RSS 1.0Brockport Biology Master’s Theses
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Taxa Determination by the Polymerase Chain Reaction: a SurveyThe objective of this research was to survey a number of DNA samples from various organisms, using different primers, to determine what differences exist between organisms relative to each primer. This survey would help to contribute information for the following: 1. Educational purposes. A student receiving an unknown DNA should be able to identify the organism to particular taxa. 2. Determine the presence of PCR products from DNA and primers that would not typically be used together. That is, a survey of unconventional combinations. 3. Determine what differences exist between the snails, L. saxatalis and L. compressa using this particular battery of primers. The results of this work indicate that: 1) A student would be able to narrow the choices if given one of these 12 DNAs as an unknown and this particular battery of primers. See Appendix D for an example. 2) The Universal bacterial primers amplified some eukaryotic DNA. 3) The human Alu primers amplified firefly and snail DNA as well as H. sapiens. 4) The Universal animal primers amplified V fischeri DNA and were not truly universal with the 8 DNA samples used here. H. sapiens DNA was not amplified by these primers. The DNA that was amplified gave fragments that were of variable size and not equivalent to the positive control. 5) The primers for the Histone 3 gene, one of the most highly conserved proteins, gave variable results and amplified DNA from V fischeri which does not contain histone proteins. 6) The Lux primers, specific for Vibrio fischeri amplified DNA from another bioluminescent organism, which was eukaryotic. 7) The Mitocox primers, specific for mitochondrial cytochrome C oxidase of insects, worked for both prokaryotes as well as all of the eukaryotes in the survey. The major 710 base pair fragment was present in all 12 organisms used and variable minor bands were seen in three organisms. 8) The Nrd primer, specific for the E. coli nucleotide reductase gene, proved to be specific for E. coli in this survey. This primer was used as a marker for coliform contamination such as suspected with C. elegans. 9) Additionally, L saxatalis and L. compressa do have a different fingerprint with these primers. These two snails have identical results with all the primers except Alu and Analu. L. compressa is amplified with the Alu primer while L. saxatalis is not. The Analu primer gives two bands for both DNAs, one of identical size and the other of a different length.
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Stopover of Neotropical and Temperate Landbird Migrants on the South Shore of Lake Ontario during Spring MigrationSelected Neotropical and long-distance migratory landbirds were studied during spring migration at a known stopover site on the southern shore of Lake Ontario. A total of 1277 individuals were captured by mist net from 1 May through 1 June 1995, and 22 April through 1 June 1996, for banding and measurement of morphological characteristics. Poor energetic condition was typical of the long-distance migratory landbirds captured at this site during this study (80.6% fell in the lowest 3 out of 6 fat classifications). Few birds were recaptured after the day of their initial captures. No significant differences in initial condition index or fat class were found in recaptured birds, compared to those captured only once, in three species with adequate sample sizes. Individuals were found to lose, maintain, or gain weight during their stay, and showed no significant correlation between length of stay and change in mass. A total of 775 individuals representing 14 species were examined for outward physiological indication of male breeding readiness and grouped latitudinally into two categories based on relative distance from the study site to the southernmost limit of the species' geographic nesting range. Two individuals from the near-nesting group exhibited a cloacal protuberance. No individuals of the far-nesting group exhibited protuberance. No significant difference in the proportion of males exhibiting a cloacal protuberance was found between the two groups. Cloacal lavage samples were taken from 22 Whitethroated Sparrows (Zonotrichia albicollis) and 26 Swainson's Thrushes (Catharus minimus). None of the migrating individuals lavaged exhibited presence of cloacal sperm regardless of species, sex, or date of capture. Flying insects (Diptera) were abundant throughout the two study periods while Lepidopteran larvae were not present until the final week of May in both 1995 and 1996. Numbers of Diptera were significantly higher at nearshore sampling sites during the final weeks of each study period. Results of my two year study suggest that: 1. Most Neotropical migrants reaching Braddock Bay continue migrating after stopping only one day. 2. Neotropical migrants reaching Braddock Bay are in relatively poor condition, as indicated by predominance of low fat class scores. 3. But most migrants seem to be able to improve condition adequately for another night's flight during a single day. 4. There is no obvious relationship between body condition, as indicated by fat class score or condition index at initial capture, and length of stay of migrants reaching the stopover site at Braddock Bay. 5. Neotropical migrants may concentrate near Lake Ontario not only due to the geographic barrier imposed but also to take advantage of abundant food resources and safe cover. 6. Depleted energetic stores may explain the lack of early sperm production or release in migrants at this stopover site.
