MEES Theses and Dissertations

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Subject: search.filters.subject.Biology, Molecular

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    Behavioral and Neuroendocrine Correlates of Sex Change in the Gilthead Seabream (Sparus aurata)
    (2009) Reyes-Tomassini, Jose J.; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Sequential hermaphroditism is the most radical form of environmental sex determination observed in fish: functional adult males or females retain their ability to change sex even as adults. Among the factors that affect sex change in these species, the least understood is the social environment. Here, I studied the influences of social context on sex change in the Gilthead Seabream, Sparus aurata, by using the individual‟s dominance rank as an indicator of social status. To understand the role that the brain might play in sex change, I also studied the two main neuroendocrine factors that serve as the sexually differentiated axes of neural plasticity in most teleost species: AVT and GnRH. To do this, I first developed a set of tools designed to address the challenges associated with observing the behavior of aquacultured species. Using these tools, I provide the first in-depth study of seabream captive behavior, including the results of size-matched and sex-matched paired encounters. I found that females are more aggressive than males, but this difference is influenced by gonadal developmental status. I also showed that small but young males are more aggressive than bigger but older females. I cloned the AVT mRNA in seabream, and validated a quantitative assay to measure total brain AVT levels together with GnRH-1, GnRH-2, and GnRH-3 levels. I found that AVT and GnRH-3 levels rise during the onset of the hypothesized sex-change window, and drop to pre-quiescent levels until spawning, when all of these factors seem to increase their expression levels again. I also show for the first time, that GnRH-2 and dominance rank are strongly correlated in seabream during the spawning season but not during quiescence. GnRH-1 was strongly correlated to rank during quiescence but not during spawning. Finally, neither dominance rank nor size were a good predictor of the outcome of sex change, which seems to contradict what has been documented in sequential hermaphrodite reef fishes. I provide a model that accounts for this apparent contradiction and conclude that the Gilthead seabream remains true to the size-advantage hypothesis of sex allocation theory, if size and dominance are seen as proximate, rather than ultimate, factors.
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    Reproductive Physiology of the Female Blue Crab, Callinectes sapidus: Spawning Induction and Vitellogenesis
    (2009) Bembe, Sarah Elizabeth; Chung, J. Sook; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In aquaculture, spawning is the baseline for production; therefore, the optimization of spawning conditions will directly increase production. The current study aims to optimize spawning conditions for Callinectes sapidus using environmental manipulations of photoperiod and temperature for induction while monitoring the physiological vitellogenin (VtG) levels during ovarian development and maturation. The photothermal manipulations for this study resulted in increased spawning events in 21°C temperatures (compared to 11°C and 15°C) and complete darkness (0L:24D; compared to 8L:16D, 16L:8D, and 24L:0D) while 24L:0D and 11°C suppressed spawning. When assessing the VtG levels in the hemolymph prior to, during, and after all spawning events, the VtG showed a decrease prior to spawning, and significant VtG activity was seen in 21°C for all photoperiods. Overall, spawning and vitellogenesis are temperature dependent events with 67% of the females spawning in 21°C. Photoperiod also has an effect on spawning, but not on vitellogenesis.
