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 Department: Department of Biological Sciences
Stress Response by Alternative Σ-factor, Rpoh, and Analysis of Posttranslational Modification of the Heat Shock Protein, Dnak, in Escherichia Coli
Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.
Characteristics of Primary Cilia and Centrosomes in Neuronal and Glial Lineages of the Adult Brain
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Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.
Cytotoxicity and Functional Toxicity of Mefloquine and Search for Protective Compounds
Mefloquine hydrochloride is an antimalarial agent that has been used for the past 40 years. Numerous reports of neurological side effects have recently led the FDA to issue a strong warning regarding long-term neurological effects. This warning lead to the U.S. Army’s Special Forces and other components to discontinue its use in July of 2013. Despite reported adverse side effects, mefloquine remains in circulation and is recommended to travelers going to specific Asian countries. Mefloquine has been used as a treatment for those already infected with the malaria parasite (blood concentrations ranging from 2.1 to 23 µM), and as prophylaxis (blood concentrations averaging 3.8 µM) (Dow 2003). The purpose of this study was to quantify Mefloquine’s toxicity using spontaneously active nerve cell networks growing on microelectrode arrays in vitro and to identify compounds that alleviate or reduce toxic effects. The current literature on mefloquine toxicity is lacking electrophysiological data. These data will contribute to research on the mechanism of adverse side effects associated with mefloquine use. Sequential titration experiments were performed by adding increasing concentrations of mefloquine solution to cultured neurons. Network responses were quantified and reversibility was examined. In each network, activity decreases were normalized as a percent of reference activity yielding a mean IC50 value of 5.97 ± 0.44 (SD) µM (n=6). After total activity loss, no activity was recovered with two successive medium changes. To test for network response desensitization resulting from sequential applications over 5-6 hr periods, one-point titrations at varying concentrations were conducted with fresh networks. These experiments yielded a single concentration response curve with an IC50 value of 2.97 µM. This represents a statistically significant shift (p < 0.0001) to lower concentrations of mefloquine, demonstrating that sequential applications result in network desensitization. After mefloquine exposures, cells were evaluated for irreversible cytotoxic damage. Over a 12-hour period under 6 µM mefloquine, process beading and granulation of somal cytoplasm were observed. At 8 µM mefloquine cell stress was apparent after only 10 minutes with major glial damage and process beading at 120 minutes. In this study, quinolinic acid served as a neuroprotectant at 20 µM. There have been multiple studies on the endogenous concentrations of quinolinic acid and current literature is quite variable. Immunocompromised individuals have some of the highest blood levels of quinolinic acid (up to 20 µM). With 30 min pre-applications of quinolinic acid, the mefloquine IC50 value shifted from 5.97 ± 0.44 µM (n=6), to 9.28 ± 0.55 µM (n=3). This represents a statistically significant change to higher mefloquine concentrations and demonstrates neuroprotection.
The Effect of Curcumin Supplementation on Physical and Biological Indices of Delayed Onset Muscle Soreness and Inflammation Following Muscle Injury
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In this project, the effects of dietary polyphenols on exercise-induced muscle damage and vascular health are examined. Dietary polyphenols exert well-known anti-inflammatory effects; however, how these effects are realized with respect to vascular health and EIMD is relatively unknown. I begin by reviewing the available literature surrounding the impact of three dietary polyphenols (curcumin, catechins, and quercetin) on inflammation associated with EIMD. It is well established that their primary means of anti-inflammation is through alterations of NF-κB and AP-1 transcription activities. Given this, their inclusion into training strategies seems reasonable. Consistent evidence is presented making a case for the anti-inflammatory effects of dietary polyphenols following EIMD. I follow this review up by completing an in-depth study on the consumption of curcumin prior to EIMD. I found curcumin (1000 mg/day) can reduce subjective soreness and decrease inflammation compared to placebo controls. To further understand the effects of dietary polyphenols on health, I investigate the effects of a four-week supplementation period of cocoa (catechins) on vascular. I concluded that atherogenic risk in obese women is reduced after consumption of cocoa. In addition to these experimental projects, I developed two novel methods that can be used to investigate vascular health (EMP concentration) and intracellular protein and mRNA production using flow cytometry.
The Microbial Retting Environment of Hibiscus Cannabinus and Its Implications in Broader Applications
Fiber-yielding plants is an area of increased interest due to the potential use in a variety of green-based materials. These biocomposites can be incorporated into multiple uses; for example, to replace building materials and interior vehicular paneling. The research here aims to focus in on the crop Hibiscus cannabinus for utilization into these functions. H. cannabinus is economically attractive due to the entire process being able to be accomplished here in the United States. The plant can be grown in a relatively short growth period (120-180 days), and then processed and incorporated in a biocomposite. The plant fiber must first be broken down into a useable medium. This is accomplished by the retting process, which occurs when microbial constituents breakdown the heteropolysaccharides releasing the fiber. The research aims to bridge the gap between the primitive process of retting and current techniques in molecular and microbiology. Utilizing a classical microbiological approach, which entailed enrichment and isolation of pectinase-producing bacteria for downstream use in augmented microbial retting experiments. The tracking of the bacteria was accomplished by using the 16S rRNA which acts as “barcodes” for bacteria. Next-generation sequencing can then provide data from each environment telling the composition and microbial diversity of each tested variable. The main environments tested are: a natural environment, organisms contributed by the plant material solely, and an augmented version in which pectinase-producing bacteria are added. In addition, a time-course experiment was performed on the augmented environment providing data of the shift to an anaerobic environment. Lastly, a drop-in set was performed using each isolate separately to determine which contributes to the shift in microbial organization. This research provided a much needed modernization of the retting technique. Previous studies have been subject to simple clone libraries and growth plate assays and next-generation sequencing will bring the understanding of microbial retting into the 21st century.
