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Title: Evaluation of marine sponge metabolites for cytotoxicity and signal transduction activity.
Creator: Longley, Ross E., McConnell, O. J., Essich, E., Harmody, Dedra K., Harbor Branch Oceanographic Institute
Date Issued: 1993
PURL: http://purl.flvc.org/FCLA/DT/3318961
Subject Headings: Marine metabolites, Sponges --Research, Cell-mediated cytotoxicity, Cellular signal transduction
Format: Document (PDF)

Title: Characterization of the motor protein, KIF9, in mammalian cell mitotic progression.
Creator: Hoke, Jordan, Harriet L. Wilkes Honors College
Abstract/Description: The kinesin family of microtubule motors is divided into subfamilies based on structure and function. KIF9, founder of the Kinesin-9 family, has been found to interact with the GTPase Gem. Subsequent studies have shown that KIF9 is vital for flagellar movement and podosome regulation. Previous work has proposed KIF9 is required for microtubule organization as well as proper mitotic entry, progression and completion. In this study, I examined the function of KIF0 in mitotic progression using...
Show moreThe kinesin family of microtubule motors is divided into subfamilies based on structure and function. KIF9, founder of the Kinesin-9 family, has been found to interact with the GTPase Gem. Subsequent studies have shown that KIF9 is vital for flagellar movement and podosome regulation. Previous work has proposed KIF9 is required for microtubule organization as well as proper mitotic entry, progression and completion. In this study, I examined the function of KIF0 in mitotic progression using shRNA-mediated knockdown, and overexpression. In knockdown cells, I saw a significant delay in mitotic progression as well as an increase in multipolarity and multinuclearity, suggesting a failure of cytokinesis. Overexpression of KIF9 produced similar effects on mitotic progression, as well as a marked increase in chromosome distance during anaphase. Taken with previous results, my research indicated that KIF9 is required for normal mitotic progression and completion, possible via regulation of the contractile ring.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3359298
Subject Headings: Cells, Motility, Protoplasmic streaming, Cell organelles, Cellular signal transduction, Cytoskeletal proteins
Format: Document (PDF)

Title: Devising a noncancerous model system to study multipolar spindle formation.
Creator: Nagarsheth, Nisha., Harriet L. Wilkes Honors College
Abstract/Description: Aneuploid tumor cells have characteristically unstable genomes which can be caused by mitotic defects such as multipolar spindles. Multipolarity relies upon the presence of extra centrosomes to form. However, some cells, both cancerous and noncancerous are able to avoid the formation of multipolar spindles through centrosomal clustering. Previous research has shown that there are a large number of genes whose activity contributes to the clustering activity, making analysis of individual...
Show moreAneuploid tumor cells have characteristically unstable genomes which can be caused by mitotic defects such as multipolar spindles. Multipolarity relies upon the presence of extra centrosomes to form. However, some cells, both cancerous and noncancerous are able to avoid the formation of multipolar spindles through centrosomal clustering. Previous research has shown that there are a large number of genes whose activity contributes to the clustering activity, making analysis of individual components of the process difficult. In order to better understand centrosomal clustering in cancer cells, we induced supernumerary centrosomes in a genomically normal cell line, RPE, to observe how the normal cells cope with extra centrosomes. Using colcemid to induce extra centrosomes in the RPE cell line, we observed an intact clustering mechanism in fixed cells. Further manipulation of the cells has allowed us to induce multipolarity in this cell line using various disrupters of cell-cycle checkpoint and dynein function.
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Date Issued: 2010
PURL: http://purl.flvc.org/FAU/3335107
Subject Headings: Centrosomes, Research, Cancer, Genetic aspects, Cellular signal transduction, Cell division
Format: Document (PDF)