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    Archaeal Transcriptional Regulation of Catabolic Carbon Monoxide Dehydrogenase in Methanosarcina species
    (2009) Anderson, Kimberly Lynn; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In Archaea, the basal transcription machinery is eukaryotic-like, but some components, such as activator and repressor proteins, are bacteria-like. To further gain knowledge into cellular processes of Archaea, the genome of Methanosarcina thermophila was searched for helicase genes. A homolog of yeast RAD25, a gene with helicase and nucleotide excision repair (NER) abilities, was isolated. M. thermophila rad25 has the domains for helicase activity, but the C-terminal end is truncated, indicating that this protein mostly likely does not function in NER. After overexpression, helicase activity assays of Rad25 indicated that it might have helicase activity; however, there appeared to be contaminating proteins in the purification, so it was not possible to assign the activity only to Rad25. Additional work is necessary to characterize this protein. To investigate transcription, catabolic gene regulation was studied, specifically regulation of carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) from Methanosarcina species. The regions upstream of the transcriptional start site, as well as the 5' leader region of cdhA, were investigated for trans factors and cis elements that might be involved in regulation. Experiments revealed that regulation of cdhABCDE does not appear to involve trans factors upstream of the transcriptional start site. However, deletion analysis indicated that the 5' leader region does have a role in regulation. Comparing the protein levels to the mRNA levels revealed there was no significant difference between the two, indicating that translational regulation was not a factor. Other experiments ruled out differential mRNA stability as a factor in regulation. A region located between +358 and +405 was important in transcriptional regulation, indicating that regulation occurred at the level of transcription elongation. A model for regulation of catabolic CODH/ACS by differential elongation is proposed. Although 5' leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential elongation. Identifying regulatory mechanism(s) of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by the methanoarchaea to efficiently direct carbon and electron flow during biomass conversion to methane.
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    The Gonadotropin Releasing Hormone-3 System in Zebrafish: Early Development and Regulation
    (2008-12-15) Abraham, Eytan; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The objective of this study was to expand our understanding of the early development of forebrain Gonadotropin Releasing Hormone (GnRH) neurons in vertebrates in general and in fish in particular. The correct migration during early development of the hypophysiotropic GnRH neurons from the olfactory region to the hypothalamus is crucial for normal gonadal development and reproduction. We developed a Tg(GnRH3:EGFP) zebrafish line in which EGFP is specifically expressed in GnRH3 neurons. Using this line, we have studied in detail the early spatiotemporal development of the GnRH3 system in vivo. In addition, we have studied various factors, including GnRH3, Netrins and Hedgehog to better understand some of the mechanisms that mediate this complex axophilic neuron migration event. Lastly, we have conducted targeted GnRH3 neuron ablation experiments in view of determining the embryonic origin of POA-hypothalamic GnRH3 neurons and the effect of lack of GnRH3 neurons in the CNS. Our findings show that: 1) GnRH neurons first differentiate and express GnRH3 at 24-26 hours post fertilization (hpf) and immediately thereafter begin to extend fibers. 2) GnRH3 neurons project a complex network of fibers, prior the GnRH3 soma migration, to various CNS regions, and to the pituitary. 3) GnRH3 soma begin migrating towards the hypothalamus at 3 days post fertilization (dpf), passing through the terminal nerve (TN), lateral telencephalon, and reaching the hypothalamus by 12 dpf. 4) expression of GnRH3 itself is necessary for the normal early differentiation and fiber extensions of GnRH3 neurons. 5) Netrin1a is directly involved as a chemoattractant in GnRH3 fiber organization and subsequently, in GnRH3 soma migration to the hypothalamus. 6). Netrin2 is required for normal early ZF embryogenesis. 7). Sonic hedgehog a does not serve as a specific factor in the development of the GnRH3 system. 8). GnRH3 neuron regeneration capacity is temporally limited. 9). Successful ablation of olfactory GnRH3 neurons during development results in lack of GnRH3 neurons in the entire sexually mature brain as well as abnormal gonadal development and inability to reproduce. This study expands our understanding vis-à-vis the early events that occur during GnRH3 system development and that regulate this complex process. In a broader sense these findings augment current knowledge regarding the regulation of long range tangential neuron migration during development.
