Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function …

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.

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 …

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 …

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) …

The goals of this dissertation work were to assess the bioaccumulation potential of carbamazepine and amiodarone, two widely used ionizable pharmaceutical compounds that possess mid-range and high LogD values, respectively, and to evaluate alternative methods to assess chemical accumulation in bluntnose minnows, catfish, and tilapia. Results indicated that carbamazepine does not appreciably bioaccumulate in fish tissue with BCFk and BAF carbamazepine values < 10. Amiodarone, however, with a log D of 5.87 at pH 7.4, accumulated in fish tissues with kinetic BCF values <2,400. Collectively, the data suggest that full and abbreviated laboratory-derived BCFs, BCFMs derived from S9 loss-of-parent assays, as well as field BAF values are similar for each of the two drugs. In summary, the results from this dissertation indicated: 1) The reduced design BCF test is a good estimate for the traditional OECD 305 test. 2) In vitro S9 metabolism assays provide comparable BCF estimates to the OECD 305 test. 3) Metabolism may play a large role in the accumulation of drugs in fish. 4) Reduced BCF tests and in vitro assays are cost effective and can reduce vertebrate testing.

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 …

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 …

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, …

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 …

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases …

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.

Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of …