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Routine Determination of Mirex and Photomirex in Fish Tissue in the Presence of Polychlorinated BiphenylsA procedure for the routine determination of Mirex and Photomirex in fish tissue is described which provides rapid analysis and confirmation using conventional gas chromatographic/electron capture detection (GC/ECD) methods. Coeluting intereferences (i.e. PCB's) are nitrated al lowing for simple separation from Mirex analogs by column chromatography. In Chinook SaImon tissue (Oncorhynchus tshawytscha), PCB removaI averaged 78% and Mirex and Photomirex recoveries were 91% and 86%, respectively. The method has been used successfully for trace analysis of Mirex levels as low as 100 pg.
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Culture of Black Bullhead (Ameriurus melas) in Cages and Open PondsBlack bullhead (Ameiurus melas) fingerlings were stocked in three 0.1 ha ponds at a rate of 900 fish/0.1 ha with 150 fish stocked in 1.2 m3 cages (three cages/pond) and 450 fish stocked free-swimming in each open pond. Fish were simultaneously cultured in cages and open ponds between 4 June 1991 and 28 September 1991. After 117 days, survival rate in all treatments did not differ. Black bullhead reared in open ponds exhibited superior growth and feed conversion when compared to fish reared in cages. Caged fish took longer to double their weight than free-swimming fish. Water quality parameters (D.O., temperature, pH, alkalinity, total ammonia-nitrogen, and Secchi disk visibility) did not differ significantly among cages and open ponds. Results of this study indicate that black bullhead cultured in open ponds outperform bullhead cultured in cages.
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Characterization of TbLpn in Trypanosoma bruceiTrypanosoma 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.
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A New Functional Zebrafish Gastrointestinal Motility AssayGastrointestinal (GI) motility is the coordinated contractions of smooth muscles resulting in mixing and propulsion of material through the GI tract. GI motility is influenced by smooth muscle, enteric neurons, and Interstitial cells of Cajal (ICC). New model systems for GI motility are needed because regulation of motility is poorly understood, and the number of drugs which can assist in GI motility disorders is insufficient. The Kit receptor is required for ICC development and maintenance in mammalian model systems and in humans. Mutations in the Kit receptor are associated with GI motility disorders. The objective of these experiments was to determine motility patterns in wildtype zebrafish and compare them to motility patterns in the zebrafish kita null mutant, sparse (spab5). Contraction frequency was measured from 7 minute digital video recordings of anesthetized larvae immobilized in 1.2% agar. Contraction frequency averaged 0.586 ± 0.222 contractions per minute in wildtype larvae at 7 dpf, and 0.329 ± 0.158 contractions per minute in 7 dpf sparse. The apparent contraction intensity was also scored, with a score from 0, no contractions, to 2, complete occlusion of the lumen. Contraction intensity averaged 1.250 ± 0.444 in wildtype larvae and 1.619 ± 0.498 in sparse larvae at 7 dpf. At 11 dpf contraction intensity decreased to 1.000 ± 0.000 and 1.125 ± 0.354, respectively. A functional motility assay was developed to quantify GI motility that contributes to propulsive movement of intestinal contents. Larvae were fed FITC labeled microspheres, washed, and fluorescence intensity of the GI tract was