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    Chitinase-An Evolutionary Duality
    (2008-08-29) Tang, Qiong; Place, Allen R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The purpose of this thesis was to characterize the molecular properties, gene copy number and gene organization for the chitinase enzyme in a lower vertebrate. Rainbow trout (Oncorhynchus mykiss) has one of the highest chitinase activities in all the fishes examined. I also aimed to clarify the evolutionary origin and associated regulatory control of the chitinase gene in this species. The cDNAs from two chitinases (Onmy-Chit.01 and Onmy-Chit.02) were successfully cloned and characterized from rainbow trout tissues. Onmy-Chit.01 is predominately expressed in the stomach, with high mRNA expression in the gastric gland. Its protein secretes along mucosal folds to the stomach lumen. Onmy-Chit.02 is primarily expressed in immunity related organs, such as kidney, spleen, and liver, as well as in reproductive organs. From in situ hybridization and flow cytometry analysis, I show that Onmy-Chit.02 is constitutively expressed in the myeloid cell lineage of the rainbow trout immune system. These two enzymes share many similarities with their mammalian orthologs. Their predicted proteins all have classic chitinase protein structures. In addition, they all have O-glycosylation sites but with different pH optimas. Fluorescent in situ hybridization (FISH) shows that both chitinases are located on Chromosome 17 of the rainbow trout genome. Upon full sequencing of two BACs containing Onmy-Chit.01 or .02, I found two copies of Onmy-Chit.01with almost identical coding regions, but with very different promoter regions. The two copies are apporoximately 9Kb apart and sit in a head to tail arrangement. Only one copy of Onmy-Chit.02 was found in the rainbow trout genome, and its distinct promoter regions are distinct from both copies of Onmy-Chit.01. Phylogenetic analyses revealed that the chitinase gene family fits an evolutionary birth and death model with the chitinase genes derived by duplication of an ancestral chitinase gene. Further gene duplication and loss of chitinolytic activity in mammals gave rise to chitolectins. Hence, I postulate that the function of chitinase is two fold: 1) it is a key element in the first line defense of the innate immunity repertoire; and 2) it serves as a gastric digestive enzyme for chitin containing food items.
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    Host preference of Perkinsus species: epizootiological, environmental, and molecular aspects
    (2007-07-10) Pecher, Wolf Thomas; Vasta, Gerardo R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Perkinsus species are protistan parasites of mollusks. In Chesapeake Bay, P. marinus, P. chesapeaki, P. andrewsi, and a Perkinsus isolate from the hard clam Mercenaria mercenaria [Perkinsus sp. (M. mercenaria)] are sympatric. In vitro experiments by others suggest a preference of P. marinus for the eastern oyster Crassostrea virginica. Studies conducted in this dissertation on the distribution of Perkinsus species in C. virginica and M. mercenaria from Virginia to Maine using PCR-based detection assays provided further evidence for a host preference of P. marinus. While P. marinus was the most prevalent species in C. virginica, its prevalence was significantly lower in M. mercenaria. Interestingly, the assay designed to be specific for Perkinsus sp. (M. mercenaria) also amplified P. andrewsi. Characterization of the rRNA gene loci of both Perkinsus species revealed the presence of a second rRNA gene unit in P. andrewsi with high percent sequence identity to the unit of Perkinsus sp. (M. mercenaria), explaining the cross-amplification. Furthermore, DNA samples of M. mercenaria inhibited PCR amplification, which was overcome by adding bovine serum albumin and dimethyl sulfoxide to the PCR reaction mixture. P. marinus resides in oyster hemocytes scavenging and/or inhibiting the production of reactive oxygen species (ROS) usually generated by oyster hemocytes. ROS scavenging enzymes include superoxide dismutases (SODs) and peroxidases such as catalases. SODs have been characterized in P. marinus, but peroxidases have not been detected. Results from the present study suggest that, while lacking catalase, P. marinus has ascorbate dependent peroxidases usually found in plants. Alternatively, P. marinus may suppress the production of ROSs by enzymes such as phosphatases. A secreted acid phosphatase activity reported earlier in P. marinus was further characterized in the present study, and its purification attempted. Furthermore, a search of a genome database yielded several phosphatase-like genes, including a putative protein phosphatase 2C predicted to be secreted. Further analysis could neither confirm its secretion nor its involvement in host preference. However, the approaches implemented throughout this research represent a strategy for processing additional phosphatase and other gene sequences identified in genome databases that will further the understanding of the biology of Perkinsus species.
