Cannabinoid (CB) receptors have been found in most vertebrates that have been studied. The location of various CB receptors in the body and brain are known, but their physiological functions are not fully understood. The effects CBs have on the cardiovascular system have been of growing interest in recent years. Increasing reports from emergency departments and law enforcement agencies detail acute cardiovascular and psychological effects from synthetic CB intoxication, such as JWH-018. This major health concern is substantiated by governmental agencies like the CDC and NIDA. This pilot study investigates the acute toxic effects of the synthetic CB, JWH-018, on the cardiovascular and neuroendocrine systems in Ictalurus punctatus (channel catfish). Research in organisms besides the traditional mammal models can provide new insights into CB function and physiology. Ictalurus punctatus lend multiple benefits as a model organism that permits researchers to investigate in vivo effects of both cardiovascular and neuroendocrine systems without much influence from traditional sampling methods, and further more provide ample size and tissue to perform specific cardiovascular experiments. Multiple methods were used to assess cardiovascular function and sympathetic nervous system activation. Two different doses, low (500 µg/kg) and high 1,500 µg/kg, of JWH-018 were evaluated in the study. Delivery of JWH-018, via dorsal aorta cannulation, was administered to channel catfish in order to measure cardiovascular functions and sample blood. Plasma levels of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) biomarkers; ACTH, cortisol, epinephrine, and norepinephrine, were measured using ELISAs. Myocardial and neural tissue was collected after the exposures for rt-PCR analysis on β2 adrenergic and glucocorticoid receptor density change. Acute exposure of JWH-018 in undisturbed channel catfish yielded several findings: (1) High dose of JWH-018 was responsible for cardio depressor effects in catfish with a tendency to produce tachycardia, (2) rt-PCR results showed a 2.7 fold increase of glucocorticoid receptor mRNA density …
Oxygen availability is critical for survival for most organisms. The nematode, C. elegans, has been useful for studying genetic regulation of anoxia tolerance due to the oxygen deprivation response mechanisms shared with other metazoans. Studies examining long-term anoxia (72h, LTA) tolerance have only been conducted at adult day 1. To investigate the effect of aging on anoxia tolerance wild-type and mutant strains were exposed to LTA between adult day 1 and day 9. Wild-type isolates and daf-16(mu86) (FOXO transcription factor regulated by insulin-signaling) and aak-2(gt33) (catalytic subunit of AMP-activated protein kinase) strains were anoxia sensitive at day 1 and displayed increased LTA tolerance with aging correlated with reproductive senescence followed by a decline in survivorhsip through day 9. The daf-2(e1370) (insulin receptor homologue of C. elegans), glp-1(e2141) (a lin-12/Notch receptor) and fog-2(q71) (required for spermatogenesis) strains were LTA-tolerant through day 5. I conclude that aging influences LTA-tolerance in a strain- and age-dependent manner. In addition to being LTA-tolerant the daf-2(e1370) and glp-1(e2141) strains have a longevity phenotype that is suppressed by loss of kri-1 or daf-12. While loss of kri-1 did not suppress the LTA-tolerant phenotype of glp-1(e2141) at day 1 the portion of impaired survivors increased at day 3 and by day 5 tolerance was suppressed. Similarly, when exposed to 4 days of anoxia the glp-1(e2141);daf-12(rh41rh611) double mutant had a reduced survivor rate at all ages analyzed compared to glp-1(e2141) controls. To better understand formation of an anoxia-tolerant physiology I exposed adults to one or more 24h bouts. Recurrent bouts increased LTA tolerance in wild-type hermaphrodites in a dose-dependent manner. Bout-treated daf-16(mu86) animals had increased survival rate compared to controls yet maximum survival remained below age-matched wild-type. Anoxia bouts decreased LTA-tolerance in aak-2(gt33) mutants, indicating the requirement for ATP regulation in establishing an LTA-tolerant phenotype. These data support the …
The purpose of my research work was to assess a variety of Northern Thailand plants tissue extracts for antibacterial and anti-platelet aggregation activity. The Minimum Inhibitory Concentration assay method was used to assess antimicrobial activity of plant extracts, while the Zebrafish Platelet Aggregation Assay and the in vitro Whole Human Blood Impedance Aggregation Assay were used to study anti-platelet activity. Forty one plant extracts harvested from the tissues of 26 plants collected from Northern Thailand were assessed. Thirty-four plant extracts were found to have antibacterial activity against the Gram positive bacteria Staphylococcus aureus and/or Bacillus subtilis, while six plant extracts demonstrated activity against the Gram negative bacterium, Escherichia coli. Thirteen plant extracts exhibited anti-platelet aggregation activity better than the positive control. Two crude plant extracts, twigs from Garcinia sp. and twigs from Goniothalamus chilensis were selected for fractionation. Five of the 12 fractions showed anti-platelet activity. Four fractions (two from each plant extract) were selected for further sub-fractionation. Fourteen of 35 sub-fractions were selected for further testing of anti-platelet aggregation activity with 12 sub-fractions demonstrating positive antiplatelet activity. Positive sub-fractions were analyzed using liquid chromatography-mass spectrometry to determine their chemical properties. Three compounds that possessed anti-platelet activity were characterized by nuclear magnetic resonance. The compounds, all isolated from Garcinia sp., were identified as cambogin, isoxanthochymol and guttiferone F.
The Cape Horn Biosphere Reserve, within the remote Sub-Antarctic ecoregion is a reservoir of expressions of biological and cultural diversity. Although it is considered one of 24 wilderness areas remaining in the world, it is not free from local and global threats, such as invasive species, and climate change. Field biologists and philosophers associated to the Sub-Antarctic Biocultural Conservation Program and the Omora Ethnobotanical Park, have worked to describe the region’s biocultural diversity, linking ecological and philosophical research into education, ecotourism, and conservation, through a methodology called field environmental philosophy (FEP), which integrates ecological sciences and environmental ethics through a 4-step cycle consisting of: 1) interdisciplinary research; 2) composition of metaphors; 3) design of field activities with an ecological and ethical orientation; and 4) implementation of in situ conservation areas. In this context, the purposes of this dissertation were to: 1) provide a comprehensive review of publications regarding the conservation status of aquatic and terrestrial insects at a global scale and with an emphasis in southern South America; 2) study the distribution of benthic macroinvertebrates through the sharp altitudinal gradient of the Róbalo River watershed; 3) describe the life histories of Gigantodax sp (Simuliidae: Diptera) and Meridialaris chiloeense (Leptophlebiidae: Ephemeroptera) in the Róbalo River and to assess the potential effects of climate change on their phenology; and 4) to apply FEP methodology in order to better understand and communicate the intrinsic and instrumental values of freshwater invertebrates in the Cape Horn Biosphere Reserve.
Through the use of natural history records, published literature, and personal sampling (2011-2016) a total of 454 caddisfly species represented by 24 families and 93 genera were documented from the south-central United States. Two Hydroptilidae species were collected during the 2011-2016 collection efforts that are new to the region: Hydroptilia scheringi and Mayatrichia tuscaloosa. Eightteen species are endemic and 30 are considered species of concern by either federal or state agencies. The majority of each of these groups is Hydroptilidae, or microcaddisflies. Trichoptera community structure, by minimum number of species, was analysed in conjunction with large-scale geographical factors to determine which factor illustrated caddisfly community structure across the region. Physiographic provinces compared to other geographic factors analyzed best-represented caddisfly communities with a minimum of 10 or more species. Statistically, Hydrologic Unit Code 4 (HUC 4) was the most significant geographical factor but low number of samples representing this variable rendered it less representative of caddisfly community structure for the study area.