Title: Manipulation of normal cells to produce a cancer-like mitotic phenotype.
Creator: Luffman, Christina., Harriet L. Wilkes Honors College
Abstract/Description: Most tumors contain multiple karyotypes due to genomic instability gained through chromosomal segregational defects. The variability of genomic changes within a population makes it difficult to study specific processes without the existence of confounding mutations. My project is to create a model system for observation of mitotic defects, specifically multipolar spindles, in a normal cell line, where the genome is intact. Induction of centrosome amplification is required for formation of...
Show moreMost tumors contain multiple karyotypes due to genomic instability gained through chromosomal segregational defects. The variability of genomic changes within a population makes it difficult to study specific processes without the existence of confounding mutations. My project is to create a model system for observation of mitotic defects, specifically multipolar spindles, in a normal cell line, where the genome is intact. Induction of centrosome amplification is required for formation of multipolar spindles. Treatments with colcemid showed a 10% increase in abnormal centrosome numbers over control. However, treatment with hydroxyurea and transfection of hMPSl showed little increase. Extra centrosomes are insufficient to drive multipolarity, therefore, I am using siRNA-mediated knockdown of Nek2 or HSET to decluster the extra centrosomes. Successful declustering will preferably show an increase in multipolar frequency, allowing us to study the formation and resolution of these structres to better understand how they contribute to aneuploidy and tumor progression.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/3325079
Subject Headings: Cell division, Karyokinesis, Cancer, Genetic aspects, Genomics, Cellular signal transduction, Centrosomes
Format: Document (PDF)

Title: Deamplification of supernumerary centrosomes by centrosomal clustering.
Creator: Thomas, Ezekiel., Harriet L. Wilkes Honors College
Abstract/Description: Supernumerary centrosomes can arise in a cell through a variety of methods. The presence of supernumerary centrosomes has been observed in nearly all types of cancer and promotes chromosomal instability, with rates of incident increasing as the cancer progresses. An oral squamous cell carcinoma line was treated with hydroxyurea to induce supernumerary centrosomes in the cells. NuMA was then knocked down using shRNA to promote centrosomal clustering and bipolar mitotic division in cells with...
Show moreSupernumerary centrosomes can arise in a cell through a variety of methods. The presence of supernumerary centrosomes has been observed in nearly all types of cancer and promotes chromosomal instability, with rates of incident increasing as the cancer progresses. An oral squamous cell carcinoma line was treated with hydroxyurea to induce supernumerary centrosomes in the cells. NuMA was then knocked down using shRNA to promote centrosomal clustering and bipolar mitotic division in cells with supernumerary centrosomes. Immunofluorescence with an antibody against SAS 6 accuately stained the centrioles for observation. The cells exhibiting supernumerary centrosomes undergoing bipolar mitotic division were studied to look for a possible pattern in centrosomal clustering where the majority of centrosomes are at one pole with a single centrosome at the other pole. Initial results suggest the presence of such a mechanism, which would describe a previously unknown mechanism for cells to deamplify supernumerary centrosomes by centrosomal clustering.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3359328
Subject Headings: Centrosomes, Cell division, Cellular signal transduction, Cancer, Genetic aspects
Format: Document (PDF)

Title: Determination of the acid dissociation constant of cytochrome B5 reductase.
Creator: Chong, Samantha., Harriet L. Wilkes Honors College
Abstract/Description: Most living organisms transduce electron transport chains in order to obtain energy. Flavin adenine dinucleotide (FAD) is a common electron transfer cofactor found in electron transport proteins referred to as flavoproteins. In this study, the different ionization and oxidation states of this cofactor found in cytochrome b5 reductase were identified spectroscopically and quantified as a function of solution potential and pH. The large data sets obtained from these experiments were analyzed...
Show moreMost living organisms transduce electron transport chains in order to obtain energy. Flavin adenine dinucleotide (FAD) is a common electron transfer cofactor found in electron transport proteins referred to as flavoproteins. In this study, the different ionization and oxidation states of this cofactor found in cytochrome b5 reductase were identified spectroscopically and quantified as a function of solution potential and pH. The large data sets obtained from these experiments were analyzed and the acid dissociation constant for reduced cytochrome b5 reductase was determined.
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Date Issued: 2008
PURL: http://purl.flvc.org/FAU/77662
Subject Headings: Cell metabolism, Cellular signal transduction, Chemistry, Statistical methods, Electron spectroscopy
Format: Document (PDF)