Understanding Microbial Biodegradation of Environmental Contaminants
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The accumulation of industrial contaminants in the natural environments have rapidly become a serious threat for human and animal life. Fortunately, there are microorganisms capable of degrading or transforming environmental contaminants. The present dissertation work aimed to understand the genomic basis of microbial degradation and resistance. The focus was the genomic study of the following bacteria: a) Pseudomonas fluorescens NCIMB 11764, a unique bacterium with specific enzymes that allow cyanide adaptation features. Potential cyanide degradation mechanisms found in this strain included nit1C cluster, and CNO complex. Potential cyanide tolerance genes found included cyanide insensitive oxidases, nitric oxide producing gene, and iron metabolism genes. b) Cupriavidus sp. strain SK-3 and strain SK-4. The genome of both bacteria presented the bph operon for polychlorinated biphenyl (PCB) degradation, but we found differences in the sequences of the genes. Those differences might indicate their preferences for different PCB substrates. c) Arsenic resistant bacterial communities observed in the Atacama Desert. Specific bacteria were found to thrive depending on the arsenic concentration. Examples were Bacteroidetes and Spirochaetes phyla whose proportions increased in the river with high arsenic concentrations. Also, DNA repair and replication metabolic functions seem to be necessary for resistance to arsenic contaminated environments. Our research give us insights on how bacteria communities, not just individually, can adapt and become resistant to the contaminants. The present dissertation work showed specific genes and mechanisms for degradation and resistance of contaminants that could contribute to develop new bioremediation strategies.
Restoration Techniques for Northern Bobwhites
Isolated populations of northern bobwhites (Colinus virginianus) have declined causing many quail managers to attempt population restoration by releasing captive-reared bobwhites or translocating wild bobwhites. I evaluated three restoration techniques: (1) release of captive-reared bobwhites, (2) translocation of bobwhites from high densities to low densities, and (3) release of captive-reared and translocated bobwhites acclimated on site prior to release. These results show that captive-reared birds have reduced survival and fewer nesting attempts when compared to translocated birds and that acclimation time was not a factor. I hypothesized that high mortality rates were caused by captive-reared birds exhibiting different predator avoidance behavior than wild birds. Captive-reared and wild-trapped bobwhites were subjected to independent predator simulations and their responses were recorded on high definition video. Threat recognition time, reaction type, and reaction time was recorded for comparative analysis. Pen-reared birds recognized the simulated raptorial and terrestrial predator threats quicker than wild-trapped birds, but reaction times were not different among groups. However, the type of reaction was different among groups where pen-reared birds typically flushed immediately upon recognizing either simulated predator as compared to wild-trapped birds which typically ran or held when subjected to the raptorial threat and showed little to no observable reaction to the terrestrial threat. These results reveal a potential loss of a holding trait in pen-reared birds, resulting in a quicker revealing of their position in the presence of a threat, thereby increasing their risk of predation.
Manipulations of Sucrose/proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass
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Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. We propose that AVP1 localizes to the PM of phloem cells and uses PMF to synthesize PPi rather than hydrolyze it, and in doing so, maintains PPi levels for efficient Suc oxidation and ATP production. Enhanced ATP production in turn strengthens the PMF via plasma membrane (PM) ATPase, increasing phloem energization and phloem transport. Phloem-specific and constitutive AVP1 overexpressing lines showed increased growth and more efficiently moved carbohydrates to sink organs compared to WT. This suggested changes in metabolic flux but diagnostic metabolites of central metabolism did not show changes in steady state levels. This research focuses on fundamental aspects of carbon utilization and transport, and has a strong applied component, since increased H+-PPase activity enhances plant biomass, nutrient up-take capacities, and stress tolerance for as yet not fully characterized reasons.
Improving Processing Efficiency for Forensic DNA Samples
The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles <300bp. These modifications resulted in robust databasing processes with up to a 37% reduction in processing time for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated (3μl fast PCR reactions) and generated high quality STR profiles with ≥90% pass rates.
BK1 and DCD1 Act Synergistically in Subsidiary Cell Formation in Zea Mays.
Subsidiary mother cell (SMC) divisions during stomatal complex formation in Zea mays are asymmetric generating a small subsidiary cell (SC) and a larger epidermal cell. Mutants with a high number of abnormally shaped subsidiary cells include the brick1 (brk1) and discordia1 (dcd1) mutants. BRK1 is homologous to HSPC300, an ARP2/3 complex activator, and is involved in actin nucleation while DCD1 is a regulatory subunit of the PP2A phosphatase needed for microtubule generation (Frank and Smith, 2002; Wright et al. 2009). Possible causes of the abnormal SCs in brk1 mutants include a failure of the SMC nucleus to polarize in advance of mitosis, no actin patch, and transverse and/or no PPBs (Gallagher and Smith, 2000; Panteris et al 2006). The abnormal subsidiary mother cell division in dcd1 is due to correctly localized, but disorganized preprophase bands (PPBs; Wright et al. 2009). The observation that brk1 has defects in PPB formation and that the dcd1 phenotype is enhanced by the application of actin inhibitors led us to examine the dcd1; brk1 double mutant (Gallagher and Smith, 1999). We found that dcd1; brk1 double mutants demonstrate a higher percentage of aberrant SCs than the single mutants combined suggesting that these two mutations have a synergistic and additive effect on SC formation. Our observations and results are intriguing and the future step will be to quantitate the abnormal PPBs and phragmoplasts in the double and single mutants using immunolocalization of tubulin and actin as well as observations of live cells expressing tubulin-YFP.
Investigation of Strategies for Improving STR Typing of Degraded and Low Copy DNA from Human Skeletal Remains and Bloodstains
Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.
Neurotoxicity of the Industrial Solvent 4-Methylcyclohexanemethanol: Involvement of the GABA Receptor
A recent chemical spill of 4-Methylcyclohexanemethanol (4-MCHM) in West Virginia left 300,000 people without water. Officials claimed that this compound is not lethally toxic, but potentially harmful if swallowed or inhaled, and can cause eye and skin irritation. Sittig's Handbook of Toxic and Hazardous Chemical Carcinogens reports high exposures from skin contact or inhalation may cause damage to the heart, liver, kidneys, and lungs, and may result in death. However, no quantitative data seem to exist and no references can be found on neurotoxicity. We have investigated the neurotoxicity of 4-MCHM using mammalian nerve cell networks grown on microelectrode arrays. Network spontaneous activity from multiple units (range 48 – 120 per network) were used as the primary readout. Individual units were followed based on spike waveforms digitized at 40 kHz (Plexon MNAP system). Dose response curves show the effective inhibitory concentration at 50 percent decrease (EC50) to average 27.4 microM SD±6.17. However, in the presence of 40 microM bicuculline, a competitive GABAA antagonist, the EC50 shifts to 70.63uM SD ±4.3; implying that early, low concentration exposures to 4-MCHM involve GABA activation. Initial activity loss occurs without active unit loss (defined as 10 or more template threshold crossing per min), indicating functional interference with spike production. Full recovery has not been seen at concentrations above 130 microM, unless the culture was given bicuculline. Direct exposure to 400uM results in immediate, irreversible loss of spike production, followed by necrosis of glia and neurons.
