• A Series of Laboratory Exercises for Use in Undergraduate Studies

      Kline, Larry K.; Frank, Daniel Patrick; The College at Brockport (1988-09-01)
      This paper focuses on developing appropriate laboratory exercises to support learning for undergraduate students in advanced biology courses. The researcher created a series of lab exercises: isolation of rat liver mitochondria, restriction enzymes laboratory, ultraviolet repair system laboratory, bacterial transformation, and hybridoma module. Each lab exercise includes a description of the educational goals/rationale, the factors considered in lab design, background information on the task, a detailed description of the sub-tasks, and detailed methods.
    • A Study of the Sequestration of Melanin-Concentrating Hormone Receptor-1 in Caveolae: A Potential Mode of Cell Signaling Regulation by an Appetite-Stimulating Hormone

      Field, Lauren D.; The College at Brockport (2012-08-12)
      The prevalence of obesity in the United States of America has increased over the last twenty years. This prevalence has led to an increase in the study of the hormones involved control of metabolism and satiety to further understand the factors involved in obesity. One of these hormones is melanin-concentrating hormone (MCH). Many of the studies of MCH focus on the brain, while little work is done on the peripheral tissues. In adipose cells stimulation with MCH causes a release of leptin through activation of melanin-concentrating hormone receptor-1, a G-protein coupled receptor. MCHR1 becomes desensitized after activation with MCH, but the method of desensitization is unknown. ELISA studies show that internalization of the receptor is low unless proteins in the clathrin pathway are incorporated, so another method of desensitization must be occurring. Through sucrosegradient centrifugation MCHR1 co-localizes with caveolin-1, suggesting a role for lipid rafts in receptor dynamics. This thesis will examine the extent of interaction between caveolin-1 and MCHR1. The first aim will be to determine the degree of co-localization of receptor and caveolin-1 under varying conditions. The second aim will be to analyze the dynamics of the MCHR1 within caveolae after MCH stimulation and with expression of the arrestins. The goal will be to better understand the interaction between caveolae and MCHR1 and possibly provide insight into MCHR1 's mode of desensitization.
    • Amino Acid Accepting Activity for Lysine and Arginine Transfer Ribonucleic Acids in Moloney and WM1-B Strains of Murine Leukemia Viruses

      Kline, Larry K.; Evans, Jack Randall; The College at Brockport (1974-01-01)
      The object of this research was to isolate and purify Moloney and WMl-B strains of Murine Leukemia virus from (NIH) Swiss mouse embryo tissue culture and to determine transfer ribonucleic acid presence for Lysine and Arginine. Viruses were purified by ultracentrifugation and activity determined by Plaque Assay. Protein concentration and RNA content by Lowry and Orcinol assays respectfully, concludes a 2-3% total RNA content of this RNA tumor virus, similar to that reported for Avian tumor viruses and other strains of Murine Leukemia viruses. Enzymatic aminoacylation proved that Murine Leukemia viruses contain transfer RNA populations. This is the first time transfer RNA amino acid accepting activity in Murine Leukemia virus has been observed and may be a general property of RNA Tumor Viruses.
    • An Investigation of Melanin-concentrating Hormone Receptor Internalization – Or Lack Thereof

      Cook, Laurie B.; Moden, Jay I.; The College at Brockport (2012-01-01)
      Melanin-concentrating hormone (MCH), a cyclic peptide hormone involved in energy homeostasis, is known to bind to two G protein-coupled receptors (GPCRs) in mammals. These receptors, melanin-concentrating hormone receptor 1 (MCHR1) and melanin-concentrating hormone receptor 2 (MCHR2), have been a popular target for MCH antagonists in an effort to fight the ongoing epidemic of obesity. In the presence of prolonged stimulus it is common for GPCRs to undergo rapid desensitization. However, the desensitization mechanisms of MCHR1 and MCHR2 are as yet poorly understood. This study aims to create epitope-tagged expression vectors to allow for the expression of MCHR1 and MCHR2 in a tissue model. Utilizing a modified cell-based ELISA and fluorescence microscopy, the sequestration of MCHR1 and MCHR2 would be measured after agonist stimulus. Receptor interactions with GRK2 and ?-arrestins would also be measured. The over expression of both MCHR1 and MCHR2 proved to be cytotoxic to BHK570 cells. The overexpression of GRK2, ?-arrestin 1, and ?-arrestin 2 showed a relatively small but statistically significant increase in receptor internalization. Fluorescence microscopy suggests that the interaction between MCHR1 and ?-arrestins were transient in nature. These finding suggest that MCHR1 can be internalized via the clathrin-mediated pathway. It is likely MCH signaling is mediated a cell specific manner based on the cellular expression levels of GRKs and ?-arrestins.
    • An Organic Energy Budget for the New York State Barge Canal