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    The role of dehalorespiring bacteria in the reductive dechlorination of polychlorinated biphenyls in Baltimore harbor sediment microcosms
    (2007-03-29) Fagervold, Sonja Kristine; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Baltimore Harbor sediment microcosms were incubated with the 12 most predominant congeners in Aroclor 1260 and their intermediate products to identify the major dechlorination pathways. Most congeners were dechlorinated in the meta position, although some dechlorination in the para and ortho positions was observed. The major dechlorination products were tetrachlorinated biphenyls with unflanked chlorines. Specific dechlorination rates of parent and intermediate PCB congeners were determined to identify the rate limiting reactions. To identify the microorganisms responsible for the dechlorination pathways, I developed PCR primers specific for the 16S rRNA genes of known PCB dehalogenating bacteria. These PCR primers were used in conjunction with DGGE to selectively identify the microorganisms that catalyzed each dechlorination reaction. Only three phylotypes were identified that catalyze the dechlorination of Aroclor 1260, and the selective activities of these phylotypes were determined. Phylotype DEH10 had high sequence similarity to Dehalococcoides spp., while phylotype SF1 had high sequence similarity to the o-17/DF-1 group of PCB dechlorinating bacteria. The third phylotype had 100% sequence similarity to the ortho-dechlorinating bacterium o-17 described previously from Baltimore Harbor sediments. Most dechlorination reactions for all three phylotypes were growth-linked, indicating that PCB-impacted environments have the potential to sustain populations of PCB dechlorinating organisms. To investigate whether bioaugmentation would be feasible for bioremediation of PCB contaminated sites, Baltimore Harbor sediment microcosms were supplemented with known dechlorinators and their effects on PCBs dechlorination patterns determined. The addition of different dechlorinators resulted in different dechlorination patterns. Finally, novel putative reductive dehalogenases were identified from the PCB dechlorinating bacterium DF-1 using degenerate PCR primers. Comparative sequence analyses indicated that they had high sequence similarity to both confirmed and putative dehalogenases from several Dehalococcoides species. In conclusion, microorganisms that can dechlorinate Aroclor 1260 have been identified for the first time and dechlorination of congener mixtures was shown to occur by the growth-linked complementary activities of bacterial consortia within the Chloroflexi. Demonstration that bioaugmentation with these organisms can influence rates and pathways of dechlorination, combined with the development of molecular assay for monitoring their fate, provide potentially valuable tools for the development of bioremedial strategies for PCB contaminated sediments.
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    Isolation and characterization of a sponge-associated actinomycete that produces manzamines
    (2006-11-20) Peraud, Olivier; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Two Indonesian sponges, Acanthostrongylophora sp. Sponge 35 and Sponge 52, containing manzamine A were collected off the coast of Manado, Indonesia. Manzamines are a family of marine alkaloids that exhibit a complex molecular architecture and possess bioactivities including antitumour, antimicrobial, antiparasitic and insecticidal activities. Manzamines have been found in 17 different species of sponges with wide geographical distribution which has led to speculation that they may be produced by a microbial symbiont rather than by the sponges themselves. The sponges' microbial communities were investigated using 16S rRNA gene analysis and a rational culture-based microbiology approach in which specific bacterial groups were targeted. The molecular analysis of these microbial communities revealed that they were complex and diverse. Microbiological analyses were conducted on Acanthostrongylophora sp. with a particular emphasis on the isolation of actinomycetes because of the high number of actinomycete sequences in this sponge 16S rRNA gene clone library and their excellent track record as bioactive compound producers. One of the isolated actinomycetes, Micromonospora sp. strain M42, produces manzamine A and 8-hydroxy-manzamine, compounds initially detected in the sponge. A detailed analysis of Micromonospora sp. strain M42 showed that it grew on a wide range of salt concentrations with an optimal growth at 0-1% NaCl. Cultures of Micromonospora sp. strain M42 consistently produced manzamine A with a maximum yield of 1 mg/l. The genome size of Micromonospora sp. strain M42 was estimated at 6.7 Mb by pulsed field gel electrophoresis. The biosynthetic gene pathway encoding manzamine A was investigated using both biochemistry and molecular methods yet it remains elusive. Micromonospora sp. strain M42 underwent UV mutagenesis leading to isolation of mutants with yield of manzamine A improved by 3.5 fold. One of the mutants produces manzamine B, the putative biosynthetic precursor of manzamine A. A fosmid library of Micromonospora sp. strain M42 was constructed and low-pass genome sequencing gave insights into the strain's genome and revealed a high number of genes devoted to the production of secondary metabolites including polyketides and non-ribosomal peptides. The isolation of Micromonospora sp. strain M42 greatly improves the chances of manzamines becoming a drug class for treatment of malaria.