digitally imaged. Larvae were placed in clean system water, and reimaged after 24 hours. Fluorescence intensities were normalized against initial intensity of ingested microspheres at 7 dpf in the intestinal bulb of the anterior GI tract. At 7 dpf the change in fluorescence intensity in the anterior GI tract decreased by 0.371 ± 0.030 and 0.119 ± 0.021 in the posterior GI tract of wildtype larvae. At 11 dpf fluorescence intensity decreased to 0.455 ± 0.056 in the anterior GI tract, and 0.258 ± 0.046 in the posterior GI tract. Peritoneal injection of the ACK-2 anti-Kit antibody results in distention and an increased volume in the GI tract (Figure 12) (Maeda, Yamagata et al. 1992). These effects result from inactivation of Kit function by ACK-2. The size of the GI tract in sparse larvae appeared to be larger compared to wildtype, suggesting that the null kita mutant resulted in a phenotype similar to the ACK-2 effects on mice. We measured the area of a longitudinal cross section of wild type and sparse mutant larvae at 7 dpf. The average area of the GI tract in wildtype larvae was 4491 ± 754 and 5532 ± 880 in sparse larvae. Fluorescence spectroscopy was used to quantify the fluorescence intensity. Total fluorescence emission averaged 62,630 ± 23,780 counts per second immediately after ingestion in 11 dpf larvae. One day later, total fluorescence intensity averaged 53,277 ± 6,333 counts per second, a 15% decrease. 5-hydroxytryptamine (5-HT), a prokinetic agent, was used to validate the assay. One day after loading, the total fluorescence intensity decreased after application of 5-HT to 86,820 ± 21,850. 11 dpf zebrafish larvae ingest fluorescent labeled microspheres, and spectroscopic analysis showed reduced fluorescence intensity one day after loading. Functional studies showed a reduced contraction frequency and an increase in surface area between sparse mutant and wildtype larvae. The decrease in fluorescence intensity in wildtype larvae suggests aboral movement of microspheres, out of the posterior GI tract.
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The Role of Kitlb on Development of Coordinated Muscular Contractions in the Zebrafish Gastrointestinal TractGastrointestinal (GI) motility is the spontaneous rhythmic contractions of smooth muscles that mix and propel the contents of the GI tract. Regulation of the complex muscular contractions is controlled by smooth muscles, interstitial cells of the Cajal (ICC) and enteric neurons. ICC act as pacemaker cells in the GI tract and set the frequency of spontaneous contractions. Altering ICC density results in uncoordinated GI muscular contractions. Our lab examines the role of ICC in GI motility and is focused on mechanisms that regulate ICC growth and development. Expression of the Kit receptor tyrosine kinase is used to identify ICC. Kit is stimulated by Kit ligand and stimulation is necessary for the growth and development of ICC. This project specifically examines the role of Kit – Kit ligand signaling on ICC development using the zebrafish model system. The zebrafish has two Kit genes (kita and kith) that are orthologous to human KIT, and two Kit Ligand genes (kit/a and kitlh ). I will examine the role of kitlh on the development and maturation of ICC using morpholino oligonucleotides knockdown in zebrafish. Gene expression was quantified using reverse transcriptase PCR analysis. Digital imaging techniques was used to examine morphology of the GI tract. It is anticipated that continued stimulation of kith by kitlh is necessary for development of the ICC network, and maintenance of the ICC network in adult animals.