      Makarewicz, Joseph C.; Amering, Allan R.; The College at Brockport (1978-11-01)
      An annual energy budget is presented for the New York State Barge Canal, a first order man-made waterway in western New York. The ecosystem approach, in which all input and output of energy as organic matter are measured, is used to describe the energy flow in an 1130-meter segment of the canal. The annual input of energy to the system is 38.1 x 109 kcal/m2. Over 99% of this is allochthonous from upstream areas. Autochthonous input from primary producers accounts for less than 0.1% of the total energy available to the-system. Meteorologic inputs (litter and precipitation) from the adjacent terrestrial ecosystem account for less than 0.1% of annual energy input. Seventy-eight percent of the geologic input and 99% of the total energy input occur as dissolved organic matter. Approximately 7,790 kcal/m2 of organic detritus is stored within the system. The annual output of energy from the canal system is 38 x 109 kcal/m2. Ninety-nine percent of the annual energy input is exported to downstream areas in canal water. Less than 0.1% of the energy output is lost through community respiration. The New York State Barge Canal is a strongly heterotrophic system in which ecosystem efficiency and flow~through energy (0.1% and 99.9%, respectively} indicate the canal makes very little use of the energy supplied to it.
    • Arginine Methylation and Enzymatic Activity of TbLpn, a Lipin Homologue from T. Brucei

      Pelletier, Michel; Ortega, Bernardo; Shen, Rongkun; Bumstead, Andrew R.; The College at Brockport (2016-07-21)
      Trypanosoma brucei is a flagellated protozoan parasite responsible for African Trypanosomiasis. T. brucei is a deadly immune-evasive parasite which circulates the bloodstream of mammalian hosts and requires the Tsetse fly as a transmission vector. The parasite is capable of antigenic variation which allows it to change its glycoprotein coat in order to escape immune detection. Of great concern is the parasite’s growing resistance to various chemotherapeutic treatments which may allow for reemergence. Protein-arginine methyltransferases (PRMTs) are a group of proteins responsible for transferring methyl groups to arginine residues within proteins. Protein methylation causes epigenetic modification of histones or changes in protein-protein interactions which, in turn, leads to the regulation of a variety of biological functions including, but not limited to: transcriptional/translational activation or repression, signal transduction, protein localization, and cell differentiation. Several PRMTs have been discovered in T. brucei including TbPRMT1, TbPRMT3, TbPRMT5, TbPRMT6 and TbPRMT7. TbLpn, a lipin family protein, was discovered based on its protein interactions with PRMTs. Lipins act as Mg2+-dependent phosphatidate phosphatases (PAPs) which catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol (DAG). DAG is a powerful cell signaling molecule which can then be channeled into the synthesis of triacylglycerol (TAG) as well as the phospholipids phosphatidylcholine (PC) and phosphotidlyethanolamine (PE) via the Kennedy pathway. PE and PC are both core constituents of the protozoan cell membrane, and PE in particular is necessary for synthesis of the glycosylphosphatidylinositol (GPI) anchor. Importantly, T. brucei synthesizes its phospholipids de novo, ensuring that these phospholipids are produced by the Kennedy pathway and not by host scavenging. To observe any methylation interaction between TbPRMTs and TbLpn, a methylation assay was conducted using Adomet as a methyl source. The results show that TbPRMT1, TbPRMT5, and TbPRMT7 all successfully methylated TbLpn independently in vitro. TbPRMT3 and TbPRMT6 were incapable of methylating TbLpn in vitro. Furthermore a phosphatidic acid phosphatase assay was conducted to observe how effective TbLpn functions as an enzyme when methylated by TbPRMTs. This assay determined the enzymatic activity of TbLpn based on the release of organic phosphate (Pi) released during the dephosphorylation of PA to DAG. Results from the phosphatidic phosphatase assay show that the enzymatic activity of TbLpn increases greatly following methylation by TbPRMT5 or TbPRMT7, but not TbPRMT1, over control TbLpn.
    • Behavioral phenotype of Vang6 mutant Drosophila melanogaster pertaining to the Olfactory System