Women Have Higher Skin Temperature on the Back during Treadmill Exercise in a Hot, Humid Environment
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A common measurement of body temperature during exercise in a hot, humid environment is mean skin temperature collected from 3-12 sites on the body. However, such an approach fails to demonstrate localized differences in skin temperature that are likely to exist as a function of gender. The purpose of this study was to examine potential differences in skin temperature between men and women at 17 different locations on the body. Young women (21 ± 1 y; n = 11) and men (23 ± 3; n = 10) were recruited to complete a 60-min walk/jog interval protocol in a hot (34 ± 1 °C), humid (64 ± 8%) environment while skin temperature was measured. Data was analyzed using a repeated-measures ANOVA (p < 0.05) and location of interaction effects determined using a Fisher’s least squares difference test. We observed a higher change (p < 0.05) from baseline skin temperatures (ΔTsk) for women in three locations: left upper back (women: avg. ΔTsk = 4.12 ± 0.20 °C; men: avg. ΔTsk = 2.70 ± 0.10 °C), right upper back (women: avg. ΔTsk = 4.19 ± 0.07 °C; men: avg. ΔTsk = 2.92 ± 0.05 °C), and right mid-back (women: avg. ΔTsk = 4.62 ± 0.14 °C; men: avg. ΔTsk =3.55 ± 0.09 °C). Individual time differences between genders occurred after 7- (left upper back) and 15-min (right upper back, right mid-back) of exercise and were maintained until the end of exercise. Women have a greater increase in skin temperature at three locations on the back following the onset of exercise in a hot, humid environment. This report provides important information regarding the implications of women exercising in a hot, humid environment.
In Vitro Investigations of Antibiotic Influences on Nerve Cell Network Responses to Pharmacological Agents
Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.
The Role of the Actin Cytoskeleton in Asymmetric Cell Division in Maize
Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.
Evidence for Multiple Functions of a Medicago Truncatula Transporter
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Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate at the concentrations tested and MtNPF1.7/NIP/LATD does not regulate the nitrate-responsive MtNRT2.1 gene. Mtnip-1 plants have an abnormal gravitropic root response implicating an auxin defect. Together with these results, MtNPF1.7/NIP/LATD is associated with nitrate and auxin; however, it does not act in a homologous fashion as AtNPF6.3/NRT1.1(CHL1) does in A. thaliana.
Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels
Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The recombinant Z. mobilis ZM4 pAA1 and wild type ZM4 were first tested on RM medium (ATCC 1341) containing 2% cellobiose as the carbon source. Ethanol production from the recombinant Z. mobilis was three times that observed from the wild type Z. mobilis. Concomitant with ethanol production was the reduction in OD from 2.00 to 1.580, indicating the consumption of cellobiose. No such change in OD was observed from the wild type. The recombinant ZM4 was then co-cultured with C. cellulolyticum using cellobiose and microcrystalline cellulose respectively as carbon sources. Results indicate that the recombinant ZM4 acted synergistically with C. cellulolyticum to utilize 2.0 g L-1 cellobiose, producing as much as 0.40 mM concentration of ethanol whereas only 0.20 mM ethanol was detected for the wild type ZM4 co-cultured with C. cellulolyticum under the same conditions. A co-culture of the recombinant ZM4 and C. cellulolyticum using 7.5 g L-1 microcrystalline cellulose gave lower ethanol yield than when using cellobiose. In the latter case, the recombinant began producing ethanol in 5 days whereas the wild type required 10 days to produce detectable ethanol. Future efforts will concentrate on identifying the correct concentration of cellulosic substrate at which synergy will be observed using the recombinant ZM4 and other cellulose degrading microorganisms, as well as optimizing medium formulations to better support both organisms.
Optimization of Novel Culturing and Testing Procedures for Acute Effects on Acartia Tonsa and Tisbe Biminiensis
Copepods comprise an ecologically important role in freshwater and marine ecosystems, which is why they are often considered an important ecotoxicological model organism. The International Organization for Standardization’s (ISO) 14669 protocol is the only guideline for the determination of acute toxicity in three European marine copepod species: Acartia tonsa. The goal of this project was to assess the feasibility of establishing and maintaining cultures of Acartia tonsa, as well as to refine current culturing and egg separation methods. Initial culture methodology proved difficult for consistent production of eggs and collection of nauplii. The development of an airlift system for the separation of eggs from nauplii and adults, based on size, successfully increased the availability of eggs, nauplii and adults. The sensitivity and relative conditions of the copepod species was assessed by running a series of 48h acute toxicity tests with the reference toxicants 3,5-dichlorophenol, 4,4’-methylenebis(2,6-di-tert-butylphenol. The acute 48 hour median lethal dose concentration (LC50), the no observed effect concentration (NOEC), and the lowest observed effect concentration (LOEC) was analyzed for the three reference compounds for of A. tonsa.
Regulation of Alternative Sigma Factors During Oxidative and Ph Stresses in the Phototroph Rhodopseudomonas Palustris
Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.
Measuring Biomarkers From Dried Blood Spots Utilizing Bead-based Multiplex Technology
Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.
The Effects of Inbreeding on Fitness Traits in the Critically Endangered Attwater’s Prairie-chicken
The goals of captive breeding programs for endangered species include preserving genetic diversity and avoiding inbreeding. Typically this is accomplished by minimizing population mean kinship; however, this approach becomes less effective when errors in the pedigree exist and may result in inbreeding depression, or reduced survival. Here, both pedigree- and DNA-based methods were used to assess inbreeding depression in the critically endangered Attwater’s prairie-chicken (Tympanuchus cupido attwateri). Less variation in the pedigree-based inbreeding coefficients and parental relatedness values were observed compared to DNA-based measures suggesting that errors exist in the pedigree. Further, chicks identified with high parental DNA-based relatedness exhibited decreased survival at both 14- and 50-days post-hatch. A similar pattern was observed in later life stages (> 50 days post-hatch) with birds released to the wild; however, the pattern varied depending on the time post-release. While DNA-based inbreeding coefficient was positively correlated with mortality to one month post-release, an opposite pattern was observed at nine months suggesting purging of deleterious alleles. I also investigated whether immunocompetence, or the ability to produce a normal immune response, was correlated with survival; however, no significant correlation was observed suggesting that inbreeding was a more important factor influencing survival. Pairing individuals for breeding by minimizing DNA-based parental relatedness values resulted in a significant increase in chick survival. This study highlights the importance of using DNA-based methods to avoid inbreeding depression when errors exist in the pedigree.