The northern bobwhite (Colinus virginianus) population decline is a severe, rangewide phenomenon beginning >150 years ago and continuing today. In this investigation, I: 1. document the timeline of bobwhite population decline and unintended genetic consequences of attempted remedies, 2) develop a model useful for predicting possible locations of potentially sustainable bobwhite populations in semiarid rangeland in Texas and Oklahoma, and 3) examine the relationship between population monitoring data and meteorological factors. While breeding season call counts of male bobwhite have been used for >70 years to provide estimates of fall populations for hunting, most studies of call counts have focused on mathematics and statistical accuracy of the count, largely overlooking the influence of meteorological factors on call counts. Here, I present the results of >4,400 individual point counts and examine their relationship with meteorological variables recorded at each stop. Humidity was positively correlated with the number of birds recorded (ρ = 0.275, p < 0.001) and temperature was negatively correlated (ρ = -0.252, p < 0.001). The number of birds recorded was significantly higher in wet years than in drought years. There was no significant correlation between wind velocity and number of birds recorded. These results suggest that, while weather does influence call counts and efforts should be made to record meteorological conditions when collecting call count data, the influence of weather may not easily factor into the analysis. These results also provide another line of evidence for decreased breeding behavior during high temperatures. With the increased focus on bobwhite habitat management on a regional scale, there is a need for reliable methods to identify potential bobwhite habitat. To identify bobwhite habitat in semiarid rangeland, I performed classification of LANDSAT scenes of Clay County, Texas from July and December 2015. Stands of mature little bluestem provide excellent bobwhite nesting cover …
The consequences of early embryonic insults and how they affect subsequent life reflects the emerging concept of "fetal programming". The aim of this project is to study the effects of embryonic insults as they subsequently manifest themselves in adults, with emphasis on the heart and vasculature. My experiments establish that fetal programming operates on the bobwhite quail inducing similar changes as those observed in mammalians and other birds. The quail's fast development provides reliable data in a short period of time than other avian models (e.g. domestic chicken). Data on quail showed a correlation between egg mass and hatchling mass; where small eggs produce small hatchlings but a high mortality made it impractical as a stressor for this study. Hypoxia was used as a stressor during embryonic incubation, where it induced a low hatching weight in quail that was not observable in adult birds. Morphological measurements demonstrated an increased ventricular collagen content and reduced ventricular lumen in birds in adults incubated in hypoxia consistent with hypertension. The hematological analyzes showed few differences indicating organ remodeling instead of hematopoietic compensation. The assessment of vascular reactivity pointed out an impaired endothelium dependent relaxation commonly associated to hypertension in birds and mammals. Fetal programming could be a widespread response to an adverse prenatal environment in endotherms and the resulting data from this work contributes to our understanding of fetal programming in vertebrates and its long term consequences.
I used the C. elegans genetic model to examine the role of ceramide biosynthesis (sphingolipid pathway) and iron regulation and found that each process impacts germline development and function. Using a sphingolipid specific antibody mAb15B4, I found that sphingolipids are associated with germ granules (P granules) within C. elegans and zebrafish; thus, suggesting conservation of macromolecules associated with germ granules. Phenotype analysis of ceramide biosynthesis mutants in C. elegans revealed that this pathway is essential for normal germline function in the aging adult hermaphrodite; specifically, precocious germline senescence was observed. Furthermore, I found that disruption of ceramide biosynthesis, via the hyl-2 deletion mutation, negatively impacts mAb15B4 localization at the P granules. Through genetic suppression analysis, I determined that insulin signaling and lipid biosynthesis can modulate the mAb15B4 localization to P granules. Additional, phenotype analysis showed that ceramide biosynthesis dysfunction decreased fecundity, and led to germline structure defects and uterine tumors. Through suppression analysis, I determined that modulation of the insulin signaling pathway suppressed the precocious germline senescence due to ceramide biosynthesis dysfunction. Since the presence of uterine tumors is associated with reproductive senescence I concluded that ceramide biosynthesis has a role in germline maintenance in the aging of the germline (germline senescence). The other important fate of a germ cell is programmed cell death. Apoptosis, which occurs through a highly conserved molecular pathway, is a normal component of growth and homeostatic processes. I used C. elegans to gain a greater understanding of the cisd gene function. The C. elegans genome has three previously uncharacterized cisd genes which code for CISD-1 (homology to vertebrate mitoNEET/CISD1 and NAF-1/CISD2) and CISD-3.1 and CISD-3.2 (homology to vertebrate Miner2/CISD3). I determined that independent disruption of the cisd genes resulted in a significant increase in the number of cell corpses within the adult hermaphrodite germline. Genetic …
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 …
Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive seasonally desiccating ponds due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause II, which precedes the onset of drought. While in this arrested state embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25ºC. Furthermore, when raised at 30ºC, embryos escape the entrance to diapause II and go on to develop directly. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. In this study I have developed methods to analyze changes in histone modifications in the context of diapause II. Histone modifications were chosen due to their extreme conservation and well characterized role as modulators of gene expression in other systems. Results utilizing adapted immunobased assays show significant changes in the global amount of H3S10P, H3K27me and H3K4me, as the embryos progress from early embryogenesis through the exit of diapause. Additionally, it is revealed that there exists a degree of phenotypic plasticity with regards to the entrance into diapause II which is modulated by the environment (severe hypoxia 0.1% O2). This work builds a foundation for future histone modification studies and contributes the development of several tools to the field. This study contributes to a greater molecular understanding of the cue(s) which influence the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.
The primary objective of this work was to determine the tissue-specific bioconcentration factors (BCFs) of the selected pharmaceuticals: norethindrone (NET), ibuprofen (IBU), verapamil (VER), clozapine (CLZ) and fenofibrate (FFB) in two freshwater fishes: fathead minnow and channel catfish. BCF tests on fathead followed the standard OECD 42-day test while a 14-day abridged test design was used in catfish exposures. Additional objectives included a) comparing the measured BCFs to the US EPA's BCFWIN model predicted values, b) comparing the BCF results from the standard and reduced tests, and c) prediction of chronic risk of the pharmaceuticals in fish using the human therapeutic plasma concentrations. Each test included uptake and depuration phases to measure tissue-specific kinetic BCFs. The results indicated that all the pharmaceuticals, except IBU, have the potential for accumulation in fish. Estimated BCFs for NET, VER and FFB may not be significant in view of the current regulatory trigger level (BCF ≥ 2000); however, CLZ's BCF in the liver had approached the criterion level. Significant differences were noticed in the tissue-specific uptake levels of the pharmaceuticals with the following general trend: (liver/kidney) > (gill/brain) > (heart/muscle) > plasma. IBU uptake was highest in the plasma. When compared to the measured BCFs, predicted values for NET, IBU, VER and FFB were slightly overestimated but did not differ largely. However, the measured BCF of CLZ in the liver was approximately two-orders of magnitude higher than the predicted level. The tissue-BCFs for the two species were not widely different indicating the potential usefulness of the reduced BCF test. Comparison of fish and human plasma levels indicated that NET, CLZ and VER have the potential to cause chronic effects in fish.