      Hing, Huey; Pelletier, Michel; Sia, Rey; Austin, Sarah Lynn; State University of New York College at Brockport (2016-01-21)
      There are many proteins that aid in the development of the olfactory system, specifically Wnt pathway and planar cell polarity (PCP) pathway proteins. It has been shown that Vang6 mutant flies have distinct olfactory abnormalities, as do Wnt5 mutant flies. In addition, Drosophila melanogaster (Drosophila) Wnt5 mutants have an improper olfactory response compared to wildtype Drosophila. After using a T-maze to explore the behavioral tendencies of Vang6 mutant Drosophila and wildtype WT1118 flies, it was shown that there is no significance between wildtype and Vang6 mutant Drosophila selecting air (control component) or Carbon Dioxide (CO_2) (test component).
    • Causes and consequences of patchy spatial distribution in male and female fairy shrimp, Eubranchipus bundyi

      Harris, Patricia; Roosa, Brian Robert; The College at Brockport (2002-12-06)
      In this thesis, I investigated fairy shrimp (E. bundyi) distribution and some possible effects they might have on the temporary pond community, focusing mainly on behavioral responses to environmental variables. Lab and field experiments, as well as transect data, suggest E. bundyi may be attracted to dim light and seek shade and structure within the water column (vegetation, sticks, roots, the bases of trees, rocks, etc.) when light levels are high. At midday, fairy shrimp seem to cluster among shaded structured regions of the pond; for instance, a thick mass of floating bark casting shade in an otherwise open patch of water may shade hundreds of fairy shrimp during the day, whereas few fairy shrimp are found in such locations at night. As evening sets in, the shade aggregations start to break up, and at night the fairy shrimp are common in deep, open, unstructured regions of pools. Overall, females tend to be less mobile, less attracted to light and deeper in the water column than males. The patchy distribution of fairy shrimp (E. bundyi) may be the result of egg hatching cues, microhabitat preferences in regards to light level, and the cryptic behavior of females. Behavioral differences between the sexes may expose males and females to different predators and food resources. The diel migration of both sexes may be responses to predation and/or UV photodamage. The community effects of fairy shrimp distribution, migration, and the different activity levels of the sexes, however, may be dampened because of abundant food resources and habitat disturbance (drying of the pond) truncating a trend towards a competition and predator oriented community. While no previous study has taken the comprehensive lab/field approach that I describe in this thesis, my results are similar to the few other studies of the effects of light, shade, and gender on other species in habitats considerably different from E. bundyi's. Diel migrations and responses to physical microhabitat parameters may be as widespread among anostracans as it is among cladocerans, and may turn out to be as useful for understanding ecology of temporary ponds as it has been for understanding the ecology of the limnetic zone of lakes (Hutchinson 1967, Wetzel 1983, Wetzel and Likens 1991).
    • Characterization of FMP35: A novel gene and its role in mitochondrial DNA stability

      Cornelius, Chad A.; The College at Brockport (2006-01-21)
      Mitochondria are essential organelles for all eukaryotic organisms with very few exceptions. The life-giving processes contributed by mitochondria are the end result of many proteins that are encoded within the mitochondria. Many nuclear encoded proteins give mitochondrial DNA (mtDNA) the high stability needed so that life can thrive. Saccharomyces cerevisiae (baker's yeast) has historically been a model organism for mitochondrial function studies. These yeast are categorized as facultative anarobes; meaning that they are able to respire or ferment depending on media available. Functional mitochondria allow baker's yeast to thrive on a 3-carbon medium (p+), while mitochondrial dysfunctions due to mtDNA defects do not allow growth on the same medium (p-). The ease of visualizing this phenotype and culturing these organisms has made S. cerevisiae an important tool for mitochondrial studies. Nuclear encoded proteins such as Abf2p and Ilv5p have been implicated in offering a degree of stability to mtDNA. Many nuclear proteins have been localized to mtDNA, creating an essential DNA-protein complex called a nucleoid. One protein that has been defined as a mitochondrial protein is Fmp35p. This is a novel protein that remains uncharacterized. A fmp35?::URA3 gene knockout yields a p- phenotype as illustrated by a respiration loss assay. Furthermore, a significant decrease in direct repeat recombination has been described by this study. A less significant increase in polymerase slippage within microsatellites has also been documented. It is the conclusion of this study that Fmp35p plays a role in a recombination pathway that gives rise to wild type yeast with a full complement of functional mtDNA. When this gene is defective and the protein is not produced yeast will not thrive.
    • Characterization of in vitro and in vivo enzymatic activity of TbLpn and its role in phospholipids biosynthesis in Trypanosoma brucei

