconclusive on this point. Total RNA was isolated from bacteria grown with urine, using Qiagen kits. The level of expression of each gene was determined using a one step procedure for quantitative (real-time) PCR. Preliminary data have shown little expression of esp, higher levels of ace, and much higher levels of ribosomal RNA. Ace RNA expression was detected by cycle thirty-two, and ribosomal RNA expression during cycle eighteen.
The Effect of Crowding on the Sex and OMP Expression
The effect of the environment on the development of an organism is an increasingly interesting area of research. Environmental factors, such as temperature, existing sex ratio, and pH are known to effect the development of many organisms. However, the effects of a crowded environment on the development are relatively unknown. Our preliminary experiments indicate that restricting the space available to embryos of the teleost fish medaka (Oryzias laptipes) interferes with their development. We are using various PCR techniques to investigate the effects of crowding on the sex determination and olfactory development in medaka in order to determine if crowding does in-fact affect the development of this fish.
Understanding the Effects of Caffeine on C. elegans
Caffeine is often referred to as the most widely used behavior altering drug. Its popularity among college students has led us to study its effects on the model organism Caenorhabditis elegans. Previous research has shown that C. elegans experience paralysis after ingesting caffeine. This response has been attributed to the ryanodine receptor which is involved in muscle contraction. Caffeine as well as ryanodine are agonists of the ryanodine receptor and when bound cause an overwhelming calcium influx, leading to continuous muscle contraction. ATP is needed to pump calcium back out of the muscle cell resulting in muscle relaxation. Wild type and ryanodine receptor mutants were treated with caffeine and ryanodine then muscle contraction was measured by counting body bends per minute. We predicted that coupling caffeine or ryanodine treatment with ATP or adenosine may result in recovery from paralysis. Assays were developed to test this hypothesis, and the results of these experiments will be presented.
Effects of Different Media and Aerobic vs. Anaerobic Growth Conditions
Enterococcus faecalis is a Gram-positive, cocci-shaped bacteria associated with nosocomial infections. It is one of the leading causes of hospital acquired secondary infections. Adherence of E. faecalis is primarily due to the expression of the ace and esp genes. Our research examined the conditions which best facilitate bacterial adherence. For this study, we examined two strains of E. faecalis, H36762 and T33123. The strains were grown under different culture conditions by varying the media composition (TSA or BHI) and/or using aerobic vs. anaerobic environments. Total RNA was isolated and quantified. The levels of gene expression were tested by using QPCR. Early data shows that the bacteria grown in an anaerobic environment have greater expression of the ace gene. Furthermore, it shows that the ace gene is more expressed in TSA than in a BHI media. Further results will be presented.
Identification of Great Salt Lake Microbes Associated with Artemia franciscana
Most research regarding the microorganisms associated with Artemia fransicana has been limited to feeding studies concerning Artemia used for aquaculture. We focused on identifying the Bacteria and Archaea associated with adult and encysted Artemia embryos in order to understand the role of prokaryotes in this invertebrate’s life cycle and to consider the role of prokaryotes in the Great Salt Lake (GSL) food web. Artemia cysts and adults were first washed in an attempt to remove any external microorganisms. DNA was then extracted and amplified by PCR using primers that amplify either Bacterial or Archaeal 16S rDNA. The amplified Bacterial or Archaeal DNA fragments were purified, cloned, and sequenced. 16S rDNA sequences were compared to known sequences using the NCBI BLAST program. Using these methods we have identified Bacterial and Archaeal species in encysted and adult GSL Artemia and have speculated on their importance to Artemia and the GSL food web.
Resistance to Ultraviolet Irradiation and Desiccation
Great Salt Lake (GSL), a natural hypersaline ecosystem, is home to salt-tolerant Bacteria and Archaea called extreme halophiles, which live at 3-5M NaCI. In addition to salt tolerance, GSL archaeal halophiles exhibit resistance to both ultraviolet (UV) irradiation and desiccation. First, with respect to UV resistance, we propose that these organisms employ photoprotective mechanisms such as pigmentation. Indeed, many halophile species have high levels of carotenoid production, which are linked to UV resistance, but the mechanism by which the pigments work is not understood. To test the hypothesis that carotenoids prevent DNA damage from occurring, we designed an immunoassay to detect thymine dimers in DNA from pigmented strains and pigment-deficient strains. UV resistance data will be presented as well as relative thymine dimer levels in these strains. Secondly, to understand desiccation resistance, we sought to determine the diversity of GSL halophiles capable of surviving halite crystal formation, dormancy, and dissolution. Recently formed halite crystals were collected from GSL and dissolved in media. Surviving microorganisms were cultured and isolated. For each isolate, genomic DNA was extracted and the archaeal 1 6S rDNA gene amplified. These sequences were compared to gene databases for determination of closest matched species. In addition to an examination of desiccation potential, salt crystal dissolution is a novel method for isolating microbial communities from an extreme environment. Evidence for both UV and desiccation resistance in these salt-tolerant GSL halophiles makes them well-suited as models for Astrobiological studies which pursue questions about life off earth.
The Upregulation of Thioredoxin Reductase in Artemia
Great Salt Lake is becoming polluted with selenium. To show how the brine shrimp, Artemia, are surviving in the presence of high selenium, gene expression of this organism was investigated. We are looking closely at levels of gene products in pathways involving selenium. RNA was isolated and some made into CDNA to perform PCR. The primers that were used are sequences within the thioredoxin reductase gene, which is known to be involved in selenium resistance. The rest of the RNA will be tested by PCR using primers from flies whose genes upregulate in response to an increase in selenium. In addition, selenium toxicity exponents will be discussed.