Metagenomics is the study of the totality of the complete genetic elements discovered from a defined environment. Different from traditional microbiology study, which only analyzes a small percent of microbes that could survive in laboratory, metagenomics allows researchers to get entire genetic information from all the samples in the communities. So metagenomics enables understanding of the target environments and the hidden relationships between bacteria and diseases. In order to efficiently analyze the metagenomics data, cutting-edge technologies for analyzing the relationships among microbes and communities are required. To overcome the challenges brought by rapid growth in metagenomics datasets, advances in novel methodologies for interpreting metagenomics data are clearly needed. The first two chapters of this dissertation summarize and compare the widely-used methods in metagenomics and integrate these methods into pipelines. Properly analyzing metagenomics data requires a variety of bioinformatcis and statistical approaches to deal with different situations. The raw reads from sequencing centers need to be processed and denoised by several steps and then be further interpreted by ecological and statistical analysis. So understanding these algorithms and combining different approaches could potentially reduce the influence of noises and biases at different steps. And an efficient and accurate pipeline is important to robustly decipher the differences and functionality of bacteria in communities. Traditional statistical analysis and machine learning algorithms have their limitations on analyzing metagenomics data. Thus, rest three chapters describe a new phylogeny based machine learning and feature selection algorithm to overcome these problems. The new method outperforms traditional algorithms and can provide more robust candidate microbes for further analysis. With the frowing sample size, deep neural network could potentially describe more complicated characteristic of data and thus improve model accuracy. So a deep learning framework is designed on top of the shallow learning algorithm stated above in order to further …
This dissertation has two overall objectives: first, to demonstrate the utility of paleozoological data for ongoing and future mussel-conservation efforts in Texas and second, to evaluate whether simple measures of habitat (e.g., water depth, velocity and particle size) are important for demonstrating the within-habitat spatial separation of mussels. Although these topics may seem disparate, both are important for increasing our understanding of unionid ecology and biogeography. Chapters 1 through 3 examine the use of paleozoological data for mussel conservation. Although these types of data are not new they have rarely been used in mussel conservation efforts within Texas. This is unfortunate because paleozoological data can provide an excellent record of the mussel fauna prior to wide-scale modern impacts and in areas where historical survey data are lacking. Chapter 4 examines whether assessments of microhabitat for mussels using simple measures of habitat (e.g., water velocity, depth and particle size) are useful. Recent studies have suggested that these measures do not explain the mussel distribution in flowing streams. If this is correct, instream flow studies using this approach need to be revised. Results of Chapter 4 indicate that mussels in the lower Brazos River basin are constrained in distribution by the availability of heterogenous substrate. Appendix A, details the first account of a living population of Truncilla macrodon, which is a candidate species for the Endangered Species Act (ESA). The population was found while conducting mussel instream flow studies in the lower Brazos River basin.
Extreme climatic events such as droughts are known to eliminate aquatic biota and alter community structure and function. Perennial headwater springs provide important drought refugia to benthic macroinvertebrates and an important source of colonists via drift or aerial adults to intermittent streams post-drought. During a supra-seasonal drought in North-central Texas summer and fall 2006, benthic macroinvertebrates from persistent groundwater-dependent macrohabitats of varying hydrological connectivity and riparian shading were studied: perennial riffles, connected pools, shaded disconnected pools, and full sun disconnected pools. Riffles were a distinct habitat with significantly higher taxa richness, proportion of lotic taxa, diversity and evenness than other macrohabitats. Macrohabitats were found to be important refugia for 106 benthic macroinvertebrates and 4 microcrustacean taxa. Throughout the extreme drought, perennially flowing habitats were refugia to 19 taxa (17.9% total taxa) not collected in disconnected pools. Shaded disconnected pools contained lotic taxa not previously known to be able to complete their lifecycles in lentic habitats, emphasizing the importance of groundwater effluent and shading. With the resumption of flow at a downstream intermittent site of Ash Creek in mid-October 2006, an annual recolonization study was conducted comparing the perennial headwaters’ benthic macroinvertebrate taxa richness, densities and community ecology with the downstream intermittent site. The headwaters supported higher mean taxa richness than the intermittent site over the duration of the study (ANOVA P < 0.001). However, the unexpected result of overall decreasing taxa richness at the perennial headwater site from August 2006 to April 2008 appears to reflect lag effects of the supra-seasonal drought combined with effects of multiple spates of 2007, which are factors confounding the point of recovery for taxa richness. Recovery of taxa richness at the intermittent site took 9 months compared to 1 to 2 months reported in other arid and semi-arid streams in the United States recovering …
The purpose of this project was to further elucidate the effects post-exercise alcohol ingestion. This project had many novel aspects including using a resistance exercise (RE) only exercise design and the inclusion of women. To our knowledge, we are the first to investigate the effect of post-RE alcohol ingestion in women. In the first chapter of this project, information on the prevalence of alcohol use and the importance of skeletal muscle as a dynamic and metabolic tissue was provided. In chapter two, the effects of post-RE alcohol ingestion in men and women are detailed. The major findings of this study was that although RE elicited similar mTORC1 signaling both in men and in women, alcohol ingestion appeared to only attenuate RE-induced phosphorylation of the mTORC1 signaling pathway in men. The third chapter focused on examining the effects of post-RE alcohol ingestion on acute testosterone bioavailability. The primary findings of this study was that alcohol substantially elevated serum total and free testosterone concentrations during recovery from a bout of resistance exercise. The fourth chapter detailed factors that contribute to bone density in men. The major findings of this study was that young adult male long-distance runners who participated in resistance training at least once per week had greater bone mineral density than their non-resistance trained and non-exercise trained peers.
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.
Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.