Acute Effects of the Antibiotic Streptomycin on Neural Network Activity and Pharmacological Responses
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The purpose of this study is to find out that if antibiotic streptomycin decreases neuronal network activity or affects the pharmacological responses. The experiments in this study were conducted via MEA (multi-electrode array) technology which records neuronal activity from devices that have multiple small electrodes, serve as neural interfaces connecting neurons to electronic circuitry. The result of this study shows that streptomycin lowered the spike production of neuronal network, and also, sensitization was seen when neuronal network pre-exposed to streptomycin.
Developing a Forest Gap Model to Be Applied to a Watershed-scaled Landscape in the Cross Timbers Ecoregion Using a Topographic Wetness Index
A method was developed for extending a fine-scaled forest gap model to a watershed-scaled landscape, using the Eastern Cross Timbers ecoregion as a case study for the method. A topographic wetness index calculated from digital elevation data was used as a measure of hydrologic across the modeled landscape, and the gap model modified to have with a topographically-based hydrologic input parameter. The model was parameterized by terrain type units that were defined using combinations of USDA soil series and classes of the topographic wetness index. A number of issues regarding the sources, grid resolutions, and processing methods of the digital elevation data are addressed in this application of the topographic wetness index. Three different grid sizes, 5, 10, and 29 meter, from both LiDAR-derived and contour-derived elevation grids were used, and the grids were processed using both single-directional flow algorithm and bi-directional flow algorithm. The result of these different grids were compared and analyzed in context of their application in defining terrain types for the forest gap model. Refinements were made in the timescale of gap model’s weather model, converting it into a daily weather generator, in order to incorporate the effects of the new topographic/hydrologic input parameter. The precipitation model was converted to use a Markov model to initiate a sequence of wet and dry days for each month, and then daily precipitation amounts were determined using a gamma distribution. The output of the new precipitation model was analyzed and compared with a 100-year history of daily weather records at daily, monthly, and annual timescales. Model assumptions and requirements for biological parameters were thoroughly investigated and questioned. Often these biological parameters are based on little more than assumptions and intuition. An effort to base as many of the model’s biological parameters on measured data was made, including a new technique for estimating optimal volumetric growth rate by measuring tree rings. The gap model was set up to simulate various terrain types within the landscape.
Cloacal Microbiota of Captive-bred and Wild Attwater’s Prairie-chicken, Tympanuchus Cupido Attwateri
The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.
Effects of a Water Conservation Education Program on Water Use in Single-family Homes in Dallas, Texas
The City of Dallas Environmental Education Initiative (EEI) is a hands-on, inquiry-based, K-12 water conservation education program that teaches students concepts about water and specific water conservation behaviors. Few descriptions and evaluations, especially quantitative in nature, of water conservation education programs have previously been conducted in the literature. This research measured the quantitative effects and impacts of the education program on water use in single-family homes in Dallas, Texas. A total of 2,122 students in 104 classrooms at three schools in the Dallas Independent School District received hands-on, inquiry-based water conservation education lessons and the average monthly water use (in gallons) in single-family homes was analyzed to measure whether or not there was a change in water use. The results showed that over a period of one calendar year the water use in the single-family homes within each school zone and throughout the entire research area in this study experienced a statistically significant decrease in water use of approximately 501 gallons per home per month (independent, t-test, p>0.001). Data from this research suggests that EEI is playing a role in decreasing the amount of water used for residential purposes. Additionally, this research demonstrates the use of a quantitative tool by which a water conservation education program’s effect on behavior change can be measured. This research shows great promise for reducing use and increasing the conservation of our world’s most precious resource.
Biodiversity of Dragonflies and Damselflies (Odonata) of the South-Central Nearctic and Adjacent Neotropical Biotic Provinces
The south-central United States serves as an important biogeographical link and dispersal corridor between Nearctic and Neotropical elements of western hemisphere odonate faunas. Its species are reasonably well known because of substantial collections, but there has been no concerted effort to document the extent of biodiversity and possible geographic affinities of dragonflies and damselflies of this region. The recent discoveries of Argia leonorae Garrison, Gomphus gonzalezi Dunkle and Erpetogomphus heterodon Garrison from southern and western Texas and northern Mexico suggest that Odonata species remain to be discovered in this area, particularly from far south Texas and northern Mexico. I have documented a total of 12,515 records of Odonata found in 408 counties within the south-central U.S. A total of 73 species of damselflies and 160 species of dragonflies was revealed in the region. The 233 (197 in Texas) Odonata species are distributed among 10 families and 66 genera. Illustrated family, generic, and species-level keys are provided. Since the beginning of this work in the Fall of 1993, one species has been added each to the Louisiana and Oklahoma faunas, and 12 species have been added, previously unreported from Texas, including four new to the U.S. The area of highest Odonata biodiversity overall (161 spp.) is in the Austroriparian biotic province. The greatest degree of faunal similarity between the south-central U.S. and other intra-continental regions was observed for the eastern (64%) United States. Diversity is a function of area, and as expected, the numbers of breeding birds and Odonata, in each contiguous U.S. state are positively correlated (r=0.376, n=33, p=0.031). There is, however, no strong correlation between land area and species diversity within the region, but those natural biotic provinces (Austroriparian, Texan, Balconian) where aquatic systems and topographic heterogeneity are the greatest provide a broader spectrum of potential Odonata habitats and thus support a greater number of Odonata species.
Isolation and Characterization of a New Capsule-Forming Bacterium
A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.
Comparative Biochemistry and Evolution of Aspartate Transcarbamoylase from Diverse Bacteria
Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.
