Almost five million barrels of south Louisiana crude oil were released into the environment following the explosion of the Deepwater Horizon oil platform on April 20, 2010, however, little is known of the effects of the spill to native species of the northern Gulf of Mexico. Field studies provide a context for ecological realism but laboratory- based studies can connect the specific cause to a specific biological response. A previous field study, completed in 2010, tracked genome expression responses in native killifish resident in oiled and several non-oiled marshes in the northern Gulf of Mexico. Those data suggested significant biological effects from contaminating oil. As a post hoc companion to field studies, we sought to determine the genome expression response of the same native killifish to oil under controlled laboratory conditions to directly link a causal agent to a specific biological response. A concentration response experiment was conducted using experimentally-weathered surrogate oil to further characterize the genome transcriptional response, and to test for additional impacts on health. Transcriptome-wide gene expression responses were determined from the gill and liver tissues of fish exposed to experimentally weathered surrogate oil across a range of concentrations and throughout a time-course of exposure. Transcriptional responses to oil in the laboratory were predictive of the transcriptional response observed in the field study that coincided with the timing and location of oil contamination. These responses included increased expression in the genes activated by the aryl hydrocarbon receptor signaling pathway, including phase 1 and phase 2 metabolism genes, among others. Additionally, network and pathway analyses implicated the effects of transcription, centrosome, cell cycle progression, RNA processing, DNA damage, and apoptosis on gene regulation. These genome expression profiles offer additional scope for interpreting genome expression responses observed in the field, and offer additional insight into consequences of oil exposures in this important native Gulf of Mexico coast species.

The biological processes in macroevolutionary transformations, which result in the origin of new species and supraspecific taxa, are not directly observable in organisms with long reproductive generations and need to be extrapolated and reconstructed from physiological, anatomical, and microevolutionary processes. This dissertation reconstructs the anatomical and biomechanical changes that affected the head-neck-shoulder apparatus during the macroevolutionary transformation of a quadrupedal mammal to a bipedal one by studying two model organisms, namely the human and the cat. I hypothesize that the anatomical differences in the head-neck-shoulder apparatus of the two organisms are caused by different force regimes that act on them. I first show that the head-neck-shoulder apparatus of humans suspends the shoulders from the skull. I then demonstrate that the head-neck-shoulder apparatus of cats suspends the head from the thorax. A comparison of the two head-neck-shoulder apparatus shows that the changes necessary to modify a head suspension apparatus to a shoulder suspension apparatus are much more modest than what is usually expected to have taken place during macroevolutionary changes. Thus, it is evident that (1) small structural and configurational modifications can have significant functional and biomechanical consequences; and (2) macroevolutionary transformations of complex systems within complex organisms are amenable to analysis and evolutionary reconstruction.

Analysis of integrative physiological responses is a valuable approach to studying the biological impacts of natural and anthropogenic stressors in biota. By measuring fluctuations in multiple physiological characteristics, we are able to predict the impact that exogenous stressors may have on organismal health. Laboratory studies are useful in studying the effects of a single or multiple stressor interactions in a controlled environment, although field-based studies allow for the understanding of how fish are affected in situ, where multiple natural and anthropogenic stressors exist. Using laboratory and field-based experimental design, physiological indices of fish health were measured in fish exposed to natural and anthropogenic stressors in two unique incidences. First, controlled laboratory-based studies were performed where fish were exposed to larval freshwater mussels, a natural stressor found in North American freshwater environments. Unionid freshwater mussel larvae, called glochidia, must attach to a host fish in order to metamorphose into their juvenile state. This obligate stage in the unionid freshwater mussel life-cycle was found to cause a stress response in fish infected at high concentrations, where elevated cortisol in stressed fish was found to be coincident with an increase in glochidia metamorphosis to the juvenile stage. Second, both laboratory and field studies were conducted to understand the effects of the Deepwater Horizon oil spill on populations of Gulf killifish (Fundulus grandis) in oil-impacted areas in Louisiana, compared to reference sites in Louisiana, Mississippi, and Alabama. We show evidence of exposure to crude oil coincident with contamination from the Deepwater horizon oil spill in field-collected fish collected in 2010 and 2011 from oil-impacted sites, which had divergent gene and protein expression patterns compared to fish from unoiled locations. Further, controlled laboratory exposures of Gulf killifish embryos to field-collected sediments from oiled locations revealed that fish exposed to heavily oiled sediments as embryos had reduced cardiovascular defects, delayed hatching, reduced overall hatching success, pericardial edema, and were smaller at hatch. Together, these data suggest that fish were exposed to toxins in crude oil for two successive breading seasons, indicating, that contaminating oil from the Deepwater Horizon oil spill impacts organismal fitness, which may be predictive of long-term effects on Gulf killifish populations, and other biota that inhabit these areas, and also highlights the utility of combining laboratory and field-based techniques to predict the effects of natural and anthropogenic stressors on fish.