Promising results from the use of problem-based learning (PBL) as a teaching method in medical programs have encouraged many institutions to incorporate PBL into their curricula. This study investigates how applying hybrid-PBL (H-PBL) in a microbiology laboratory impacts students' higher-order thinking as compared to applying a lecture-based pedagogy. The experimental design compared the learning outcomes of two groups of students: the control group and the H-PBL group, for whom PBL cases comprised 30% of the curriculum. Both groups were taught basic skills for the microbiology lab by the same instructor. Using the traditional teaching style for the control group, the instructor offered each student what they needed for their experiments. The H-PBL group practiced experimental design, data analysis, theory proposal, and created research questions by using six study cases that were closely linked to the area of study. The outcome was measured using a pre- and post- assessment consisting of 24 questions that was designed by following Bloom's taxonomy of learning levels. A one-way ANOVA was used to analyze the data. The results showed that for the first three levels of Bloom's taxonomy— knowledge, comprehension, and application—there were no statistically significant differences between the H-PBL and control group gain scores as determined by a one-way ANOVA. For the knowledge level, f (1, 78) = .232, and p = .632; for the comprehension level, f (1, 78) = .004, and p = .951; and for the application level f (1, 78) =. 028, and p =.863. On the other hand, the gain scores for the three higher levels—analysis, evaluation, and creativity—improved for the H-PBL group. The analysis level showed statistically significant differences, with f (1, 78) = 4.012, and p = .049. Also, there were statistically significant differences in students' performance at the evaluation level, with f (1, 78) = 11.495, …
Glial cells maintain homeostasis that is essential to neuronal function. Injury to the nervous system leads to the activation and proliferation of glial cells and pericytes, which helps to wall off the damaged region and restore homeostatic conditions. Sonic hedgehog is a mitogen which is implicated in injury-induced proliferation of glial cells and pericytes. The mitogenic effects of sonic hedgehog require primary cilia, but the few reports on glial or pericyte primary cilia do not agree about their abundance and did not address effects of injury on these cilia. Primary cilia are microtubule-based organelles that arise from the centrosome and are retracted before cells divide. Depending on cell type, proteins concentrated in cilia can transduce several mitotic, chemosensory, or mechanosensory stimuli. The present study investigated effects of stab wound injury on the incidence and length of glial and pericyte primary cilia in the area adjacent to the injury core. Astrocytes, polydendrocytes and pericytes were classified by immunohistochemistry based on cell-type markers. In normal adult mice, Arl13b immunoreactive primary cilia were present in a majority of each cell type examined: astrocytes, 98±2%; polydendrocytes, 87±6%; and pericytes, 79±13% (mean ± SEM). Three days post-injury, cilium incidence decreased by 24% in astrocytes (p< 0.008) and 41% in polydendrocytes (p< 0.002), but there was no significant effect in pericytes. Polydendrocytes labeled with the cell cycle marker Ki67 were less likely to have cilia compared to resting, Ki67- polydendrocytes. Considering post-injury rates of proliferation for astrocytes and polydendrocytes, it appears that resorption of cilia due to cell cycle entry may account for much of the loss of cilia in polydendrocytes but was not sufficient to account for the loss of cilia in astrocytes. Under normal conditions, astrocytes rarely divide, and they maintain non-overlapping territories. However, three days after injury, there was a 7-fold increase in …
The Deepwater Horizon oil spill released millions of barrels of oil into the Gulf of Mexico, impacting economically and ecologically important fishes. Polycyclic aromatic hydrocarbons (PAHs) present in the oil have been shown to cause developmental impairments in early life stage fishes, such as morphological and behavioral changes related to eye formation and visual processing following PAH exposure. Prior research reported reduced eye growth in open water, pelagic species, as well as reduced photoreceptor-specific transcription factors associated with eye development following exposure to crude oil. Though changes in transcriptomic-level pathways associated with vision and visual processing have been reported, it has yet to be determined how these changes relate to physiological or behavioral-level effects in fish. Therefore, the present studies evaluated the effect of weathered crude oil on eye development and visual function in mahi-mahi, red drum, sheepshead minnow, and zebrafish larvae. Fish were assessed through several visually-mediated behavioral assays, analyzed histologically and immunohistologically, along with subsequent transcriptomic analyses and associated gene expression changes. Larvae exposed to crude oil experienced significantly reduced abilities to exhibit optomotor or optokinetic responses relative to controls, with associated reductions in retinal development. Furthermore, genes associated with eye development and phototransduction were downregulated, with subsequent decreases in the immunofluorescence of neurological connections within the retina and a choroid-specific increase in apoptotic activity. We related oil-induced transcriptomic-level effects to morphological, physiological, and behavioral-level impairments in larval teleost fishes.
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.
Primary cilia are ubiquitous sensory organelles found on most cell types including cells of the dorsal root ganglia (DRG). The DRG are groups of peripheral neurons that relay sensory information from the periphery to the CNS. Other cell types in the DRG include a type of glial cell, the satellite glial cells (SGCs). The SGCs surround the DRG neurons and, with the neurons, form functional sensory units. Currently are no reports describing the numbers of DRG cells that have cilia. We found that 26% of the SGCs had primary cilia. The incidence of cilia on neurons varied with neuron size, a property that roughly correlates with physiological characteristics. We found that 29% of the small, 16% of the medium and 5% of the large neurons had primary cilia. Primary cilia have been shown to have a role in cell proliferation in a variety of cell types. In some of the cells the cilia mediate the proliferative effects of Sonic hedgehog (Shh). In the CNS, Shh signaling through primary cilia affects proliferation during development as well as following injury, but no studies have looked at this function in the PNS. The SGCs and neurons of the DRG undergo complex changes following peripheral nerve injury such as axotomy. One marked change seen after axotomy is SGC proliferation and at later stages, neuronal death. We found that following axotomy there is a significant increase in the percentage of SGCs with primary cilia. We also found a significant increase in the percentage of medium-sized neurons with primary cilia. In other experiments we tested the idea that Shh plays a role in SGC proliferation. When Shh signaling was blocked following axotomy we found decreased proliferation of SGCs. This is the first report of a change in the percentage of cells with cilia following injury in …
Pharmaceuticals have routinely been detected in the environment resulting in a growing concern about whether these drugs could elicit effects on aquatic organisms. The concerns are centered on the highly conserved nature of mammalian therapeutic targets in fish. These pharmaceuticals are found at very low levels in the environment, which can result in sub-lethal effects in aquatic organisms. Therefore, 28 d early-life stage studies were conducted on six pharmaceuticals to assess their impacts on survival and growth fathead minnow larvae. Two pharmaceuticals tested, carbamazepine and fenofibrate, resulted in no alterations to survival and growth. However, amiodarone, clozapine, dexamethasone, and levonorgestrel (LNG) reduced survival at concentrations tested with LNG being the most potent at 462 ng/L. Survival was increased with amiodarone and clozapine; however LNG significantly decreased growth at 86 ng/L. Therefore, the most potent pharmaceutical tested was the synthetic progestin LNG with survival and growth impacts at concentrations less than 1 μg/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 d ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, and FSH were significantly down-regulated following 28 d exposure to both 16.3 and 86.9 ng/L LNG. Also, CYP19a expression was significantly down-regulated at 86.9 and 2392 ng/L LNG. Subsequently, a second study examined time periods that may be most sensitive (e.g., windows of sensitivity) for FHM larvae exposed to LNG. Larvae were exposed to a single concentration of LNG (i.e. LOECgrowth of 86.2 ng/L as determined in the 28 d ELS study) for different time periods starting with fertilized egg through 28 dph. Growth and mRNA expression of the four differentially expressed transcripts from the first study were measured. Regardless of the duration of exposure, LNG significantly decreased growth in fathead minnow larvae at day 28. For both 20β-HSD and CYP19a, …
This study determined the effect of hypoxia on air-breathing onset and physiological and morphological characters in larvae of the air breathing fishes Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently (12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization. Survival, onset of air breathing, wet body mass, O2, Pcrit were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet mass, and labyrinth organ respiratory surface of Betta, but did not affect these factors in Trichopodus. Hypoxic exposure increased O2 by 50-100% at each day throughout larval development in Betta, but had no effect on larval Trichopodus. Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%. Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1 to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects. These interspecific differences are likely due to ecophysiological differences: Betta is a non- obligatory air-breather after 36 dpf with a slow lifestyle reflected in its low metabolism, while Trichopodus is an obligatory air-breather past 32 dpf with an athletic fast lifestyle and accompanying high metabolism.