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Dynamics of Steroid Secretions in RatsIn spite of the vast information available on hormone secretions and their levels in blood, there is a paucity of information in age related changes in the dynamics of steroid metabolism in male rats. Also, few investigations exist concerning the metabolic clearance rates (M.C.R.) of steroids in blood of female rats. This thesis consists of two parts, the first part deals with changes in M.C.R. in aging male rats and the second part in pregnant rats. It examined these changes relative to testosterone (T), estradiol (E_2), and progesterone (P), using the non-equilibrium single dose injection method. 3H-steroids were administered via the cannulated jugular vein. Sequential blood samplings were obtained up to 150 min. 3H-steroids were extracted, processed by Sephadex LH-20 column chromatography and the radioactive steroids were quantified with liquid scintillation spectrometer. Results were analysed by the computer using the 'peel-off' method for a two-compartment model. In aging studies M.C.R. and production rate of T were found significantly higher in young male rats when compared with aged rats. Half-life (t ½ ?) in the outer pool was significantly higher in the old rats than in young rats while t ½ ? in the inner pool did not change significantly between both aging groups. However, the M.C.R. for E_2 and P remained unchanged in both groups. Chronically castrated animals of both age groups showed significant decline in M.C.R. of T and increased in t ½ ? when compared with their respective intact controls. M.C.R. of T in young or old castrated rats previously injected with T were restored to values similar to those young intact rats. From these results it is concluded that the age-related changes in M.C.R. of testosterone are due to an interaction of testicular dysfunction and androgen status of animal. In the second part of the study, non-pregnant, pregnant (D-10 and D-20) and post-partum rats were used. The M.C.R. of E2 in D-20 rats was found to be significantly higher when compared with other groups. In post-partum rats the M.C.R. of E2 was significantly higher than those of non-pregnancy or D-10 of pregnancy. All values for t ½ ? of E_2 were found to be similar for all groups. t ½ ? of E_2 in D-20 was greatly lower than both non-pregnancy and D-10 gestation. However, the M.C.R. of P remained unchanged in all female groups. The fast component (t ½ ?) of P was significantly higher in post-partum than in D-10, meanwhile it did not change in other groups. The slow component (t ½ ?) of P was higher in non-pregnant rats compared with D-10, other groups remained unchanged. The M.C.R. of T was significantly lower in non-pregnant rats than in post-partum rats, other groups remain unchanged. t ½ ? of T was significantly higher in post-partum rats than in D-20 and D-10 of pregnancy. There was no change in t ½ ? of T between all groups. It is evident that changes in metabolic clearance rates of different steroids vary according to their respective roles in pregnancy reflecting physiological demands.
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Evaluation of the Basis and Effectiveness of Habitat Assessments in Wetland Functional Assessment MethodsI studied the basis and effectiveness of wetland assessment methods in providing habitat assessments. While it is well understood that wetlands and riparian areas provide important ecological functions and habitat for a wide variety of wildlife species, much is still to be learned about providing meaningful, accurate and repeatable methods for assessing them. I examined and evaluated four assessment methods to determine their accuracy and usefulness in assessing a site's provision of habitat. One hypothesis I tested is that if the assessment methods studied provide an accurate assessment of wetland functions, then the resulting site scores for the methods should be correlated. The second hypothesis is that there is a correlation between the site scores and an independent measure of function, specifically the number of riparian-associated bird and butterfly species observed at each site. Biological and physical data collected from 47 riparian sites in California's Central Valley were used to calculate site scores using Habitat Assessment Technique (HAT), Rocky Mountain Riparian Hydrogeomorphic (HGM), Southern California Riparian Model, and Reference Wetland assessment methods. The rankings of these site scores were also calculated for each method. Correlation coefficients (r) were calculated between the site scores of the four methods, as well as between the site scores and the numbers of riparian-associated bird and butterfly species for each plot. The site scores were mostly uncorrelated. Only one statistically significant correlation was demonstrated between the site scores for the Southern California Riparian Model and Reference Wetland methods (df = 46, r = 0.46, p = 0.00103, with Bonferroni correction). With Bonferroni corrections (p < 0.00625), the site scores were also uncorrelated with the numbers of riparian-associated bird and butterfly species. Without Bonferroni corrections, only two statistically significant correlations were demonstrated: between the number of riparian-associated bird species and the HAT score (df = 46, r = 0.37, p = 0.0095) and the number of riparian-associated butterfly species and the Reference Wetland score (df = 46, r = 0.38, p = 0.0092). I rejected both original hypotheses, which demonstrated that the assessment tools currently available do not consistently produce relatively precise, or reproducible results. Possible reasons for these problems include attempting to assess a function that is too broadly defined, inappropriately or subjectively selected variables, subjectively assigning values to variables, or inappropriately selecting reference sites. The existing attempts at assessing wetland or riparian function are important steps in the right direction toward assessment of wetland and riparian sites and achievement of "no net loss," but functional assessment must be considered a work in progress.
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Characterization of Melanin-concentrating Hormone Receptor DesensitizationMelanin-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.