      Munini, Dominic Nyamai (2012-06-20)
      Trypanosoma brucei belongs to a group of parasitic protozoan called flagellates. It causes African sleeping sickness and is transmitted by tsetse fly of genus Glossina. African sleeping is a devastating vector-borne disease which threatens over 60 million people in 36 Sub-Saharan Africa. It causes over 70,000 deaths annually and is fatal unless treated. Reemergence of the disease in recent years has been a concern for scientists. There is concern on design of new drugs for the treatment of the disease because of the toxic effects of drugs designed around 50 years ago. Trypanosomes have great importance over the other parasitic organisms. They are able to synthesize phospholipids de novo. This makes the trypanosome phospholipid biosynthesis mechanism a very attractive target for design of new drugs. TbLpn which has been identified in T. brucei is a protein homolog to yeast and mammalian Lipins. Yeast and mammalian lipins are phosphatidate phosphatases involved in membrane biogenesis, energy metabolism and adipose tissue development. The lipin protein family in mammals and yeast catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol, which is in tum used to synthesize phospholipids. Two conserved domains as well as the two aspartic acid residues necessary for enzymatic activity in lipin protein family have also been identified in TbLpn. This is a clear indication that TbLpn might represent functional homolog of lipin proteins. The goal of this research project is to demonstrate in vivo and in vitro enzymatic activity of TbLpn. Recombinant His-TbLpn expressed in Escherichia coli was purified over Nickel column and used to test the enzymatic activity of TbLpn. Native TbLpn was also immunoprecipitated from T. brucei cytosolic extract and used to carry out the dephosphorylation of phosphatidic acid.
    • Characterization of Melanin-concentrating Hormone Receptor Desensitization

      Cook, Laurie B.; Goodspeed, Andrew E.; The College at Brockport (2013-06-01)
      Melanin-concentrating hormone (MCH) receptor 1-knockout mice have limited incidence of diet-induced obesity. This makes the MCH signaling pathway a potential pharmacological target to fight human obesity. MCHR1 is a G-protein coupled receptor (GPCR) that activates multiple signaling pathways, including ERK phosphorylation. Overstimulation of GPCR signaling is a hallmark of many diseases. Likewise, inadequate desensitization of MCH signaling could potentiate the obese phenotype. GPCR desensitization typically involves agonist-induced internalization of activated receptors, and subsequent degradation or receptor recycling. The broad aim of this study was to determine the length and intensity of ERK phosphorylation and it's desensitization to MCHR1 activation by MCH. In order to measure this, we maximally stimulated MCHR1-transfected BHK-570 cells with 100 nM MCH for 10 min, then following three washes in serum-free media and a 30 min recovery period, cells were stimulated again. Western blots of lysates for phosphorylated-ERK and total ERK were performed. ImageJ was used to normalize activation levels. MCH was unable to signal a second round of ERK signaling unless we waited 70 minutes, indicating that the MCH signaling pathway is desensitized during this period. We hypothesized that MCHR1 internalization was responsible; however using a cell-based ELISA, we only measured a 15% loss of surface MCHR1 after 30 min of MCH treatment. We tested the hypothesis that G protein-coupled receptor kinases were limiting factors in preventing agonist-mediated endocytosis of MCHR1 however none showed significant gains in internalization. We conclude that MCHRl can undergo receptor-mediated endocytosis, but the fraction of available receptors on the plasma membrane does not account for the extensive loss of ERK signaling observed. We also tested the effect that a GRK2 dominant negative would have on MCHR1 desensitization. In a co-transfected BHK-570 model, we did not observe desensitization if GRK2 is not present. This suggests that GRK2 is necessary for MCHR1 desensitization at the plasma membrane. We have also observed similar ERK desensitization following both isoproterenol treatment and MCHR2 activation which could suggest that simply the ERK pathway desensitizing is being observed which could be independent of the agonist. This study suggests that MCH-mediated ERK signaling desensitizes while MCHR1 is at the plasma membrane, rather than via removal of the receptor from the cell surface. Future experiments will be aimed at determining whether this ERK pathway desensitization is homologous or heterologous in addition to observing downstream pathways of MCHR1 activation other than ERK.
    • Characterization of RNA Associated with Rat Liver Plasma Membranes