Postmortem interval (PMI) estimation is a necessary but often difficult task that must completed during a death investigation. The level of difficulty rises as time since death increases, especially with the case of skeletonized remains (long PMI). While challenging, a reliable PMI estimate may be of great importance for investigative direction and cost-savings (e.g. suspect identification, tailoring missing persons searches, non-forensic remains exclusion). Long PMI can be estimated by assessing changes in the organic content of bone (i.e. collagen), which degrades and is lost as the PMI lengthens. Visible-near infrared (VNIR) spectroscopy is one method that can be used for analyzing organic constituents, including proteins, in solid specimens. A 2013 preliminary investigation using a limited number of human cortical bone samples suggested that VNIR spectroscopy could provide a fast, reliable technique for assessing PMI in human skeletal remains. Clear separation was noted between "forensic" and "archaeological" specimen spectra within the near-infrared (NIR) bands. The goal of this research was to develop reliable multivariate classification models that could assign skeletal remains to appropriate PMI classes (e.g. "forensic" and "non-forensic"), based on NIR spectra collected from human cortical bone. Working with a large set of cortical samples (n=341), absorbance spectra were collected with an ASD/PANalytical LabSpec® 4 full range spectrometer. Sample spectra were then randomly assigned to training and test sets, where training set spectra were used to build internally cross-validated models in Camo Unscrambler® X 10.4; external validations of the models were then performed on test set spectra. Selected model algorithms included soft independent modeling of class analogy (SIMCA), linear discriminant analysis on principal components (LDA-PCA), and partial least squares discriminant analysis (PLSDA); an application of support vector machines on principal components (SVM-PCA) was attempted as well. Multivariate classification models were built using both raw and transformed spectra (standard normal variate, …
Cardiovascular disease (CVD) is the leading cause of death for people of most ethnicities on a global scale, and countless research efforts on the pathology of CVD has been well-characterized over the years. However, advancement in modern technologies, such as nanotechnology, has generated environmental and occupational health concerns within the scientific community. Current investigation of nanotoxicity calls into question the negative effects nanomaterials may invoke from their environmental, commercial, and therapeutic usage. As a result, further research is needed to investigate and characterize the toxicological implications associated with nanomaterial-exposure and CVD. We investigated the toxicity of multi-walled carbon nanotubes (MWCNT) and titanium dioxide (TiO2), which are two prominently used nanomaterials that have been previously linked to upregulation of inflammatory and atherogenic factors. However, the mechanistic pathways involved in these nanomaterials mediating detrimental effects on the heart and/or coronary vasculature have not yet been fully determined. Thus, we utilized two different routes of exposure in rodent models to assess alterations in proinflammatory and proatherogenic signaling pathways, which are represented in contrast throughout the dissertation. In our MWCNT study, we used C57Bl/6 mice exposed to MWCNTs (1 mg/m3) or filtered air (FA-Controls), via inhalation, for 6 hr/d for 14d. Conversely, intravenous TiO2 was administered to F344 male fisher rats, following 24h and 28d post-exposure to a single injection of TiO2-NPs (1 mg/kg), compared to control animals. MWCNT-exposed endpoints investigated the alterations in cholesterol transport, such as lectin-like oxidized low-density lipoprotein receptor (LOX)-1 and ATP-binding cassette transporter (ABCA)-1, inflammatory markers [tumor necrosis factor (TNF)-α], interleukin (IL)-1β/IL-6, nuclear-factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signaling factors involved in activation of the pathway, as well as intracellular/vascular adhesion molecule(s) (VCAM-1, ICAM-1), and miRNAs (miR-221/-21/-1), associated with CVD, were analyzed in cardiac tissue and coronary vasculature. Cardiac fibrotic deposition, matrix-metalloproteinases (MMP)-2/9, and reactive oxygen species …
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.
Horizontal gene transfer enables acquisition and dissemination of novel traits including antibiotic resistance and virulence among bacteria. Frequently such traits are gained through the acquisition of clusters of functionally related genes, often referred to as genomic islands (GIs). Quantifying horizontal flow of GIs and assessing their contributions to the emergence and evolution of novel metabolic traits in bacterial organisms are central to understanding the evolution of bacteria in general and the evolution of pathogenicity and antibiotic resistance in particular, a focus of this dissertation study. Methods for GI detection have also evolved with advances in sequencing and bioinformatics, however, comprehensive assessment of these methods has been lacking. This motivated us to assess the performance of current methods for identifying islands on broad datasets of well-characterized bacterial genomes and synthetic genomes, and leverage this information to develop a novel approach that circumvents the limitations of the current state-of-the-art in GI detection. The main findings from our assessment studies were 1) the methods have complementary strengths, 2) a gene-clustering method utilizing codon usage bias as the discriminant criterion, namely, JS-CB, is most efficient in localizing genomic islands, specifically the well-studied SCCmec resistance island in methicillin resistant Staphylococcus aureus (MRSA) genomes, and 3) in general, the bottom up, gene by gene analysis methods, are inherently limited in their ability to decipher large structures such as GIs as single entities within bacterial genomes. We adapted a top-down approach based on recursive segmentation and agglomerative clustering and developed a GI prediction tool, GEMINI, which combined compositional features with segment context information to localize GIs in the Liverpool epidemic strain of Pseudomonas aeruginosa. Application of GEMINI to the genome of P. aeruginosa LESB58 demonstrated its ability to delineate experimentally verified GIs in the LESB58 genome. GEMINI identified several novel islands including pathogenicity islands and revealed the …
The worldwide rise of diabetes and obesity has spurred research investigating the molecular mechanisms that mediate the deleterious effects associated with these diseases. Individuals with diabetes and/or obesity are at increased risk from a variety of health consequences, including heart attack, stroke and peripheral vascular disease; all of these complications have oxygen deprivation as the central component of their pathology. The nematode Caenorhabditis elegans has been established as a model system for understanding the genetic and molecular regulation of oxygen deprivation response, and in recent years methods have been developed to study the effects of excess glucose and altered lipid homeostasis. Using C. elegans, I investigated transcriptomic profiles of wild-type and hyl-2(tm2031) ( a ceramide biosynthesis mutant) animals fed a standard or a glucose supplemented diet. I then completed a pilot RNAi screen of differentially regulated genes and found that genes involved in the endobiotic detoxification pathway (ugt-63 and cyp-25A1) modulate anoxia response. I then used a lipidomic approach to determine whether glucose feeding or mutations in the ceramide biosynthesis pathway or the insulin-like signaling pathway impact lipid profiles. I found that gluocose alters the lipid profile of daf-2(e1370) (an insulin-like receptor mutant) animals. These studies indicate that a transcriptomic approach can be used to discover novel pathways involved in oxygen deprivation response and further validate C. elegans as a model for understanding diabetes and obesity.
An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous to human genes that are differentially regulated in response to metabolic diseases, suggesting that there may be conserved gene expression responses between C. elegans supplemented with glucose and a diabetic and/or obese state observed in humans. These findings support the utility of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-induced sensitivity to oxygen deprivation) and identify potentially novel regulators of common complications seen in hyperglycemic and T2D patients (e.g., macrovascular complications).