Biogeographic Relationships of Pocket Gophers (Geomys breviceps and Geomys bursarius) in the Southeastern Portion of Their Ranges
This research utilized population genetic analyses (protein starch-gel electrophoresis and DNA sequencing of the cytochrome b mtDNA gene), host-parasite specificity (lice coevolution), remote sensing of satellite data, and geographic information systems (GIS) to characterize newly discovered populations of pocket gophers (genus: Geomys) in Arkansas. These populations are isolated and occur in seemingly unsuitable habitat in the Ozark Mountains of Arkansas. Analyses of electrophoretic and ectoparasite data suggested the populations in the Ozark Mountains represented isolates allied to Geomys bursarius, a species not known to occur in Arkansas. Comparison of mitochondrial DNA sequence data of the cytochrome b gene with that of other taxa and morphometric analyses confirmed that these populations are most closely allied to G. bursarius occurring to the north in Missouri. Moreover, these mtDNA sequence analyses indicated a degree of differentiation typical of that between other subspecies of pocket gophers. Therefore, these populations represent a distinct genetic entity in an intermediate stage of speciation and should be designated as a new subspecies, Geomys bursarius ozarkensis. Molecular clock analysis revealed a time of lineage divergence for this new subspecies as approximately 511,000 YBP. Due to the isolated nature and limited distribution of this subspecies, an evaluation of critical habitat needs was initiated. Remote sensing and GIS technologies were used to identify and describe suitable habitat Computerized classification of satellite imagery of suitable vegetation, integrated with ancillary digital information on soil associations, roads, and water systems, revealed that human activity had played a positive role in the establishment and dispersal of pocket gophers in this area. This research represents an initial combination of classical systematic tools with remote sensing and GIS to investigate biogeographic patterns and evolution. This project establishes a framework for using an interdisciplinary approach to studying organisms with limited distributions, determining evolutionary status, and providing recommendations for conservation.
Dalbergia and Albizia: Plantlet Production via Tissue Culture, Karyological Evaluation, and Seed Anatomy with Scanning Electron Microscopy
A publication by the National Academy of Sciences, USA (1979) outlined some of the research need for a great variety of economically important woody species whose remaining genetic resources need urgently to be collected and conserved. A viable regeneration system was established via tissue and cell suspension culture for Albizia falcataria and A. lebbeck, two important wood yielding leguminous tree species. The culture medium was standardized after several trials to obtain callus from the leaflet explants of these two tree species. The optimum use of casein hydrolysate (w/v) and coconut milk (v/v) in addition to 6-Benzylaminopurine and Indole-3-butyric acid could induce morphogenesis and somatic embryogenesis in the cultured tissue. This reports the first observation on somatic embryogenesis ofA. lebbeck using leaflets as the explants. Scanning Electron Microscopy and histological studies were done on the different stages plant development following standard techniques. Embryogenesis in suspension culture followed regeneration of plantlets in A. lebbeck. In A.falcaaria the regenerative process followed via organogenesis from the shoot buds developed on the leaf explants. After hardening the regenerated plants were transferred to the greenhouse. Some of the trees grew more than 25 feet tall within a few months outside the greenhouse. Karyotype of the three leguminous trees Albizia lebbeck, A. falcataria, and Dalbergia sissoo was analyzed. In D. sissoo, various chromosomal anomalies were observed in the cultured tissue. The abnormality indices and ploidy level varied with the age and the frequency of the subculture. In the aged culture the regenerative potential declined but was reinstated to some extent with the addition of two complex growth factors, coconut milk and casein hydrolysate. Seed anatomy of 26 species of 4 leguminous genera was studied with SEM. The main distinguishing anatomical features observed in the seed sections were uniseriate or multiseriate epidermis, epidermal projections, and number of rows and nature of columns of hypodermal layer, especially the nature of endosperm. Three species of Dalbergia, Acacia and Cassia and two species of Albizia are difficult to distinguish externally even with seed coat study under SEM, but this study with cross sections provided enough characteristic features to distinguish one from the other.
Role of α-Keto Acids In Cyanide Detoxification and Assimilation by Pseudomonas Bacteria
Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.
Mutagenized HLA DNA Constructs: Tools for Validating Molecular HLA Typing Methodologies
This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.
Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations
Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.
Distribution of a Novel Gram Negative, Capsule-Forming Bacterium
A novel Gram negative, capsule-forming bacterium was previously isolated in Dr. G. Roland Vela's laboratory. The distribution of this bacterium in soils from various locations was investigated. Soil samples from 188 locations around the world were examined. Isolates of the bacterium were obtained from 50 of these soils, with 48 of the isolates found in soils from the southwestern United States and northern Mexico. This suggests that this region is the natural habitat of the bacterium. The other two isolates were obtained from Madrid, Spain and Taipei, Taiwan. None were found in soils from South America or Australia. A lack of variation in morphology and physiological properties in the isolates suggests that a homogeneous population exists, even from widespread geographical locations.
A Multimedia Atlas of Dissection for Comparative Anatomy of the Vertebrates
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This interactive multimedia content is part of the thesis that was prepared for the degree of Master of Science in Biology: Traditional methods of teaching the laboratory course for Comparative Anatomy of the Vertebrates could be improved by applying current computer technology to construct an interactive, multimedial atlas of dissection. Five specimens used in comparative anatomy courses at most institutions were chosen as representative members of the Phylum Chordata: amphioxus, lamprey, dogfish shark, mud puppy, and cat. Specimens were dissected according to the modified method of Wischnitzer, 1993, and each stage was photographed with a Kodak DC120 digital zoom camera. These images were processed on a Power Macintosh 7600 computer with Adobe Photoshop v. 5.0. The atlas was constructed from these images using Macromedia Authorware v. 4.0.3. Each image contains a series of interactive objects that display a highlight and descriptive text as the cursor passes over each object.
Development of Enabling Technologies to Visualize the Plant Lipidome
Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism in cellular (perhaps even subcellular) context with substantial metabolic engineering implications. To visualize metabolite distributions, a free and customizable application, Metabolite Imager, was developed providing several tools for spatially-based chemical data analysis. These tools collectively enable new forms of visualizing the plant lipidome and should prove valuable toward addressing additional unanswered biological questions.