      Kline, Larry K.; DeBellis, John J.; The College at Brockport (1977-06-01)
      RNA was extracted from purified rat liver plasma membranes. The RNA was characterized in terms of molecular weight distribution (electrophoresis) and base composition. Plasma membrane RNA was shown to have a major 28S species, several minor 15-22S species and another minor 4S species. There was no difference between plasma membrane RNA and ribosomal RNA in terms of base composition. The intact plasma membrane was incubated in the presence of RNAse as well as varying concentrations of NaCl. These results demonstrate that the RNA associated with the plasma membrane is partially digested with RNAse, while 0.15 M NaCl seems to have little effect on the plasma membrane RNA content. This may indicate that the plasma membrane RNA is protruding from the plasma membrane but is attached to the plasma membrane in some manner. Incubation of intact plasma membranes with 0.30 M NaCl (final concentration) removes 63.2% of the RNA associated with the plasma membrane. The species of RNA released is unknown.
    • Characterization of TbLpn in Trypanosoma brucei

      Frainier, Alyssa S.; The College at Brockport (2012-05-30)
      Trypanosoma brucei, is a flagellated, unicellular, parasitic protozoan transmitted by the tsetse fly. It is the source of African sleeping sickness in humans. African sleeping sickness has two different stages, the bloodstream and central nervous system stages, each characterized by different symptoms. Problems with treatment result from severe side effects of the drugs used to treat African sleeping sickness. No vaccine is available due to high antigenic variation. T. brucei exists as two forms. The procyclic fly form relies on oxidative phosphorylation, expresses procyclin as its surface protein, and is morphologically long and slender. In contrast, the mammalian bloodstream form expresses the surface protein VSG, and is characterized as short and stumpy. In T. brucei, gene regulation is controlled primarily at the post-transcriptional level, thus RNA binding proteins play a role in gene regulation. Some RNA binding proteins serve as substrates for enzymes known as protein arginine methyltransferases (PRMTs). These enzymes specifically methylate arginine residues on proteins. A yeast two hybrid approach was used to identify proteins interacting with TbPRMT1 in T. brucei. Among the proteins shown to interact with TbPRMT1, one is a homolog of yeast and mammalian lipin proteins. This protein, which is termed Tblpn, has 2 conserved domains characteristic of lipin proteins. In addition, 2 aspartic acid residues were conserved in T. brucei. Lipin is involved in adipocyte development in mice. A mutation of lipin causes decreased adipocyte development associated with fatty liver dystrophy. Overexpression of the protein results in obesity in mice. Lipin also plays an important role in fatty acid synthesis and signaling in yeast, but possibly relates to the development of important phospholipids in T. brucei, specifically phosphatidylethanolamine and phosphatidylcholine. The objectives of my project were to determine where TbLpn is localized in the cell, to determine whether TbLpn interacts with TbPRMT1 in vivo, and finally to determine if TbLpn is methylated in vivo.
    • Characterization of Transfer RNA Associated with Plasma Membranes

      Kline, Larry K.; Mancusi, Vincent Joseph; The College at Brockport (1976-06-01)
      RNA extracted from purified rat liver plasma membranes was found to contain transfer RNA. Amino acid acceptor activity was detected for arginine and lysine, a confirmation of previous reports. In addition, acceptor activity was also detected for tryptophan and proline. Isoaccepting tRNA species for lysine and arginine were chromatographed using Benzoylated DEAE-Cellulose column chromatography. No major difference was found between cytoplasmic and membrane isoaccepting species for lysine and arginine transfer RNAs.
    • Expression Level of Kita, Kitb, Kitla, and Kitlb in Zebrafish Gastrointestional Tract