Cardiovascular activities may increase the brain blood flow improving neuronal activities leading to improved cognition. Consequently, the effects of an acute bout of moderate intensity aerobic exercise on brain hemodynamics and its correlation with cognitive color-word Stroop task performance were tested. The Stroop tasks were congruent (color matches word) and incongruent (color does not match word). Prefrontal (PFC) and motor cortex (MC) blood flow was recorded by fNIRS (functional near-infrared spectroscopy) while the subject was performing the Stroop tasks before and after the 30 minutes of exercise or equivalent time of rest controls (checking for practice effects). Ninety human subjects of age 24± 6, 20 ADHD (attention-deficit hyper-activity disorder), 27 High-BMI (>25), 29 males were recruited. Reaction time ‘RT' decreased (p<0.05) after exercise for both the congruent (12%) and incongruent (10%) Stroop tasks, compared to 8% with practice alone. Accuracy did not change after practice or exercise. HR changes after exercise correlated (p<0.05) with better accuracy and faster RT for the incongruent Stroop task. In general, a metabolic lag occurred in the neuronal deoxy- hemoglobin (Hb) signals behind the systemic oxy-Hb signals. PFC showed the highest effect sizes of Stroop task-responsive systemic hemodynamic changes compared to baseline irrespective of rest or exercise. Yet, PFC showed most significant (p<0.001) neuronal hemodynamic changes between the before and after exercise sessions, and these changes were opposite for right and left PFC, and opposite for congruent and incongruent Stroop tasks. Correlating the RT and mistakes with hemodynamics for both the Stroop tasks revealed that, after exercise, neuronal hemodynamic changes occurred at both PFC and MC associated with faster RT (p<0.05), and systemic hemodynamic responses occurred at PFC correlated (p<0.05) with mistakes. Overall, it was concluded that exercise changed the neuronal hemodynamic changes affecting speed; however, neuronal metabolic changes did not occur sufficiently to help …
Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and PKU animals based on PAH allele inheritance. Ocular and developmental phenotypes in the PAHenu2 colony match those described in previous IGF-1 studies. Understanding the IGF-1 inheritance discrepancy will enable better osteopenia research using PAHenu2 mice and allow breeding of a healthier mouse colony for continued research. Collectively the results from this work describe a new therapeutic approach for treatment of PKU as well as a better understanding of the PAHenu2 mouse model to study this disease.
Crocodilians and birds comprise sister taxa of archosaurs, the development of these vertebrates occurs within an egg case that leaves developing embryos susceptible to fluctuations in the nesting environment. Studies suggest that sub-optimal conditions alter morphological growth and cardiovascular physiology. Regulation of the cardiovascular system is immature in the subjects studied, and embryos may rely on humoral rather than neural control of the cardiovascular system. The primary focus of this dissertation was to assess regulatory mechanisms responsible for maintenance of arterial pressure and heart rate. Dehydration stress had marked effects on embryo growth, and altered baseline cardiovascular parameters, while leaving the response to humoral regulator, angiotensin II (Ang II), unaffected. However, dehydrated alligator embryos developed cholinergic tone on heart rate. Hypoxic incubated chicken embryos were reduced in embryo mass, and altered response to humoral regulatory components Ang I and adenosine in addition identifying a novel regulatory component of the cardiovascular response to acute hypoxia. Collectively, these studies add to the existing knowledge of cardiovascular physiology in embryonic archosaurs and suggest that some components of cardiovascular regulation are plastic following developmental stress.
It is well established that continual consumption of a diet high in fat leads to the development of chronic conditions such as obesity, cardio metabolic syndrome, and atherosclerosis that are associated with high incidence of cardiovascular disease. Recent studies have identified endotoxin-derived inflammation as a major diving force for the development of these conditions. Our laboratory has recently demonstrated that consumption of a single high-fat meal results in acute postprandial endotoxemia and alters monocyte cell surface adhesion molecule expression and scavenger receptor CD36 expression. These collective projects describe our efforts to understand the physiological significance of these postprandial changes and if supplementation with spore-based probiotics are able to provide any form of protection against these responses that are associated with the onset of atherogenesis.
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.
This dissertation examines the ability of in vitro biotransformation assays to provide an indication of metabolic potential. The potential for xenobiotic compounds to bioconcentrate in aquatic organisms is expressed through the bioconcentration factor (BCF). The metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions, microsomes and cell suspensions. Metabolic transformation rates (kM) were extrapolated from in vitro intrinsic clearance of parent compound (CLm) and integrated into a refined BCF model. In general, CLm of test compounds was greater in liver S9 fractions and hepatocytes. However, the influence of hepatic metabolism on kM and BCF was limited by hepatic blood flow (20-25%) compared to gill blood flow (~100%). A significant difference was noted between BCF solely based on KOW and BCF including kM. These studies indicate that the inclusion of kM in BCF models can bring predicted bioconcentration estimates closer to in vivo values. Primary cell suspensions are preferred over subcellular fractions as cell suspensions possess both phase I and phase II enzyme activity. Further study was conducted on ibuprofen biotransformation pathways. As fish do not contain the same cytochrome P450 (CYP) 2C homologs known to metabolize ibuprofen in mammals, it cannot be assumed that piscine biotransformation is similar. Metabolite analysis found 2-hydroxy-ibuprofen as the major metabolite in S9 and microsomal fractions. Additional assays involving the induction and inhibition of specific CYP isozymes support CYP1A2 as an alternative metabolic pathway.
The aim of the study was to identify the factors that have an influence on the completion rates of undergraduate students in the fields of science, technology, engineering and mathematics (STEM). Using Tinto's retention rate theory as the theoretical foundation, data were collected from freshman who were enrolled in the years 2005 to 2008. Results showed gender and first-generation status were significant predictors of STEM completion and time taken to complete the degree. Institutional bias played a role in race/ethnicity not being a factor affecting completion rates, as this study was conducted at a Predominantly White Institution. SAT scores and first and second-year college GPA showed to have the most prominent influence on both STEM completion rate and time taken to complete the degree. Females with higher first-year college GPA and higher high school rank finished faster. Similar results were found with first-generation students as well. Students belonging to ethnic minority groups with higher SAT scores and college GPA had greater success in STEM fields as well. The study results can be used to increase completion rates of underrepresented students in the STEM fields, given what we know about the interactions between underrepresented student groups and the most important predictors.