The Effect of Natural Gas Well Setback Distance on Drillable Land in the City of Denton, Texas
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Municipalities protect human health and environmental resources from impacts of urban natural gas drilling through setback distances; the regulation of distances between well sites and residences, freshwater wells, and other protected uses. Setback distances have increased over time, having the potential to alter the amount and geographical distribution of drillable land within a municipality, thereby having implications for future land use planning and increasing the potential for future incompatible land uses. This study geographically applies a range of setback distances to protected uses and freshwater wells in the city limits of Denton, Texas to investigate the effect on the amount of land remaining for future gas well development and production. Denton lies on the edge of a productive region of the Barnett Shale geological formation, coinciding with a large concentration of drillable land in the southwestern region of the study area. This region will have the greatest potential for impacts to future municipal development and land use planning as a result of future gas well development and higher setback standards. Given the relatively high acreage of drillable land in industrially zoned subcategory IC-G and the concern regarding gas well drilling in more populated areas, future drilling in IC-G, specifically in IC-G land cover classes mowed/grazed/agriculture and herbaceous, would have the least impact on residential uses and tree cover, as well as decreasing the potential for future incompatible land uses.
The Effects of Glyphosate Based Herbicides on Chick Embryo Development
Glyphosate based herbicides are among the most widely used herbicides in the world. The purpose of this study was to determine developmental toxicity of glyphosate, the active ingredient in the common herbicide Roundup, on developing chicken embryos. Few studies have examined toxic effects of glyphosate alone versus the full compound formulations of Roundup, which include adjuvants and surfactants. Adjutants and surfactants are added to aid in solubility and absorption of glyphosate. In this study chicken embryos were exposed at the air cell on embryonic day 6 to 19.8 or 9.9 mg / Kg egg mass of glyphosate in Roundup or glyphosate only. Chickens treated with 19.8 and 9.9 mg / Kg glyphosate in Roundup showed significant reduction in survivability compared to glyphosate alone treatments and controls. On embryonic day 18, embryos were sacrificed for evaluation of developmental toxicity using wet embryo mass, dry embryo mass, and yolk mass as indicators. Morphology measurements were taken on liver mass, heart mass, tibiotarsus length and beak length. Embryos treated with 19.8 mg / Kg glyphosate and 9.9 mg / Kg glyphosate in Roundup showed significant reductions in wet and dry embryo mass and yolk mass. Tibiotarsus length in 9.9 mg / Kg glyphosate in Roundup treatments were significantly reduced compared to 9.9 mg / Kg glyphosate treatments. Beak length was significantly reduced in 9.9 mg /Kg glyphosate in Roundup treatments compared to all other groups.
Effects of Natural/anthropogenic Stressors and a Chemical Contaminant on Pre and Post Mycorrhizal Colonization in Wetland Plants
Arbuscular mycorrhizal fungi, colonizing over 80% of all plants, were long thought absent in wetlands; however, recent studies have shown many wetland plants harbor arbuscular mycorrhizae (AM) and dark septate endophytes (DSE). Wetland services such as biodiversity, shoreline stabilization, water purification, flood control, etc. have been estimated to have a global value of $14.9 trillion. Recognition of these vital services is accompanied by growing concern for their vulnerability and continued loss, which has resulted in an increased need to understand wetland plant communities and mycorrhizal symbiosis. Factors regulating AM and DSE colonization need to be better understood to predict plant community response and ultimately wetland functioning when confronting natural and human induced stressors. This study focused on the effects of water quality, hydrology, sedimentation, and hurricanes on AM and DSE colonization in three wetland species (Taxodium distichum, Panicum hemitomon, and Typhal domingensis) and plant communities of coastal wetlands in Southeast Louisiana and effects of an antimicrobial biocide, triclosan (TCS), on AM (Glomus intraradices) spore germination, hyphal growth, hyphal branching, and colonization in fresh water wetland plants (Eclipta prostrata, Hibiscus laevis, and Sesbania herbacea) from bottom land hardwood forest in north central Texas. The former, mesocosm studies simulating coastal marsh vegetation ran for five years. In the latter studies, AM spores and wetland plants were exposed to 0 g/L, 0.4 g/L, and 4.0 g/L TCS concentrations in static renewal and flow through exposures for 21 and 30 days, respectively. AM and DSE colonization was significantly affected by individual and interactions of four independent variables in mesocosm experiments. Similarly, spore germination, hyphal growth, hyphal branching, and AM colonization in selected wetland plants were significantly lowered by exposure to the TCS at environmentally relevant concentrations. However, levels of effects were plant species and fungal propagules specific. My results showed that natural and human induced alterations in environmental factors and chemical contaminants can significantly impact levels of mycorrhizal spore germination, colonization, and spore density in coastal and freshwater wetland plants. The resulting impacts on plant community structure and ecosystem function require further study.
Tissue-specific Bioconcentration Factor of the Synthetic Steroid Hormone Medroxyprogesterone Acetate (Mpa) in the Common Carp, Cyprinus Carpia
Due to the wide spread occurrence of medroxyprogesterone acetate (MPA), a pharmaceutical compound, in wastewater effluent and surface waters, the objectives of this work were to determine the tissue specific uptake and bioconcentration factor (BCF) for MPA in common carp. BCFs were experimentally determined for MPA in fish using a 14-day laboratory test whereby carp where exposed to 100 μg/L of MPA for a 7-day period followed by a depuration phase in which fish were maintained in dechlorinated tap water for an additional 7 days. MPA concentrations in muscle, brain, liver and plasma were determined by liquid chromatography/mass spectrometry (LC/MS). The results from the experiment indicate that MPA can accumulate in fish, however, MPA is not considered to be bioaccumulative based on regulatory standards (BCF ≥ 1000). Although MPA has a low BCF value in common carp, this compound may cause reproductive effects in fish at environmentally relevant concentrations.
Developing a Collection Digitization Workflow for the Elm Fork Natural Heritage Museum
Natural history collections house immense amounts of data, but the majority of data is only accessible by locating the collection label, which is usually attached to the physical specimen. This method of data retrieval is time consuming and can be very damaging to fragile specimens. Digitizing the collections is the one way to reduce the time and potential damage related to finding the collection objects. The Elm Fork Natural Heritage Museum is a natural history museum located at the University of North Texas and contains collections of both vertebrate and invertebrate taxa, as well as plants. This project designed a collection digitization workflow for Elm Fork by working through digitizing the Benjamin B. Harris Herbarium. The collection was cataloged in Specify 6, a database program designed for natural history collection management. By working through one of the museum’s collections, the project was able to identify and address challenges related to digitizing the museum’s holdings in order to create robust workflows. The project also produced a series of documents explaining common processes in Specify and a data management plan.