      Strouse, Jennifer B.; The College at Brockport (2011-06-01)
      Gastrointestinal (GI) motility is the muscular contractions that move intestinal contents in an anterograde (mouth to anus) direction and is necessary for nutrient absorption and elimination of waste. GI motility is highly coordinated and rhythmic contraction patterns. Interstitial cells of Cajal (ICC), enteric neurons, and smooth muscle cells all regulate GI motility. ICC function as pacemaker cells and determine contraction frequency. ICC growth and development is influenced by Kit, a tyrosine kinase receptor located on the plasma membrane of ICC. TMEM16A is a calcium activated chloride channel which contributes to the slow wave in the GI tract. Constipation, delayed gastric emptying, and bloating have been correlated with deficits of ICC in GI tissues. A functional Kit receptor and stimulation of Kit with Kit ligand is necessary for ICC growth and development. However, little is known about ICC development in adults or in developing GI tissue. The objective for this project is to determine the relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb in the zebrafish model system at several developmental time points. Understanding the temporal and relative expression pattern of these genes is the first step towards a more complete understanding of ICC development and turnover. The zebrafish model system is anatomically similar to the human GI tract and at early time points the zebrafish is transparent. One advantage to this model system is that GI motility may be examined in the intact larvae. RNA was isolated from dissected zebrafish GI tissues and used as template for reverse transcriptase reactions to make eDNA. Relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb was determined at 5 days post fertilization (dpf), 7 dpf, 11 dpf, 28dpf, and in adult gut tissues using eDNA as template for real time PCR. Kita and Kitla were confirmed as a functional receptor/ligand pair which was first identified in melanocyte migration19. The relative expression data suggests that Kitb and Kitlb are also a functional receptor/ligand pair. Temporal expression data shows high expression of Kitb early in development (5dpf). Besides the early high expression of Kitb, gene expression for all genes of interest peak at 11 dpf. TMEM16A (also called ANOI) was identified as a more accurate marker for gastrointestinal stromal tumors (GIST) than Kit24. RNA isolated from dissected zebrafish GI tract was used to make eDNA which became the template for reverse transcriptase (RT)-PCR and real-tin1e PCR (q-PCR). Anti-ANOI antibodies were used to identify TMEM16A in dissected, fixed zebrafish GI tract. RT-PCR showed that TMEM16A, B, and Care expressed in the zebrafish GI tract. Immunohistochemistry identifies a network of cells in the zebrafish GI tract that is similar in morphology and location to ICC stained by Kit antibodies. Relative and temporal expression was determined using samples isolated at 5, 7, 11, 28dpf, and adult time points. Expression of TMEM16B dominates TMEM16A and B at 28dpf and adult time points.
    • Melanin-Concentrating Hormone Receptor-1 is Enriched in Lipid Rafts and the Effects of Lipid Raft Integrity on Receptor Signaling

      Delorme-Axford, Elizabeth; The College at Brockport (2008-07-01)
      The melanin-concentrating hormone receptor-1 (MCHR-1) is a member of the G protein-coupled receptor (GPCR) superfamily, and functions in the regulation of food consumption and energy metabolism. MCHR-1 is expressed in neural, pancreatic, and fat tissue. Fat cells begin their journey as pre-adipocytes, culminating in the formation of mature adipocytes as differentiation occurs. Of interest to note, fat cells accumulate caveolae markers -caveolin-1 and cholesterol -during this process. Certain GPCRs and their associated downstream signaling molecules co-localize with caveolae membranes. This thesis seeks to demonstrate that MCHR-1 is enriched in lipid rafts and that a possible consequence of this may be altered signal transduction in obese individuals, such as downregulated ERK 1/2-mediated leptin transcription. Increased amounts of adipose tissue, and thus caveolae, may play a central role in the regulation of MCHR-1 signaling. The first aim of this project addressed the question of MCHR-1 localization to lipid rafts. To test this, caveolae membranes were isolated via sucrose density gradient ultracentrifugation. Subsequent fractionation and western blotting confirmed that MCHR-1 is enriched in lipid raft fractions containing caveolin-1. The second objective assessed ligand dependence on MCHR-1 localization. MCH exposure (1.0-µM) had no obvious effect on receptor localization to lipid raft fractions containing caveolin-1 over an expanded time course. The third aim examined the effect of lipid rafts on MCHR-1 signaling. Pharmacological disruption of caveolae by cholesterol depletion with methyl­ ?-cyclodextrin (M?CD) dampened MCH-mediated ERK 1/2 activation, suggesting that lipid rafts may significantly impact the regulation of MCHR-1 signaling in cells.
    • Mitochondrial DNA Stability and Maintenance in Saccharomyces cerevisiae