Rapidly accumulating genomic data as a result of high-throughput sequencing has necessitated development of efficient computational methods to decode the biological information underlying these data. DNA composition varies across structurally or functionally different regions of a genome as well as those of distinct evolutionary origins. We adapted an integrative framework that combines a top-down, recursive segmentation algorithm with a bottom-up, agglomerative clustering algorithm to decipher compositionally distinct regions in genomes. The recursive segmentation procedure entails fragmenting a genome into compositionally distinct segments within a statistical hypothesis testing framework. This is followed by an agglomerative clustering procedure to group compositionally similar segments within the same framework. One of our main objectives was to decipher distinctive evolutionary patterns in sex chromosomes via unraveling the underlying compositional heterogeneity. Application of this approach to the human X-chromosome provided novel insights into the stratification of the X chromosome as a consequence of punctuated recombination suppressions between the X and Y from the distal long arm to the distal short arm. Novel "evolutionary strata" were identified particularly in the X conserved region (XCR) that is not amenable to the X-Y comparative analysis due to massive loss of the Y gametologs following recombination cessation. Our compositional based approach could circumvent the limitations of the current methods that depend on X-Y (or Z-W for ZW sex determination system) comparisons by deciphering the stratification even if only the sequence of sex chromosome in the homogametic sex (i.e. X or Z chromosome) is available. These studies were extended to the plant sex chromosomes which are known to have a number of evolutionary strata that formed at the initial stage of their evolution, presenting an opportunity to examine the onset of stratum formation on the sex chromosomes. Further applications included detection of horizontally acquired DNAs in extremophilic eukaryote, Galdieria sulphuraria, which …
Mercury (Hg) is a ubiquitous environmental contaminant, which is capable of global atmospheric transport. As a result, even the most pristine aquatic ecosystems are affected by atmospheric Hg deposition, following which microbial transformation yield organic Hg forms, the most concerning of which is methylmercury (MeHg). Methylmercury is capable of bioaccumulation and biomagnification in food webs, resulting in potentially toxic body burdens due to regular dietary exposure in long-lived organisms at higher trophic levels. It is also a molecular mimic of some endogenous amino acids, providing a route of transfer from mother to offspring via large amino acid transporters. Exposure during neurodevelopment can lead to serious, irreversible neurological dysfunction, associated with a variety of cognitive and motor abnormalities across species. The present studies evaluate the effects of maternally-transferred dietary MeHg, at environmentally relevant concentrations on early life stage fathead minnows (Pimephales promelas). Embryos were collected from adult fatheads exposed to one of three diets with varying concentrations of MeHg for 30 days. Adult reproductive metrics were also monitored over the course of the study, with results indicating no effects on spawning frequency, clutch size, or total egg output. In embryos, Hg concentration was a function of female diet and the duration (number of days) of female exposure. Offspring spawned in tanks administered the low Hg diet displayed altered embryonic movement patterns (hyperactivity), decreased time to hatch, decreased mean larval size, and alterations to several metabolite abundances when compared with controls. Significantly altered metabolites include those associated with cellular energetics, fatty acid metabolism, and polyamine synthesis, indicating current environmental exposure scenarios are sufficient to disrupt important cellular pathways. Dysregulation of the dopaminergic system of embryos is also characterized, and may be a possible mechanism by which hyperactive behaviors are observed in these embryos. Offspring from tanks administered the high Hg diet exhibited …
An avian embryo is ectothermic, with body temperature determined by environmental temperature. Upon hatching, the neonate begins a conversion so that endothermic capacity becomes feasible and body temperature becomes independent of environment. Whole animal metabolic rate and ventilation response, cardiovascular development, and maturation of muscle mitochondrial flux were the focus of this dissertation because of the direct role in shivering thermogenesis. Precocial ducks and altricial Double-crested Cormorants exhibit increasing hematocrit and disproportionate increases in fractional heart mass resulting in greater oxygen delivery capacity and increased capacity of muscles to utilize oxygen compared with ectothermic American Alligator and Common Snapping turtles. By selecting for faster growth and higher meat yield in the domestic chicken, differences in whole-animal, tissue, cellular, and regulatory responses are evident between broiler and layer type birds. In the altricial red-winged blackbird, despite appearance of a whole animal endothermic response sometime after 7 dph, capacity of skeletal muscles involved in shivering thermogenesis peaks prior to that time. Thus, full development of endothermy is delayed in this species, allowing the altricial nestling to allocate energy towards growth rather than metabolic maintenance. Hypothyroidism in neonate red-winged blackbirds results in delayed maturation of the cardiovascular system and mitochondrial oxidative capacity of skeletal muscle. Such deficiencies were quickly recovered once the animals returned to a normothyroid state, apparently at the cost of increasing body mass. Insights into onset of thermoregulation provide a more thorough understanding of metabolic and physical transitions a hatchling bird must undergo to reach the adult endothermic phenotype. Endothermic capacity will continue to be at the forefront of physiological research because of the significance of changes between the energetic relations of an animal that must occur with its environment.
Using a high throughput closed respirometry method to measure oxygen consumption, I determined metabolic rates in asexual and sexual Schmidtea mediterranea and Girardia dorotocephala, as a function of temperature, taxon, stressors, reproductive mode, age, regeneration, and specific dynamic action. This study has shown that oxygen consumption can reliably be measured in planaria using optode closed respirometry, and also provided a reliable method for measuring wet mass in planaria, which has been a challenge to researchers in the past. This research revealed that oxygen consumption in S. mediterranea is 1.5-2.1X greater in the sexual strain over the asexual strain at 13-18°C. Within the sexual strain, oxygen consumption is 1.5 -2.2X greater in sexually mature adults over the sexually immature groups (hatchlings, juveniles, and regenerating sexuals). Furthermore, I was able to quantify differences in sexual morphology between these groups exhibiting significant differences in oxygen consumption. The results of this research supports a theory of higher metabolic costs with sexual maturity in S. mediterranea. Therefore, this study has established sexual and asexual S. mediterranea as simple, yet attractive models for investigating energetic costs between sexual and asexual phenotypes. This research also provided quantitative values for specific dynamic action in planaria, with a maximum increase in oxygen consumption of 160% induced by feeding, as well as metabolic relationships in planaria involving temperature, age, and regeneration. These values establish planaria as one of the simplest animal models in which common metabolic patterns, such as SDA and poikilothermic temperature sensitivity, have been demonstrated. Therefore, this research has contributed to the overall knowledge of the basic physiology in this animal, providing the framework for future metabolic studies in planaria involving environmental factors, reproduction, regeneration, development, and aging. Information from this study may supplement interpretation and understanding of modern cellular, molecular, and genomic studies in planaria.
The Cape Horn Biosphere Reserve is located at the southern tip of South America. This large archipelago is considered one of the last pristine areas left on the world. Despite it being an unpopulated area with most of the native forest cover intact, it has not been exempt from biological invasions, one of the major drivers of biodiversity loss. Three species that naturally interact in their native range in North America – American beavers (Castor canadensis), muskrats (Ondatra zibethicus), and American mink (Neovison vison) – were independently introduced in this remote region. In my dissertation, I investigated (i) the hypothesis of niche expansion in the invasive mink population on Navarino Island towards terrestrial habitats; (ii) potential mink impact on breeding success of forest-bird populations; (iii) habitat selection of small-rodent species and their perception on the mink's novel predation risk; and (iv) the dynamics of multiple-species invasions under the hypothesis of an invasional meltdown. Additionally, I worked within the framework of environmental philosophy. I provide an example of combining ecological and cultural dimensions within the International Long-Term Ecological Research network to disentangle the ethical dilemmas that surround the management of invasive species. I finally proposed a management plan based on the idea of multiple invasive species management, discussing potential solutions to overcome the challenges that the control of invasive species represent and to more effectively protect the biological integrity of the native ecosystems.