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    Comparative genomic analysis of Vibrio cholerae O31: capsule, O-antigen, pathogenesis and genome
    (2006-11-21) Chen, Yuansha; Morris, J Glenn; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Vibrio cholerae is the causative agent of cholera. In order to understand the genetic basis underlying the emergence of novel epidemic strains of V. cholerae, the genetics of surface polysaccharide biogenesis, and the role of lateral gene transfer in the evolution of this species, we investigated. NRT36S and A5 are both NAG-ST producing, cholera toxin negative, serogroup O31 V. cholerae. NRT36S is encapsulated and causes diarrhea when administered to volunteers; A5 is acapsular and does not colonize or cause illness in humans. The structure of the capsular (CPS) polysaccharide in NRT36S was determined by NMR. The gene cluster of CPS biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data. The CPS gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. The LPS biogenesis regions in A5 were similar to NRT36S except that a 6.5 kb fragment in A5 replaced a 10 kb fragment in NRT36S in the middle of the LPS gene cluster. The genome of NRT36S was sequenced to a draft containing 174 contigs plus the superintegron region. Besides confirming the existence of NAG-ST, we also identified the genes for a type three secretion system (TTSS), a putative exotoxin, and two different RTX genes. Four pili systems were also identified. Therefore, the genome of non-O1 Vibrio cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from O1 V. cholerae. We conclude that lateral gene transfer plays a critical role in the emergence of new strains. The co-location of CPS and LPS could provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. Our data also highlights the apparent mobility within the CPS/LPS region that would provide a basis for the large number of observed V. cholerae serogroups and the emergence of novel epidemic strains.
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    Gonadal and steroid feedback regulation of the hypothalamus-pituitary axis in striped bass (Morone saxatilis)
    (2006-09-18) Klenke, Ulrike; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The objective of the present study was to expand our understanding of the mechanisms of gonadal steroid feedback regulation of the hypothalamus-pituitary (HP) axis during several reproductive stages (juvenile, pubertal, adult) throughout the life cycle of the striped bass. Towards this end, we investigated effects of bilateral gonadectomy and steroid replacement on the endocrine correlates of the HP axis in vivo. We also developed a brain-slice culture method and utilized pituitary cell cultures to investigate direct effects of estrogen on these correlates at the level of the brain and the pituitary in vitro. Our findings indicate that: 1) During their development, the gonads play an important role in providing feedback to the HP axis. These feedback patterns change during the transformation from the juvenile to the adult and throughout the adult reproductive cycle. The pathways involved use both non-steroidal and steroidal pathways as regulatory mechanisms. 2) Gonads, through their steroids, become more involved in regulating the HP axis during reproductive development and their main feedback target appears to be gene transcription in the pituitary. 3) The observed changes in gonadal feedback throughout the adult reproductive cycle probably reflect the physiological requirements of gametogenesis. 4) The responsiveness of the HP axis towards steroids initially appears during puberty and further increases in adult females. In adults, steroids solely affect the pituitary in early stages of gametogenesis, while in later stages GnRH expression in the brain is also regulated by steroids. However, the nature of the feedback is dependent on estrogenic and/or androgenic pathways. 5) Our in vitro studies showed that estrogens act directly at the levels of the brain and the pituitary in female adult fish. Based on these findings, it appears that the activity along the endocrine reproductive web of striped bass intensifies with age, and that prior cycles of oocyte development may prime the HP axis to respond faster and more vigorously in subsequent years. This study has provided an improved resolution and a broader perspective on mechanisms involved in gonadal steroid feedback regulation of GnRH neural activity and its targets at the level of the pituitary in striped bass.