Due to the positive academic results in numerous studies on cooperative learning and the need and desire to improve academic results in East Feliciana High School Chemistry classes, the implementation of a cooperative learning structure called numbered heads together was studied during the spring 2013 semester at this rural, low performing high school. Numbered heads together was utilized during three units of a Chemistry class with 24 students and three units of an AP Chemistry with 11 students after completion of two units taught without the use of any type of cooperative learning structure. Using pre- and post-tests, learning gain differences were analyzed using a Wilcoxon matched-pairs test to determine the effectiveness of numbered heads together versus the use of individualized learning only for whole classes, varying levels of academic performance, and gender. Results indicated the use of numbered heads together was more effective than individualized learning for boys in the Chemistry class and those students classified as weaker performing students in the Chemistry class. The use of numbered heads together was as effective as individual instruction for all other groups of students. Student surveys indicated more enjoyment and engagement in their Chemistry or AP Chemistry class using numbered heads together as opposed to individualized learning.

Photosynthetic microalgae optimize the utilization of inorganic carbon by active uptake and concentration of inorganic carbon (Ci) around the carbon-fixing enzyme Rubisco. This process, mostly induced under limiting carbon conditions is called the Carbon Concentrating Mechanism or CCM. The photosynthetic green alga Chlamydomonas reinhardtii serves as an excellent model organism for the study of eukaryotic CCMs. However, unlike the prokaryotic cyanobacterial CCM, which has a well-characterized Ci uptake system, the Ci uptake and transport system of the C. reinhardtii CCM is not well understood. Since CO2 is a small neutral molecule, it is believed to be able to passively diffuse into the cell with or without any assistance from membrane bound transport proteins. However, the charged HCO3- ion needs transporters to facilitate its uptake across each membrane barrier. The hydrophobic barriers posed by the plasma membrane, chloroplast envelope and thylakoid membranes in the path of the charged HCO3- ions ultimate destination to the thylakoid lumen has led to the proposed existence of one or more transport proteins at each of these membrane locations. The roles of the C. reinhardtii NAR gene family, showing sequence homology to the Formate/Nitrite transporter family, were investigated for any changes with respect to the induction of the CCM. NAR1.2 was found to be the most interesting NAR protein with respect to the CCM. NAR1.2 was localized to the chloroplast envelope and is believed to be part of the chloroplastic Ci uptake system. The previously identified putative Ci transporter, LCI1 was localized to the plasma membrane. The expression of this protein in a LCI1 deficient background showed a significant increase in Ci uptake and Ci affinity of cells even in the absence of a functional CCM. However, the RNAi mediated knockdown of the protein failed to show any growth deficiencies or changes in photosynthetic rates at different pH levels. LCI1 is a transporter that is part of the plasma membrane Ci uptake system. In the quest for mutations in either previously identified or novel Ci transporters, an insertional mutagenesis project was also undertaken. The sequenced wild-type strain, CC-503, was found to be a natural mutant for the periplasmic carbonic anhydrase, CAH1.

Dissertation under the direction of Professor Bruce H. Appel Oligodendrocytes are specialized glial cells in the vertebrate central nervous system (CNS) that extend processes to wrap axons in myelin sheaths. Their number and distribution must be regulated to ensure uniform axon myelination for rapid conduction of nerve impulses. Elevated levels of Notch signaling activity can block neurogenesis and promote formation of glial cells, but the mechanisms that limit Notch activity to balance formation of neurons and glia from neural precursors are poorly understood. By screening for zebrafish mutations that disrupt oligodendrocyte development we found one allele, called vu56, which produced excess oligodendrocyte progenitor cells (OPCs). Positional cloning revealed that vu56 is a mutation of fbxw7 (f-box protein and wd repeat domain-containing 7), which encodes the substrate recognition component of a ubiquitin ligase that targets Notch for degradation. To investigate the basis of the mutant phenotype we performed in vivo, time-lapse imaging, which revealed that the increase in OPC number resulted from production of extra OPCs by ventral spinal cord precursors and not from changes in OPC proliferation or death. Notch signaling activity was elevated in spinal cord precursors of fbxw7 mutant zebrafish and inhibition of Notch signaling suppressed formation of excess OPCs. Our data indicate that Fbxw7 helps attenuate Notch signaling during zebrafish neural development thereby limiting the number of OPCs.

