Dusp4 and Ppme1 have been identified as novel genes in skeletal muscle that are upregulated in response to neurogenic atrophy in a mouse model. Overexpression (OE) of Dusp4 wild-type (Dusp4-WT), as well as a Dusp4 Dominant Negative (Dusp4-DN) in C2C12 cells inhibits proper muscle differentiation potentially through its attenuation of ERK-MAPK signaling. Co-immunoprecipitation analysis shows Dusp4-DN associating with ERK1/2 but not p38, suggesting Dusp4 is specific for ERK1/2 but not p38. Quantitative PCR as well as Western blot analysis confirm that Ppme1 is expressed uniformly during muscle cell proliferation and differentiation. Interestingly, Ppme1 mRNA levels appear to decrease as differentiation proceeds whereas the protein levels remain constant throughout proliferation and differentiation. Transcriptional regulation of Ppme1 was observed by cloning proximal promoter fragments of the gene. The Ppme1 promoter is highly active on its own but when myogenic regulatory factors are ectopically expressed they repress promoter activity. Furthermore, mutation of a conserved E-box sequence inhibits full induction of the Ppme1 reporter gene , suggesting this E-box element is necessary for full Ppme1 expression. Additionally, inhibition of Ppme1 using a pharmacologic inhibitor results in delayed muscle cell differentiation and significant attenuation of AP-1 reporter activity. However, Ppme1 inhibition does not result in significant effects on phosphorylation of ERK1/2, c-Jun, or AKT. Additionally, C2C12 cells were treated with ERK1/2 and MEK1/2 inhibitors to compare results to that of Ppme1 inhibition and Dusp4 OE. While all of these conditions exhibit attenuation of AP-1 reporter activity, western blotting showed Dusp4 OE and Ppme1 inhibition do not mimic the results of ERK1/2 and MEK1/2 inhibition. Thus, Dusp4 and Ppme1 likely do not just block phosphorylation but act through more complex protein-protein interactions.

Algae are a group of highly diverse photosynthetic organisms found in variety of habitats. As the primary energy base in ecosystems, knowledge of the diversity and presence of certain algal lineages is paramount to our understanding of the trophic state of aquatic habitats. In recent years, the state of Florida has seen an increase of both marine and freshwater algal blooms. Similarly, filamentous algae have begun outcompeting vascular macrophytes throughout many of Florida’s springs as nutrient enrichment from anthropogenic sources increases. Traditionally, the Florida algal spring communities have been assessed using classic morphological methods, which may underrepresent the true biodiversity present. Therefore, the goal of this study was to conduct a more complete diversity assessment implementing next-generation sequencing techniques (NGS) with morphological analyses and culturing methods. While morphological methods identified a wide variety of algal taxa, belonging to 4 phyla (Bacillariophyta, Charophyta, Chlorophyta, and Cyanobacteria), next-generation sequencing techniques provided greater detail of the diatom community. This is particularly important as many diatom taxa are used as indicators of water quality. We noted discrepancies between these two methods, highlighting how NGS techniques may complement the use of morphological analyses when analyzing algal diversity in this system. Culturing methods also revealed the presence of two taxa new to science (Nodosilinea fontisand Brasilonema variegatus), indicating these springs may represent a potential source of novel cyanobacteria. Taken together, this study showcases Florida springs are rich in algal diversity and a combination of methods is required for more complete biodiversity assessments. Future studies implementing such methods will aid in the preservation and conservation of these ecosystems.