Impact of Morphine Metabolite Differences on Analgesia in Women and Men
This study aims to explore the pharmacokinetic factors that may contribute to gender-specific differences in response to painful stimuli and alteration of response with opioid analgesics by assessing the interaction of a parent drug with its active metabolites. This study will (1) assess the impact of morphine metabolites on analgesia in women and men during stable gonadal hormones levels, and (2) assess the impact of morphine metabolites on analgesia in women during fluctuating gonadal hormone levels. Subjects participated in the two-period crossover study (12 male and 12 female, ages 18-40). During one session, subjects receive 50 mg of ranitidine IV 30 minutes before the session, followed by a target controlled infusion of morphine maintained for 1 hour then an increased target concentration of morphine held for 1 hour. In the crossover session, each subject receives a saline infusion rather than the ranitidine dose. The results of this study will identify the role that morphine metabolites play in contributing to acute analgesia from morphine and determine the impact that gonadal steroid hormones have in modulation of these effects. This knowledge can then be used to determine the clinical implications of these factors for providing adequate analgesia in females compared to males.
Antioxidant Effects on MPP+ Induced Parkinson’s Disease
Parkinson’s disease (PD) is a neurodegenerative disease that affects roughly 40,000 people yearly. It is hypothesized that the degeneration of dopamine neurons that results in decreased mobility, is due to damage in mitochondria. Antioxidants are chemicals known to neutralize free radicals, and others have hypothesized that antioxidants may have a positive effect in the treatment of PD. The small roundworm Caenorhabditis elegans can be induced to express a PD-like phenotype when treated with the mitochondrial toxin MPP+. We chose to test the effects of antioxidants on this model of PD in worms. Standard locomotion assays that measure body bending were conducted to look for decreased mobility as a symptom of PD, and to test whether antioxidants had a positive effect. Results are forthcoming.
Using Mitochondrial DNA Segments to Determine Evolutionary Relationships Within Avian Species
Avian and other animal species are currently being classified by their DNA sequences instead of their morphology, as was done in the past. There is an advantage to using mitochondrial DNA in this classification over nuclear DNA. The nucleic acids in mitochondrial DNA mutate at a slightly faster rate then that of nuclear DNA. Our hypothesis is that one would be able to begin building an avian phylogeny based extracting and amplifying a target segment of mitochondrial DNA. Once the segments have been sequenced, a comparison of the disparity between and within a species of bird can be analyzed. The determination could be made as to whether the target segment is a reliable indicator of species’ phylogenetic relationships.
DNA Protection Mechanism in Halophiles: Carotenoid Investigation
Many microscopic organisms call the Great Salt Lake home. Salt-tolerant Bacteria and Archaea called “halophiles” make up a significant portion of the bio-population. Halophiles are exposed to intense UV radiation daily, and as a result many depend on UV-resistance to survive. Carotenoids, which exist in the cell membrane, are thought to absorb UV rays before they are able to penetrate the cell and damage the DNA. Although carotenoids have been referenced as a likely contributor to UV-resistance their role as a photoprotector has not been exclusively established. Using the Great Salt Lake species, Halorubrum salsolis, we examined the effect of differing salinities and light conditions, in hopes of obtaining variation in carotenoid levels and composition within a single species. So far cultures exposed to light have shown the highest levels of growth. We will further discuss this result along with data on other variant conditions in our presentation. Future studies will involve varying nutritional conditions and carotenogenesis inhibitors.
Synthesis of Novel West Nile Virus Inhibitor
The West Nile Virus is an emerging pathogen for which there is currently no therapeutic treatment option. Those patients affected by the virus only receive supportive therapy. The objective of this project is to develop a polyribonucleotide inhibitor of the RNA directed RNA polymerase (RdRP) of the West Nile Virus (WNV). Previous work has shown that some ribonucleic acid homopolymers with modified bases inhibit the activity of Reverse Transcriptase (RT) of HIV, an RNA directed DNA polymerase (RdDP). 1 The theory is that the inhibitor binds within the active site of the RT polymerase thus blocking the binding of the viral RNA and prohibiting viral replication. With replication halted, this will essentially block the ability of the virus to proliferate in the body. It is proposed that the homopolymers will be capable of inhibiting both the RdRP and RdDP of two viruses because the replication mechanism of the enzymes are similar. To establish proof of principle that a polyribonucleotide construct is able to inhibit the polymerase of the West Nile Virus, the polymer Poly (1-propargylinosic acid) has been synthesized through four synthesis steps. Synthesis and purification of this polymer and an imidazopurine analog will be described and results of biological activities will be presented.
Homosexuality: Science, Society & Theory
Homosexuality has been subject to much intense debate, the origins of which being discussed in the philosophical, sociological, and scientific realm. Scientific data in support of homosexuality as a biological (natural) phenomenon include assays with mature D. melanogaster males who display courtship behavior toward young males due to an identified excitatory female pheromone, which leads to young males attracting the sexual interest of mature males. The social response to homosexuality has led to homosexual students being five and a half times more likely to be threatened or injured with a weapon four or more times at school. This study will address the most current scientific data supporting the biological origins of same-sex breeding patterns, our society’s response to homosexuality, as well exploring socio-biological theories of the origins of homosexuality and the human reaction to it.