The social amoeba Dictyostelium discoideum senses the bacterial metabolite folic acid to track down bacteria. Previous studies suggested that the folic acid receptor is a serpentine receptor. In this study, I examined the transcriptional level changes of 46 putative serpentine receptor genes after folic acid stimulation. Seven candidate genes showed significantly up-regulated transcriptional levels, including fslA, fslB, fslH, fslJ, fslK, grlB, and grlD. Unfortunately, subsequent disruption of these genes did not affect folic acid sensing. Among these putative serpentine receptor gene mutants, grlB- cells exhibited delayed aggregation. Further studies suggest that GrlB is required for GABA binding. Detailed characterization of GABA metabolism indicates that GABA is used as an ancient signal during early development and culmination. Loss of GABA leads to a delay in early development and reduced detergent-resistant spores. GABA metabolism and signaling are regulated by distinct genes in different stages. In vegetative and early developmental stages, GABA is generated by the glutamate decarboxylase GadB and signals through GrlB. In late developmental stage, GABA is synthesized by the glutamate decarboxylase GadA and signals through GrlE. Mitochondrial GabT is required for GABA degradation, and DdvGAT is likely responsible for GABA secretion in culmination. Study of G proteins suggests that G protein alpha subunit Gα8 is probably involved in GABA signaling. Characterization of Gα8 also reveals that Gα8 promotes adhesion in the social amoeba. Here I propose a non-canonical model of how Gα8 works. Starvation triggers the up-regulation of both Gα8 and Gβγ. Gα8 appears to function as a heterotrimer. This heterotrimer interacts with unknown downstream effectors, which eventually promotes the expression of plasma membrane-anchored adhesion molecules or facilitates the membrane presenting of these adhesion molecules. Once the G protein heterotrimer is activated, Gα8 separates from Gβγ and no longer activates adhesion molecules. This activated Gα8 can mediate other signaling pathways, most notably the AprA or GABA signaling pathway.

Late Embryogenesis Abundant (LEA) proteins are highly hydrophilic, intrinsically disordered proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Embryos of the brine shrimp, A. franciscana, contain high titers of group 3 LEA proteins during desiccation-tolerant stages such as diapause and pre-emergence development. Here I report the sequencing of three novel variants of AfrLEA3m mRNA (Afrlea3m_47, Afrlea3m_43 and Afrlea3m_29), whose deduced protein sequences are are predicted to localize to the mitochondrion. These mRNAs are very similar to Afrlea3m, but each has a stretch of sequence that is absent in at least one of the others. In addition Afrlea3m_43 has five single nucleotide changes scattered across its sequence, and Afrlea3m_47 and Afrlea3m_43 have three single nucleotide differences in the section of sequence shared only by these two variants. Protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 is highest in diapause embryos and decreases throughout development to their lowest levels in desiccation-sensitive nauplius larvae. This pattern of protein expression is in agreement with previously reported mRNA expression for AfrLEA2 and AfrLEA3m and supports a role for LEA proteins in desiccation tolerance of embryos. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins. Investigations of protein secondary structure show AfrLEA2 and AfrLEA3m to be intrinsically disordered in solution and that they gain structure during desiccation and in the presence of the solvents TFE and SDS. I also show that during drying recombinant AfrLEA2 and AfrLEA3m confer protection to three desiccation-sensitive enzymes (lactate dehydrogenase, phosphofructokinase and citrate synthase). The degree of protective ability was found to depend on the target enzyme chosen. The strongest degree of stabilization was observed when a given LEA protein was used in the presence of the stabilizing sugar trehalose, which is naturally accumulated by A. franciscana embryos. Finally, AfrLEA2 is shown by immunohistochemistry to reside in the cytoplasm and nucleus of embryonic cells of A. franciscana, and the AfrLEA3m proteins are localized to the mitochondrion. The presence of LEA proteins in multiple subcellular compartments suggests a requirement to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by specific binding to recognition elements in the 3 untranslated regions (UTRs) of target mRNAs. In zebrafish embryonic development, miRNAs coordinately interact with many target transcripts to fin-tune overall gene expression. However, their expression profiles and critical functions during early zebrafish development remain to be elucidated. In order to understand miRNA expression and function, I first characterized the temporal expression of all miRNAs during very early zebrafish embryonic development using high-throughput sequencing and identified 8 novel miRNAs. I also identified an unexpected abundance of piRNAs during early developmental. To analyze specific targets and understand the rationale for miRNA regulation, I focused on mRNA targets for miR-153 and miR-27. I show that miR-153 regulates snap25 during synaptic transmission and motor neuron development. In addition, I also show that miR-27 targets ptk2.2 to regulate pharyngeal arch morphogenesis.

The purpose of this research is to determine whether graphic organizers foster better student achievement in science classrooms than guided note taking with PowerPoint presentations. The study was quantitative. Using approximately 69 high school Biology I students, two body systems were taught using graphic organizers as the main lesson plan tool and two body systems were taught only using a guided notes lecture with PowerPoint. A pre-test and a post-test were administered for each body system studied. Student test scores were evaluated to determine whether knowledge gains differed between the two types of lessons. It was found that the use of graphic organizer instruction was significantly better for student achievement when compared to the use of PowerPoint instruction and that there was much more interaction between student and teacher during the graphic organizer lessons. The delivery of the lesson by the use of graphic organizers seemed to promote more student success than the use of the PowerPoint lesson.