      Sia, Rey; Pelletier, Michel; Tsubota, Stuart; Ashman, Nicole; The College at Brockport (2016-06-24)
      This research involved studying mitochondrial DNA stability using Saccharomyces cerevisiae as a model organism. Gene knockout strains focused on the genes FIS1, DNM1, CLU1 and RTG1. Both FIS1 and DNM1 are involved in mitochondrial fission. The CLU1 gene encodes a protein that may associate with the core complex of eukaryotic translation initiation factor 3 (eIF3) in Saccharomyces cerevisiae. The eIF3 plays a role in initiation of mRNA translation. The specific function of the Clu1p in this process is undefined. The gene knockout of CLU1 does not affect growth or translation initiation. The knockout does however cause defects in mitochondrial distribution and organization. The RTG1 gene encodes a transcription factor (bHLH) involved in interorganelle communication. The protein also contributes to communication between mitochondria, peroxisomes, and the nucleus. Deletion strains of all four genes were studied for spontaneous respiration loss. This assay reveals if certain nuclear gene products have a role in stabilizing the mitochondrial DNA. The mitochondrial genome encodes proteins that are solely required for respiration. This assay compared the spontaneous respiration loss of a wild type yeast strain to those of the various knockout strains described above. In wild type yeast, a 3.26% spontaneous respiration loss is observed. In the fis1?, dnm1?, clu1?, and rtg1? deletion strains, the percentage of spontaneous respiration loss is 13.29%, 16.36%, ~5.55%, and 8.21%, respectively, using dextrose as a carbon source. When raffinose was used as a carbon source, the spontaneous respiration loss was higher for knockout genes involved in morphology. From this result, we can assume that the nuclear genes above are critical in maintaining mitochondrial DNA stability. Direct repeat-mediated deletion assays were also performed. These assays were performed for both wild-type and fis1? strains. Both strains contained nuclear and mitochondrial reporters. Using reporters flanked by homologous repeats we were able to quantify the recombination rates in both nuclear and mitochondrial DNA. The purpose of this assay was to compare nuclear and mitochondrial mutation rates for the wild-type and fis1? strains. Homologous recombination rates were not significantly different for fis1? strains compared to wild-type in the nuclear genome. There was an increase in the homologous recombination rate in the mitochondrial genome in fis1? strains. This result indicates that the FIS1 nuclear gene plays a role in maintaining mitochondrial DNA stability, for without it, mitochondrial DNA recombination rates increase.
    • Red Lionfish (Pterois volitans) Invade San Salvador, Bahamas: Early Population Characteristics, and Comparisons of the Coral and Fish Communities on Shallow Patch Reefs in 2001 and 2007