Immunohistochemistry of the Gills of the Channel Catfish Ictalurus Punctatus: Cells and Neurochemicals That May Be Involved in the Control of Cardioventilatory Reflexes
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In teleost fishes the neurochemicals involved in sensing and responding to hypoxia are unresolved. Serotonergic branchial neuroepithelial cells (NECs) are putative O2 chemoreceptors believed to be homologous to the neural crest (NC) derived APUD (amine-precursor uptake and decarboxylation) pulmonary NECs and carotid body type-1 glomus cells. Branchial NECs contain serotonin (5-HT), thought to be central to the induction of the hypoxic cardioventilatory reflexes. However, application of 5-HT in vivo does not elicit cardioventilatory reflexes similar to those elicited by hypoxia. But previous in vitro neural recordings from glossopharyngeal (IX) afferents innervating O2 chemoreceptors in the trout gill show the same discharge response to hypoxic conditions as does that of acetylcholine (ACh) application. This evidence strongly supports the cholinergic hypothesis of chemoreceptor impulse origin rather than a serotonergic-induced impulse origin model. We therefore hypothesized that NECs contain ACh among other neurochemicals in cells belonging to the APUD series. Although serotonergic branchial NECs did not colocalize with ACh using immunohistochemical methods, several populations of ACh and/or tyrosine hydroxylase (TH) (catecholaminergic) positive, dopamine (DA) negative, cells were found throughout the second gill arch of the channel catfish Ictalurus punctatus. In addition, the NC derivation marker zn-12 labelled the HNK-1-like epitope (Human natural killer) expressed by lamellar pillar cells’ collagen column-associated pillar cell adhesion molecules (CC-PCAMs), evidence confirming their hypothesized NC origin.
Shortened in Vivo Bioconcentration Factor Testing in Cyprinus Carpio
Bioconcentration factor testing serves as the most valuable surrogate for the assessment of bioaccumulation. The assessment of potentially harmful chemicals is crucial to not only the health of aquatic environments, but to humans as well. Chemicals that possess the ability to persist in the environment or that have the potential to bioaccumulate, pose a greater risk to organisms that are exposed to these chemicals. The Organization for Economic Cooperation and Development Guideline 305 outlines specific protocols to run an accurate and reliable aquatic flow-through test. However, since its adoption in 1996, very few changes have been made to accommodate the endeavor to lowering the amount of test species to run one of these said tests. Running an aquatic flow-through test, according to 305, takes much time and money as well as numerous amounts of fish. Such burdens can be eliminated through simple modifications to the standard protocols. In this study, we propose an abbreviated study design for aquatic bioconcentration testing which effectively alleviates the burdens of running a flow-through test. Four chemicals were used individually to evaluate the usefulness of the proposed shortened design; 4-Nonyphenol, Chlorpyrifos, Musk Xylene, and DDT. The study consisted of exposing Cyprinus carpio for 7 days followed by 7 days of depuration, for a total of a 14-day study. Our results for each of the four compounds are consistent with literature values, thus, demonstrating that BCFk can be accurately predicted in an abbreviated in vivo test.
The Effect of Menthol on Nicotine Metabolism: a Cross Species Evaluation
The effect of menthol on nicotine metabolism was examined in liver S9 fractions of four different species and in the in vivo mouse model. The purpose of this study was to investigate three parameters: (1) biotransformation of nicotine to cotinine in various species (human, mouse, rat and trout) using in vitro methods; (2) to determine if the addition of menthol with nicotine altered biotransformation of nicotine to cotinine; (3) and to assess similar parameters in an in vivo mouse model. The major findings of this study include: (1) mice appear to metabolize nicotine, over time, in a manner similar to humans; (2) menthol decreased cotinine production, over time, after a single dose in mice; and (3) menthol increased cotinine production, over time, after repeated doses, in mice.
Relationships of Benthic Macroinvertebrate Community Structure with Land-use, Habitat, In-stream Water Chemistry, Depositional Sediment Biofilm Fatty Acids, and Surfactants in the Effluent Dominated Texas Trinity River
The Trinity River is an urbanized, effluent-dominated river, and is heavily relied upon for drinking water. The benthic macroinvertebrate community has been monitored for over 20 years, with the focus of this dissertation on three studies (1987-88, 2005, and 2011). Water quality improvement following dechlorination resulted in increased benthic metrics. Overall habitat quality, in-stream cover, surface water total organic carbon, sediment total organic carbon, near-field urban land-use, near-field forested land-use, surface water surfactant toxic units, and depositional sediment biofilm fatty acids all have statistically significant relationships with benthic macroinvertebrate metrics. These relationships are better defined with increased taxonomic resolution at the genus/species level for all benthic taxa, including Chironomidae and Oligochaeta. It is recommend that benthic identifications for state and city water quality assessments be done at the genus/species level. A novel method for quantifying depositional sediment biofilm fatty acids has been produced and tested in this dissertation. Benthic metrics are directly related to fatty acid profiles, with several essential fatty acids found only at upstream sites.
Effects of Airway Pressure, Hypercapnia, and Hypoxia on Pulmonary Vagal Afferents in the Alligator (Alligator Misssissippiensis)
The American alligator (Alligator mississippiensis) is an aquatic diving reptile with a periodic breathing pattern. Previous work has identified pulmonary stretch receptors (PSR), both rapidly- and slowly-adapting, and intrapulmonary chemoreceptors (IPCs) that modulate breathing patterns in alligators. The purpose of the present study was to identify the effects of prolonged lung inflation and deflation (simulated dives) on PSR and/or IPC firing characteristics in the alligator. The effects of airway pressure, hypercapnia, and hypoxia on dynamic and static responses of pulmonary stretch receptors (PSR) were studied in juvenile alligators (mean mass = 246 g) at 24°C. Receptor activity appeared to be a mixture of slowly-adapting PSRs (SARs) and rapidly-adapting PSRs (RARs) with varying thresholds and degrees of adaptation, but no CO2 sensitivity. Dives were simulated in order to character receptor activity before, during, and after prolonged periods of lung inflation and deflation. Some stretch receptors showed a change in dynamic response, exhibiting inhibition for several breaths after 1 min of lung inflation, but were unaffected by prolonged deflation. For SAR, the post-dive inhibition was inhibited by CO2 and hypoxia alone. These airway stretch receptors may be involved in recovery of breathing patterns and lung volume during pre- and post-diving behavior and apneic periods in diving reptiles. These results suggest that inhibition of PSR firing following prolonged inflation may promote post-dive ventilation in alligators.