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Effects of Training on Atrial Rate and Sensitivity of Isolated Rat Atria to Catecholamines and AcetylcholineThirty five male rats were subjected to a treadmill running program and body weight heart weight and effects of neurotransmitters were measured. Rats engaged in training programs show a lower body weight, a lower heart rate and lower intrinsic heart rate. The response of all isolated rat atria to different drugs were observed. Epinephrine in concentrations of 1 X10-5M, 1X10-6M and 1X10-7M increased the atrial rate in trained rats by averages of 41.3%, 20.04% and 15.3% respectively, and in control animals by averages of 18.6%, 11.1% and 8.6% respectively. Norepinephrine in concentrations of 1X10-6M and 1x10-7M increased the atrial rates of trained animals by averages of 13.3% and 8.3% respectively. Acetylcholine of 1x10-6M decreased the atrial rate in trained rats averages of 48.4% and 28.2% in control animals. Atrophine in concentrations of 1x10-5M, x 10x10-6M, and 1x10-7M increased the atrial rates in all preparations, but the percent change was higher in trained rats. 1x10-5M atropine added to isolated rat atria of trained rats increased the atrial rate to a rate almost identical to the basal atrial rate of control rats. A biphasic response of atrial rate was observed when equimolar concentrations of acetylcholine and norepinephrine were added to isolated rat atria. It is concluded that trained rats have a lower resting heart rate and a lower intrinsic heart rate than control rats. The isolated rat atria of trained animals were more sensitive to catecholamines, acetylcholine and atropine. Increased stores of acetylcholine in the region of the pacemaker may account for the lowered heart rate. The negative chronotropic action of acetylcholine was blocked and the heart rate was brought to the basal rate of control animals in the presence of 1x10-5M atropine.
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Activity of Phosphofructokinase in Summer Honey Bees (Apis mellifera) of Different AgesPhosphofructokinase activity may be an indicator in the aging process of honey bees (Apis mellifera). This paper seeks to quantify phosphofructokinase activity in summer honey bees of different ages. Honey bee flight muscle was utilized in tests for enzyme activity of phosphofructokinase at sequential stages of honey bee development. Homogenized honey bee flight muscle was ultracentrifuged and spectrophotometric measurements were made. This study found that the enzyme activity of phosphofructokinase of honey bees of different ages is low at day 0-1 and high from day 4-5 through 19-20 day old honey bees. This study lays a foundation for the study of summer and winter honey bees.
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Energy Costs of Foraging by Honey Bees on Artificial Flower Patches of Variable and Constant Nectar DistributionsExperimentation with the honey bee, Apis mellifera, was performed with two artificial flower patches, located at a certain distance from an apiary. Patches were tested adjacent to each other and with a separation distance between them. Responses of foraging bees on the patches were measured by censusing at one-minute intervals in order to determine preferences by the bees for three factors which differed between patches; Nectar Distribution ("constant" or "variable" amounts per flower), Flower Color (blue or yellow), and Distance from the apiary (near or far). The bees preferred the "constant" nectar distribution and the blue flower color. Although a distance preference was not found, the data suggest that a preference for the nearer patch may be exhibited at distances greater than those used in these experiments.