      Alexander, Amanda K.; The College at Brockport (2011-08-01)
      Biological invaders are a leading contributor to global losses of biodiversity. A recent invader to the waters surrounding San Salvador, Bahamas, the red lionfish, Pterois volitans, was first reported in 2006; by 2009 they were common in waters 2 - 40 m deep around the island. Among the 5,078 fish observed on shallow patch reefs in 2007, only two were P. volitans; they were much more prevalent in deeper water along San Salvador's "wall." Captured P. volitans ranged in size from 19-32 cm, all longer than maturity length. Pallid goby ( Coryphopterus eidolon), black cap basslet ( Gramma melacara) and red night shrimp (Rynchocienetes rigens) were the most commonly identified stomach contents. My study in 2007 also collected data on coral communities and fish assemblages at three patch reef complexes (Rice Bay, Rocky Point, Lindsay Reef), during the initial phase of the invasion, and compared the results to a similar study done in 2001, before P. volitans colonized San Salvador. Scleractinian and, therefore, total coral species richness decreased significantly from 2001 to 2007; however, coral percentage cover increased significantly by ~50% from 2001 to 2007, probably due to a more precise estimation procedure rather than a real increase in coral cover. Significantly more fish species and numbers were observed in 2007 than in 2001, again probably due to a difference in counting procedures (2.25 more and increasing population of P. volitans on San Salvador's reef ecosystem are uncertain at this time; future monitoring of lionfish and potential changes in coral and fish communities on the patch reefs of San Salvador is recommended.
    • Regulation of Actin Dynamics by Melanin-Concentrating Hormone Potentiates Downstrean Signaling to Extracellular Signal-Regulated Kinase in Cultured Cells

      Portwood, Scott Michael; The College at Brockport (2011-03-18)
      Melanin concentrating hormone (MCH) binds and activates two G protein-coupled receptors involved in the control of appetite and energy expenditure in mammals. MCH receptor-I interacts with two cytoskeleton-binding proteins in vitro. One of these proteins, periplakin, has been shown to contribute to the desensitization of MCH signaling events by displacing the interacting G protein. While periplakin is an actin- and intermediate filament-binding protein, MCH is not known to signal cytoskeletal rearrangements. These studies ask whether MCH receptors mediate actin cytoskeletal rearrangements in response to hormone binding. 3T3-L l pre-adipocytes, endogenously expressing MCH receptors, were treated with MCH for varying times, fixed, and actin fibers were stained with Alexa Fluor phalloidin. Using fluorescence microscopy, cells were categorized as 1) having prominent actin stress fibers, 2) being round with many plasma membrane extensions, or 3) being small and round in blinded experiments. Pharmacological agents were used to dissect the contributions of two downstream effectors of Gq, phospholipase C and ADP-ribosylation factor 6, as well as contributions of MCHJ1 versus MCHR2 on MCH-mediated actin rearrangements. A small, but statistically significant change in actin morphology was observed after exposure to MCH for a little as 2 minutes showing MCH docs indeed to the cytoskeleton in this cell line. Phospholipase C activators mimic this response on a similar time course suggesting it is a major participant in this signal transduction pathway.
    • The Effects of KU70 on Mitochondrial Stability in the Saccharomyces cerevisiae

      Sia, Rey; Ortega, Bernardo; Tsubota, Stuart; Burkhart, Allyson (2016-06-23)
      The purpose of this experiment is to determine the role of KU70, a nuclear gene, in maintaining mitochondrial DNA in the model organism Saccharomyces cerevisiae, the budding yeast. The mitochondrion is an organelle in eukaryotes that produces much of the ATP used by a cell. ATP, or adenosine-triphosphate, is a molecule within a cell that provides energy for cellular functions via its high energy holding phosphate bonds. Mitochondria have their own genomes, separate from nuclear DNA, which encodes many proteins needed for cellular respiration. Mutations can occur in the mitochondria of humans that could result in decrease or loss of mitochondrial function, which leads to neuromuscular or neurodegenerative diseases. The KU70 gene is actually a subunit of a heterodimer that works in coordination with KU80. These genes function in the stability of the genome during DNA double-strand break (DSB) repair through nonhomologous end-joining (NHEJ) and telomere maintenance. The goal of this project is to determine the effects caused by the loss of KU70 on the mitochondrial stability. By completing two different assays, respiration loss and direct repeat-mediated deletion (DRMD), the role of the gene can be predicted. The respiration loss assay showed a 1.4-fold decrease (p=0.027) in spontaneous respiration loss compared to the wild type strain. The rate of DRMD events in the nuclear and mitochondrial genomes showed 1.42-fold decrease (p= 0.014) in spontaneous mutation rates in nuclear DNA and a 1.69-fold decrease (p=0.075) in mitochondrial DNA compared to the wild type. Finally, the induced-DRMD assay showed a 1.23-fold decrease (p=0.029) in homologous recombination events compared to the wild type. These results suggest that Ku-independent end joining may be a more efficient repair pathway and promote mitochondrial stability.