Population Dynamics of Zebra Mussels (Dreissena Polymorpha) in a North Texas Reservoir: Implications for Invasions in the Southern United States
This dissertation has two main objectives: first, quantify the effects of environmental conditions on spatio-temporal spawning and larval dynamics of zebra mussels (Dreissena polymorpha [Pallas 1771]) in Lake Texoma, and second, quantify the effects of environmental conditions on survival, growth, and reproduction of young of the year (YOY) juvenile zebra mussels. These biological responses directly influence population establishment success and invasive spread dynamics. Reproductive output of the zebra mussel population in Lake Texoma was significantly related to water temperature and lake elevation. Annual maximum larval (veliger) density decreased significantly indicating a population crash, which was likely caused by thermal stress and variability of lake elevation. In 2011, temperatures peaked at 34.3°C and lake elevation decreased to the lowest level recorded during the previous 18 years, which desiccated a substantial number of settled mussels in littoral zones. Estimated mean date of first spawn in Lake Texoma was observed approximately 1.5 months earlier than in Lake Erie, and peak veliger densities were observed two months earlier. Veligers were observed in the deepest oxygenated water after lake stratification. During a 69-day in situ experiment during summer in Lake Texoma, age-specific mortality of zebra mussels was generally high until temperatures decreased to approximately 28°C, which was observed after lake turnover in late summer. No study organism died after temperatures decreased to less than 26°C, which indicates individuals that survive high summer temperatures are likely to persist into autumn/winter. Shell length growth and soft tissue growth rates were related to temperature and chlorophyll-a concentration, respectively. Growth rates of study organisms were among the highest ever reported for D. polymorpha. Water temperature and body size influenced reproduction of YOY zebra mussels in Lake Texoma. Fecundity of females were positively related to temperature; however, sperm production was negatively related to temperature, which indicates males could be more sensitive to physiologically-stressful conditions than females and could perform better in cooler waters. YOY mussels spawned up to approximately 40,000 eggs and 3.47E+08 sperm after a single-summer growing season. Reproductive effort and reproductive mass were independent of sex. YOY individuals from each study site (n = 5) were able to spawn viable gametes capable of sperm binding and egg cleavage, which provides the first evidence that YOY zebra mussels can successfully reproduce. Individual mortality of zebra mussels will likely be high in warm waters and intermittent, extreme droughts, which are observed more frequently at lower latitudes, can significantly reduce population sizes. However, rapid growth and single-season maturation can decrease generation times and could facilitate establishment and spread of zebra mussels in warm-water environments in the southern United States.
Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene
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Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.
Modeling the Effects of Chronic Toxicity of Pharmaceutical Chemicals on the Life History Strategies of Ceriodaphnia Dubia: a Multigenerational Study
Trace quantities of pharmaceuticals (including carbamazepine and sertraline) are continuously discharged into the environment, which causes concern among scientists and regulators regarding their potential long-term impacts on aquatic ecosystems. These compounds and their metabolites are continuously interacting with the orgranisms in various life stages, and may differentially influence development of embryo, larvae, juvenile, and adult stages. To fully understand the potential ecological risks of two candidate pharmaceutical chemicals (carbamazepine (CBZ) and sertraline (SERT)) exposure on survival, growth and reproduction of Ceriodaphnia dubia in three sucessive generations under static renewal toxicity test, a multigenerational approach was taken. Results indicate that SERT exposure showed higher sensitivity to chronic exposure to C. dubia growth and reproduction than CBZ exposure. The lowest concentration to affect fecundity and growth was at 50 µg L-1 SERT in the first two generations. These parameters become more sensitive during the third generation where the LOEC was 4.8 µg L-1. The effective concentrations (EC50) for the number of offspring per female, offspring body size, and dry weight were 17.2, 21.2, and 26.2 µg SERT L-1, respectively. Endpoints measured in this study demonstrate that chronic exposure of C. dubia to SERT leads to effects that occur at concentrations an order of magnitude higher than predicted environmental concentrations indicating potential transgenerationals effects. Additionally, a process-based dynamic energy budget (DEB) model is implemented to predict the simulated effects of chronic toxicity of SERT and CBZ to C. dubia individual behavior at laboratory condition. The model‘s output indicates the ecotoxicological mode of action of SERT exposure, which acts on feeding or assimilation with an effect that rapidly saturates at higher concentrations. Offspring size decreases with the toxic effects on feeding, and offspring number is thus less affected than total investment in reproduction. Consequently, CBZ affects direclty in reproduction which are captured by DEBtox model as increased embryonic hazard and reproduction cost as well as growth and maintenance costs. Furthermore, stress factor linearly increased not only with increasing chemical concentrations but also with exposure time. The DEBtox model establishes a cumulative life history consequence of multigenerational exposure to CBZ and SERT. This approach provides a tool to which to understand the effect of chemical to the individual organism and predict the population level effects in ecological risk assessment of the emerging contaminants.
Investigation of the Pharmacokinetics of Diazepam in Juvenile Channel Catfish (Ictalurus Punctatus)
The presence of pharmaceuticals in the environment is becoming an increasing regulatory and scientific concern. Thus, the metabolic profile and bioconcentration potential of diazepam, a model benzodiazepine, were examined, as well as effects on the endocrine system in channel catfish. Through the use of specific and non-specific cytochrome P450 (CYP450) inhibitors, it was determined that CYP3A-like enzymes may play a role in the biotransformation of diazepam into temazepam; however, the isoform(s) required for the formation of other metabolites is still unknown. Overall, only around 7-8% of diazepam is biotransformed into two known metabolites. Due to the lack of inherent metabolism of diazepam in channel catfish, further analysis was conducted to determine the tissue-specific bioconcentration potential of diazepam in catfish. Various tissues were analyzed for the presence of diazepam as well as metabolites and bioconcentration factors (BCF) were calculated, which were all well below regulatory threshold values (> 2000). Additionally, modulation of the endocrine system by diazepam was examined by measuring steroid hormone concentrations and analyzing mRNA expression of selected steroidogenic enzymes and receptors. Two steroidogenic enzymes were modulated following diazepam exposure, indicating potential endocrine disrupting properties of diazepam. Together, these data suggest that diazepam exhibits low metabolic transformation rates in channel catfish, which may lead to accumulation of benzodiazepine compounds that may negatively affect the endocrine system. However, further studies should be aimed at identifying other steroidogenic enzymes and/or receptors that may be modulated following diazepam exposure.