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In Vitro Studies of Cultured Rat Trophoblast CellsThis study was designed to explore the feasibility of using rat trophoblast cells as an animal model for studies of placental growth and regulation. The structural and functional similarities between the giant cells of the rat placenta, and the syncytio-trophoblasts of the human placenta warrant such an investigation. In this study, trophoblast cells were cultured in vitro. Particular attention was paid to the cytodifferentiation of cytotrophoblasts into giant trophoblast cells, and the time at which they appeared after they were first seeded in culture. Trophoblast cells derived from the isolated basal zone of the chorio-allantoic placenta from pregnant rats sacrificed on days 12, 16, and 20 of gestation were used for experimentation. These cells were enzymatically dispersed and cultured in vitro as primary monolayers. The morphology of these cells was studied using histochemical methods under light, phase contrast, and fluorescent microscopy. Human chorionic gonadotropin (hCG) as a self regulatory/tropic agent, and diethylstilbestrol (DES) as a maternal estrogen were added to placental cell cultures to investigate their effects on steroidogenesis. Radioimmunoassay (RIA) was employed to analyze the culture media for the determination of progesterone hormone secretion by the trophoblast cells. The basal secretion of P by the placental cells was highest in day 16 trophoblast with an average of 3.0ng P/18h. Day 12 trophoblast produced the second highest amount with 0.8ng P/18h, and a marked reduction was seen in day 20 trophoblast cultures with only 0.3ng P/18h. DES treatment had no significant effect on day 16 and 20 trophoblast. However, day 12 trophoblast showed a biphasic response with an intense stimulatory effect (26.6ng P/18h) at the 10-10M; with higher concentrations causing an inhibitory effect (0.36ng P/18h) at 10-5M. HCG stimulated both day 12 (2.4ng P/18h) and day 16 (6.7ng P/18h) trophoblast with peak P production at the lowest concentration of 10mIU. However, hCG had no significant effect at any dosage level on day 20 trophoblast; while increased concentrations did not further stimulate day 12 and day 16 trophoblast to secrete P above the levels attained at 10-10 M. Morphological observations of the isolated basal zone cells revealed six distinct cell types: 1) mononucleated polygonal cells, 2) binucleated cells, 3) large multinucleated cells, 4) multinucleated giant cells, 5) mononucleated giant cells, and 6) fibroblasts. Within each of the 3 placental cell cultures we have observed that the formation of giant trophoblast cells occurs through several stages of differentiation of the mononuclear cytotrophoblast cells by means of cell and nuclear fusion. Therefore, isolated and cultured trophoblast cells are destined to become giant cells, and this differentiation can occur in the absence of maternal and fetal cues.
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Chironomid (Diptera: Chironomidae) Larvae as Indicators of Water Quality in Irondequoit Creek, NYChironomid community structure and mouthpart deformities were examined as indicators of pollution or degradation of water quality in the Irondequoit Creek watershed. Differences in Simpson's diversity, taxa richness, and chironomid abundance were assessed in upper, middle, and lower creek locations to determine changes as the creek passes through increasingly populated areas. Differences in the same measures also were assessed in vegetation, mud, and gravel habitats in order to assure that any changes observed were not due to differences in chironomid community structure in dissimilar substrates. Diversity and taxa richness were highest in the upper creek and lowest in the lower creek. Abundance was highest in the middle creek. All three measures were highest in the gravel habitats and lowest in the vegetation habitats. Slight organic pollution impacts on the creek were indicated by lower diversity and richness values in the lower locations of lrondequoit Creek and by community structure differences. Mouthpart deformity comparisons were inconclusive because affected taxa were not present at all sites. This study brings into question the feasibility of using chironomid communities and deformity rates as indicators of water quality changes in the Irondequoit Creek system. Due to the high variance in chironomid distributions, a larger number of samples is needed to detect changes in chironomid communities. Other changes in sampling methods may also be necessary.
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The Effects of Low Temperature on Metabolism and Survival of Individual Honey Bees (Apis mellifera)The honey bee (Apis mellifera) is one of the few insects capable of thermoregulation. Heat regulation of an isolated individual under the low temperature conditions normally seen in a northern temperate winter are investigated in this thesis. The factors examined are twofold: the variation in thermal output with ambient temperature and the survival potential from a cold comatose state. Individual workers attempted to maintain their body temperature through active thermoregulation. Active heating was pronounced and continuous in the range of thoracic temperatures between 19.l-29.3°C. Oxygen consumption, and corresponding heat production, showed a linear increase with a decrease in ambient temperature in the range of T_ambient from 35-12°C (V_O2 = -2.82 x T_ambient + 96.64, r2 = 0.94). Large increases in oxygen consumption were seen at temperature differences (T_thorax - T_ambient) ? 2.0°C (above the physiologic minimum.) At cabinet temperatures below 12.9°C, the oxygen consumption of individuals did not stabilize, but decreased continuously, representing an abrupt cut-off in metabolic capacity seen at the chill coma point. The survival potential of a comatose honey bee is high, with a 51% survival rate seen over all tests. In general, the number of survivors decreased with exposure time and exposure temperature. For all bees over all tests, chill coma temperature was dependent on exposure time, but was not dependent on exposure temperature. Revival time was found to be dependent on both exposure temperature and exposure time. An individual was most likely to survive chill coma if it revived in less than 4 min and under 18°C with passive exogenous heating.