Transforming growth factor beta (TGF-β) has been implicated in anterior subcapsular cataract (ASC) development. In the first section of this thesis, an in-vitro rat lens model was used to determine the role of matrix metalloproteinases during TGF-β-induced ASC. In the second part, an in-vivo TGF-β transgenic and Smad3 knockout model was used to examine the role of Smad3 signaling pathway in TGF-β-induced ASC development. Lastly, an in-vitro rat lens epithelial explant culture model was used to investigate the potential role of heat shock proteins (Hsps) in TGF-β-induced epithelial-mesenchymal transition (EMT). Optical, morphological and molecular changes were analyzed in theses studies. Results from cultured rat lenses show a significant increase of back vertex distance variability (decrease of sharpness and focus) during ASC development. Inhibition of MMPs eliminated the TGF-β-induced plaque formation. Similarly, the overexpression of TGF-β1 in transgenic mouse lenses leads to ASC formation and a decrease in lens optical quality in comparison to wild-type lenses, while TGF-β1/Smad3-/- (null) lenses show diminished TGF-β-induced effects. The plaques formed in the TGF-β1/Smad3-/- lenses are substantially smaller than in the TGF-β1/Smad3+/+ lenses. The morphological and molecular changes of TGF-β2/FGF-2 treated rat lens epithelial explants are similar to those found in the TGF-β2 treated rat lenses and transgenic TGF-β1 mouse lenses. Heat shock treatment prior to TGF-β treatment significantly reduced the effects of EMT in rat LECs. In conclusion, MMP inhibition prevented TGF-β-induced ASC formation whereas heat shock treatment and the absence of Smad3 protein expression only reduced the severity of TGF-β-induced effects.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Basigin and Embigin are members of the immunoglobulin superfamily that function as cell adhesion molecules. Studies of Basigin null mice revealed reproductive sterility, increased pain sensitivity, and blindness. It is thought that the mechanism causing blindness involves misexpression of monocarboxylate transporter 1 (MCT1) in the absence of Basigin. It is known that the transmembrane domain of Basigin interacts with MCT1. In the absence of Basigin, MCT1 does not localize to the plasma membrane of expressing cells and photoreceptor function is disrupted. Studies of the Basigin null mouse brain suggest that MCT1 is properly expressed, which suggests a separate mechanism causes the increased pain sensitivity in these animals, and also that a different protein directs MCT1 to the plasma membrane of expressing cells in mouse brain. Embigin is known to interact with MCT2 in neurons and with MCT1 in erythrocytes. It is not known, however, if Embigin normally interacts with MCT1 in the mouse brain or if Embigin acts to compensate for the lack of Basigin in the Basigin null animals. Therefore, the purpose of this study was to determine if Embigin normally interacts with MCT1, 2, or 4 in the mouse brain and if so, whether the interaction is similar to that between Basigin and MCT1. Expression of Basigin, Embigin, MCT1, MCT2, and MCT4 in mouse brain was assessed via immunoblotting and immunohistochemical analyses. In addition, recombinant protein probes corresponding to the Embigin transmembrane domain were generated for ELISA binding assays using endogenous mouse brain MCTs. It was determined that the proteins in question are rather ubiquitously expressed throughout the mouse brain, and that the cell adhesion molecules Basigin and Embigin may be co-expressed in the same cells as the MCT2 and MCT4 transporter proteins. In addition, it was determined that the Embigin transmembrane domain does not interact with the MCTs. The data therefore suggest that MCTs do not require Basigin or Embigin for plasma membrane expression in mouse brain.

Transforming growth factor beta (TGF-β) has been implicated in anterior subcapsular cataract (ASC) development. In the first section of this thesis, an in-vitro rat lens model was used to determine the role of matrix metalloproteinases during TGF-β-induced ASC. In the second part, an in-vivo TGF-β transgenic and Smad3 knockout model was used to examine the role of Smad3 signaling pathway in TGF-β-induced ASC development. Lastly, an in-vitro rat lens epithelial explant culture model was used to investigate the potential role of heat shock proteins (Hsps) in TGF-β-induced epithelial-mesenchymal transition (EMT). Optical, morphological and molecular changes were analyzed in theses studies. Results from cultured rat lenses show a significant increase of back vertex distance variability (decrease of sharpness and focus) during ASC development. Inhibition of MMPs eliminated the TGF-β-induced plaque formation. Similarly, the overexpression of TGF-β1 in transgenic mouse lenses leads to ASC formation and a decrease in lens optical quality in comparison to wild-type lenses, while TGF-β1/Smad3-/- (null) lenses show diminished TGF-β-induced effects. The plaques formed in the TGF-β1/Smad3-/- lenses are substantially smaller than in the TGF-β1/Smad3+/+ lenses. The morphological and molecular changes of TGF-β2/FGF-2 treated rat lens epithelial explants are similar to those found in the TGF-β2 treated rat lenses and transgenic TGF-β1 mouse lenses. Heat shock treatment prior to TGF-β treatment significantly reduced the effects of EMT in rat LECs. In conclusion, MMP inhibition prevented TGF-β-induced ASC formation whereas heat shock treatment and the absence of Smad3 protein expression only reduced the severity of TGF-β-induced effects.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