Factors currently at the forefront of human in vitro fertilization (IVF) that collectively influence treatment success in the form of blastocysts development were investigated during early mammalian embryology with concentration on infertile patients presenting with diminished ovarian reserve or preliminary ovarian failure. A novel experimental technique, Graft Transplant-Embryonic Stem Cells (GT-ESC) was introduced in the mouse model, as the first inclusive approach for embryo selection in IVF treatments resulting in successful graft integration of sibling cells, stage-dependent (day 4) blastocysts. E-Cadherin-catenin bonds play an integral role in trophectoderm cell viability and calcium removal, inducing disruption of cell-to-cell bonds at the blastocyst stage was detrimental to continued blastocyst development. One of the leading methods for embryo selection for uterine transfer in human IVF is application of pre-implantation genetic screening (PGS) methods such as next generation sequencing (NGS). Female patients <35 y do not benefit from this treatment when outcome is measured by presence of fetal heart beats at 10 weeks of gestation. Patients 35-37 y benefit from PGS with no significant difference of outcome based on form of PGS method utilized. Therefore, small nucleotide polymorphism array (snp-array) or targeted-NGS should be selected for this age range to lessen the financial burden of the patient. Embryos from women >40 y have a higher rate of mosaic cell lines which can be detected by NGS. Therefore NGS is most beneficial for women >40 y. Additionally, ovarian stimulation of the patient during human IVF can notably influence outcome. Anti-Müllerian hormone (AMH) is a more conducive indicator of blastocysts development per treatment compared to basal follicle stimulating hormone (FSH). Actionable variables included in a decision tree analysis determined a negative influence (0% success, n=11) of high dose gonadotropin use (>3325 IUs) in good prognosis patients (>12 mature follicles at trigger, AMH >3.15 ng/mL). A positive …
The anthropogenic alteration of riverine ecosystems has led to declines in the abundance and diversity of freshwater mussels (Bivalvia: Unionoida) worldwide. Central Texas is home to a diverse freshwater mussel fauna including three candidates for federal listing under the Endangered Species Act. Surveys conducted over the last few decades suggest many of the endemic freshwater mussel species in Texas exist in small isolated populations that may be vulnerable to the deleterious effects of genetic diversity loss. Microsatellite primers from two closely related species were used to identify a set of genetic markers that functioned in the Golden Orb (Quadrula aurea). Microsatellite markers were then applied to document the population genetic structure of Q. aurea within and among three connected river drainages in southeastern Texas. Gene flow within existing Q. aurea populations appears high indicating little potential for genetic issues stemming from isolation and inbreeding. Two weakly divergent admixed populations were identified occupying the San Antonio and Guadalupe/San Marcos rivers. Population genetic structure was related to river basin affiliation, but results for environmental factors were unresolved. Current effective population size estimates are large for the Guadalupe/San Marcos drainage and moderately large for the San Antonio drainage and there is no clear genetic evidence of contemporary population declines. Transport in the glochidial phase by a highly mobile host fish, the channel catfish (Ictalurus punctatus), may provide a mechanism for maintaining connectivity among spatially discrete mussel beds and deserves further study. Information on the occurrence and habitat associations of Q. aurea and two other threatened freshwater mussel species was documented. Quantification of the population genetic structure for Q. aurea provides important information needed for the management of this species, a baseline for understanding future changes, and insight into the factors that shape the population genetic structure of other threatened unionids in Texas.
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.
The major components of the tracheal system of insects are an extension of the exoskeleton, and the size of the exoskeleton is fixed in the adult stage, so any increase in metabolic demand that may accompany reproduction must be met by a relatively unaltered tracheal system that the female set in place at ecdysis, when entering adulthood. Acute hypoxia tends to elicit an increase in ventilation in insects, and here, I observe increased interburst VCO2 release, and a tendency towards a more continuous gas exchange pattern being preferred over discontinuous gas exchange when Blaptica dubia and Eublaberus posticus are exposed to a descending regime of hypoxia. Additionally, higher temperatures appear to increase sensitivity to hypoxia in these species, an expected result because both species, like most ectothermic animals, display a Q10 effect, increasing metabolic rates as temperature increases. The reproductive mode of B. dubia is considered to be lecithotrophic pseudoviviparity (or type A ovoviviparity), and by the time the embryos are born, they have more than doubles in volume from the time of oviposition. This gain is apparent in the wet mass of the embryo, with no change occurring in dry mass. The egg mass that can be attributed to water begins at 39% at oviposition and increased to nearly 82% at hatching. The metabolic rates of females and embryos increase as embryonic development progresses, but bomb calorimetry reveals that energy content of the embryos does not change. It is possible that these embryos gain some nutrition from the mother during embryonic development, but direct evidence remains to be demonstrated. Blaptica dubia and Eublaberus posticusare both blaberid species that display the same reproductive mode, pseudoviviparity, with incubation occurring in a brood sac. Comparisons between the reproductive traits of B. dubia and E. posticus reveal that the two species have similar …
Resistance exercise (RE) training is a well-known and effective method for promoting increases in muscle mass and strength. A single bout of RE induces physiological disturbances that require coordinated activation of the immune system and intramuscular signaling in order to return the tissue to homeostasis and adapt to the RE challenge. On the other hand, acute binge alcohol consumption can affect the immune response to an inflammatory challenge, intramuscular anabolic signaling, and muscle protein synthesis, and the effects of alcohol on these processes are opposite that of RE. Furthermore, individuals who report more frequent exercise also report a greater frequency of binge drinking. However, few investigations exist regarding the effects of binge alcohol consumed after a bout of RE on RE-induced physiological changes and performance recovery. Therefore, the overarching purpose of the investigations contained within this dissertation was to investigate the effect of alcohol consumed after RE on the RE-induced changes in mTOR pathway signaling, muscle protein synthesis, inflammatory capacity, strength recovery, and power recovery. Although RE increased mTOR pathway signaling and inflammatory capacity after exercise and reduced maximal strength and explosive power the day after exercise, we observed no effects of alcohol (1.09 g ethanol∙kg-1 lean body mass, designed to result in a peak blood alcohol concentration of approximately 0.12 g∙dl-1) consumed after RE on mTOR pathway signaling, 24-hour rates of muscle protein synthesis, inflammatory capacity, or strength and power recovery in resistance-trained individuals.
Achievement of endothernic capacity is vital for independence from ambient temperature changes, sustained activity, optimal biochemical reactions and optimization of parental care. During early avian development, the core tenets of transition from ectothermy to endothermy are development of metabolic capacity (oxygen consumption, mitochondrial bioenergetics), enhanced cardiovascular function (heart rate and cardiac output), pulmonary ventilation and thermogenic capacity. Thyroid hormones, particularly T3, are key metabolic regulators of basal metabolism, thermogenesis, pulmonary ventilation and mitochondrial respiration. Thyroid hormone fluctuation patterns during both precocial and altricial avian endothermic transition suggest a prominent role in maturation of endothermy, cardiovascular, respiratory and skeletal muscle physiology. This body of work explores effects of T3 manipulations in two avian species: the precocial Pekin duck and the altricial Red-winged Blackbird. Increased plasma T3 during late incubation resulted in increased cardiac mass, elevated resting and intrinsic heart rate, intrinsic mean arterial pressure, increased cholinergic tone and blunted alpha-adrenergic tone in the precocial Pekin duck. In both Pekin duck and Red-winged blackbird, plasma T3 levels correlated with changes in the trajectory of endothermic ontogeny, systemic oxygen consumption, thermogenesis, maturation of pulmonary ventilatory function, altered growth and effects on skeletal and cardiac mitochondrial bioenergetics. These observations support the role of thyroid hormones as metabolic and developmental regulators at the time of attainment of endothermy during the perinatal period in precocial and altricial avian species. Insights into the role of thyroid hormone as a metabolic and development regulator at the time of avian endothermic attainment provide a more thorough understanding of metabolic and physical transitions a hatchling bird must undergo to reach the adult endothermic phenotype. Such insights also deepen understanding of the complex role thyroid hormones play in homeostasis and offer implications about the evolutionary history of endothermic capacity.
Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of …
Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on thrombocyte function in zebrafish could enhance fundamental knowledge on hemostasis and may provide future target candidates for therapies.
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