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The Status of Woody Non-Native Species in the Western Half of New York StateFor woody exotics in the western half of New York state, I have compiled data from herbaria, published literature, professional judgment and field surveys to determine the naturalization status of these non-native species. Based upon herbaria collections over the last 163 years, a total of 1 19 non-native woody species were collected in western New York. Of the 119 species, collection labels indicated only 12 species were naturalized. Similarly, floras described just 18 species as naturalized. In contrast; 69 species were considered naturalized by respondents to a survey of the region's botanists, and in my field survey, naturalized populations were documented for 44 species. Furthermore, even when collection labels, literature or professionals stated a species was naturalized, this was not clear-cut evidence of naturalization because the terms naturalization and naturalized were inconsistently defined and applied. For this reason, my concluded statuses are based primarily on my field surveys and then supported by herbaria collection data, literature and expert opinion. I have concluded that 44 species now have naturalized populations, 23 are escaping, 30 are persisting and the remaining 23 species have not been collected in the past 50 years are likely only persisting in isolated areas or no longer present in the region.
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Melanin-Concentrating Hormone Receptor-1 is Enriched in Lipid Rafts and the Effects of Lipid Raft Integrity on Receptor SignalingThe 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.
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The Relationship between Habitat Variables and the Distribution of the Creek Chub, Semotilus atromaculatus, and the Hornyhead Chub, Nocomis biguttatus, In Sandy, Salmon, and Northrup Creeks, Monroe County, New YorkThe creek chub, Semotilus atromaculatus, and the hornyhead chub, Nocomis biguttatus, along with numerous other minnow (Family Cyprinidae) species, are found in streams throughout Monroe County. Interestingly, however, only creek chubs have been found in Salmon Creek, only hornyhead chubs have been found in Sandy Creek, and both chubs have been found in parts of Northrup Creek. A review of the life history of these species suggests that differences in width of stream, depth of stream, midstream current, and edge of stream current may account for this distribution. In this study a number of habitat parameters were measured at various sites within all three of the streams. The resulting data suggests that differences in midstream and edge of stream current may account for the differing distribution of creek chubs and hornyhead chubs among streams.
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Spring Thermal Fronts and Salmonine Distributions in Lake OntarioThe hypothesis that salmonine catches in Lake Ontario are greater at thermal fronts in spring and early summer was tested in 1990 by comparing catches in non-frontal water and three types of fronts (thermal bar, 4 °C; spring thermocline, 6-8 °C; thermal break, ? 9 °C). A thermal front in the spring on Lake Ontario is a rapid temperature cline across the surface of the lake (in this study defined as ? 0.15 °C/min at the standard 3.2 - 4.8 km/h trolling speed) parallel to shore that extends obliquely from the surface toward shore and the bottom. Surface temperature was recorded every 2 min during 45 hours of fishing. Only 20% of the time was spent fishing in thermal fronts where 35% of the 88 strikes occurred. Catch per unit effort (CPUE) for salmonines at thermal fronts was significantly greater than non-frontal CPUE on each of the 11 sampling dates (P < 0.001). Catches were better in thermal breaks (P < 0.002), spring thermocline (P < 0.01) and thermal bar (P < 0.05) than in non-frontal waters. The data support the hypothesis that there is a relationship between salmonine susceptibility to capture and thermal fronts. Relative to non-frontal water, coho salmon (Oncorhynchus kisutch) CPUE was greater in the spring thermocline (P < 0.01); rainbow/steelhead trout CPUE was greater in thermal breaks (P < 0.05), spring thermocline (P < 0.05) and thermal bar (P < 0.002). It appears that anglers can effectively catch specific salmonine species by fishing specific thermal structures. These results likely are applicable to other pelagic habitats utilized by salmonines.