The eyes of snakes and most geckos, as well as a number of other disparate squamate taxa, are shielded beneath a layer of transparent integument referred to as the “reptilian spectacle.” Derived from the embryonic fusion of palpebral tissues, the spectacle contains a number of specializations of the skin to benefit vision while still allowing it to function as the primary barrier to the environment. For example, in nearly all species that possess it, it is markedly thinned compared to the surrounding integument and its keratinized scale is optically transparent. While the spectacle may thus seem ideally adapted to vision in allowing the eyes to be always unoccluded, it does have a few drawbacks. One such drawback is its vascularity, the implications of which are still not fully understood, but are explored herein. As no recent synthesis exists of the body of knowledge on reptilian spectacles, the first chapter of this thesis consists of a review of spectacle anatomy, physiology, adaptive significance and evolution to help put into context the following chapters that present original research. The second chapter describes the dynamics of blood flow through the spectacle vasculature of colubrid snakes, demonstrating three main points: (1) that the spectacle vasculature exhibits cycles of regular dilation and constriction, (2) that the visual perception of a threat induces vasoconstriction of its vessels, and (3) that spectacle vessels remain dilated throughout the renewal phase. The implications of these points are discussed. The third chapter describes the spectral transmittance of the shed spectacle scale, the only keratinized structure in the animal kingdom to contribute to the dioptric apparatus of the eye, as well as its thickness. Spectacle scale transmittance and thickness was found to differ dramatically between snakes and geckos and found in snakes to vary between families. The adaptive significance of the observed variation is discussed. The fourth chapter describes biochemical analyses of the shed spectacle scales of snakes and geckos and compares their composition to other scales in the integument. Spectacle scales were found to differ significantly from other scales in their keratin composition, and gecko spectacle scales in particular were found to lack ß keratin, that hard corneous protein thought to be common to all reptile scales. The concluding chapter will discuss where this research has brought the state of our knowledge on the spectacle and offers thoughts on potentially useful avenues for further research.

The eyes of snakes and most geckos, as well as a number of other disparate squamate taxa, are shielded beneath a layer of transparent integument referred to as the “reptilian spectacle.” Derived from the embryonic fusion of palpebral tissues, the spectacle contains a number of specializations of the skin to benefit vision while still allowing it to function as the primary barrier to the environment. For example, in nearly all species that possess it, it is markedly thinned compared to the surrounding integument and its keratinized scale is optically transparent. While the spectacle may thus seem ideally adapted to vision in allowing the eyes to be always unoccluded, it does have a few drawbacks. One such drawback is its vascularity, the implications of which are still not fully understood, but are explored herein. As no recent synthesis exists of the body of knowledge on reptilian spectacles, the first chapter of this thesis consists of a review of spectacle anatomy, physiology, adaptive significance and evolution to help put into context the following chapters that present original research. The second chapter describes the dynamics of blood flow through the spectacle vasculature of colubrid snakes, demonstrating three main points: (1) that the spectacle vasculature exhibits cycles of regular dilation and constriction, (2) that the visual perception of a threat induces vasoconstriction of its vessels, and (3) that spectacle vessels remain dilated throughout the renewal phase. The implications of these points are discussed. The third chapter describes the spectral transmittance of the shed spectacle scale, the only keratinized structure in the animal kingdom to contribute to the dioptric apparatus of the eye, as well as its thickness. Spectacle scale transmittance and thickness was found to differ dramatically between snakes and geckos and found in snakes to vary between families. The adaptive significance of the observed variation is discussed. The fourth chapter describes biochemical analyses of the shed spectacle scales of snakes and geckos and compares their composition to other scales in the integument. Spectacle scales were found to differ significantly from other scales in their keratin composition, and gecko spectacle scales in particular were found to lack ß keratin, that hard corneous protein thought to be common to all reptile scales. The concluding chapter will discuss where this research has brought the state of our knowledge on the spectacle and offers thoughts on potentially useful avenues for further research.

This study sought to investigate the effects of multiple intelligences based instruction (MIBI) and learning styles on ninth graders&amp / #8217 / attitudes toward biology, biology achievement, and overall multiple intelligences. This experimental research was conducted in the second term of 2002-2003 educational year in Eregli high school. The sample involved in the study consisted of 64 students from the two different ninth grade classes taught by the same teacher. One class was assigned as the experimental group in which students were exposed to MIBI, whereas the other class was assigned as the control group in which students were exposed to traditional instruction in the unit of diversity and classification of living organisms. The data were obtained from attitudes toward biology scale, unit achievement test, multiple intelligences and Kolb&amp / #8217 / s learning style inventories, interviews with the students and the teacher, and observations in the experimental classroom during the treatment. Multivariate Analysis of Covariance was used for the quantitative data obtained from measuring tools. Results indicated that there was a significant effect of MIBI on students&amp / #8217 / attitudes toward biology and biology achievement, but there was not any significant effect of MIBI on students&amp / #8217 / overall multiple intelligences. In addition, there was not any significant effect of the students&amp / #8217 / learning styles on their attitudes toward biology, biology achievement, or overall multiple intelligences. Moreover, interpretations of the interviews with the students and the teacher, and the observations of the experimental classroom indicated that MIBI had positive contributions on teaching and learning process.