Title: Correlation between specific carcinogenic chemicals and specific mitotic defects and the restorative role of antioxidants.
Creator: Yates, Travis., Harriet L. Wilkes Honors College
Abstract/Description: The progression of cancerous cells towards a more aggressive tumor can be linked to external elements called carcinogens. The goal of this project is to examine the correlation between exposure to specific carcinogens and an increase of mitotic defects. These defects can manifest as lagging chromosomes, multipolar spindles, and anaphase bridges. Some of these instabilities are associated with the formation of reactive oxygen species (ROS), which are known to damage DNA. The potential for...
Show moreThe progression of cancerous cells towards a more aggressive tumor can be linked to external elements called carcinogens. The goal of this project is to examine the correlation between exposure to specific carcinogens and an increase of mitotic defects. These defects can manifest as lagging chromosomes, multipolar spindles, and anaphase bridges. Some of these instabilities are associated with the formation of reactive oxygen species (ROS), which are known to damage DNA. The potential for damage to the genome can be averted via antioxidants. Using the oral cancer cell line UPCI:SCC103, we established a baseline for the mitotic defects in the absence and presence of various ROS-inducing carcinogens using DAPI-stained fixed cells examined by immunofluorescent microscopy, The cells were treated with varying concentrations of the antioxidants, Vitamin C, (Sb(B-Carotene, and Vitamin E. The reactive oxygen scavengers significantly reduced the number of mitotic defects. A possible link between the carcinogens and lagging chromosomes was established.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/210007
Subject Headings: Cellular signal transduction, Genetic regulation, Antioxidants, Therapeutic use, Apoptosis, Molecular aspects, Cancer, Chemoprevention
Format: Document (PDF)

Title: Knockdown of KIF9 leads to defects in mitotic entry and progression in mammalian cells.
Creator: Alsina, Laura., Harriet L. Wilkes Honors College
Abstract/Description: Kinesin motors bind to microtubules and function in mitosis and intracellular transport depending on the position of the motor domain within the primary sequence (Hirokawa and Noda 2008). KIF9 has recently been shown to be involved in MTOC positioning and mitotic entry in Dictyostelium (Tikhonenko et al. 2009). To determine if a similar role for KIF9 exists in mammalian cells, we are using siRNA-mediated knockdown of KIF9 in COS-7 cells. Analysis of unsynchronized and cell-cycle synchronized...
Show moreKinesin motors bind to microtubules and function in mitosis and intracellular transport depending on the position of the motor domain within the primary sequence (Hirokawa and Noda 2008). KIF9 has recently been shown to be involved in MTOC positioning and mitotic entry in Dictyostelium (Tikhonenko et al. 2009). To determine if a similar role for KIF9 exists in mammalian cells, we are using siRNA-mediated knockdown of KIF9 in COS-7 cells. Analysis of unsynchronized and cell-cycle synchronized cells treated with siRNA to KIF9 reveal that the transition from G2 to M phase is delayed and that mitotic progression is also affected. Additionally, our data indicates that spindle pole function during anaphase may be abnormal in cells treated with siRNA, suggesting a role for KIF9 during that stage.
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Date Issued: 2010
PURL: http://purl.flvc.org/FAU/3334255
Subject Headings: Cells, Motility, Protoplasmic streaming, Cell organelles, Formation, Cellular signal transduction
Format: Document (PDF)

Title: Cells and cocktails: antioxidants rescue carcinogen induced mitotic defects in both chromosomally stable and unstable cells.
Creator: Griffin, Isabel Sloan., Harriet L. Wilkes Honors College
Abstract/Description: Tumor cells are characterized by an increase in genomic instability, brought about by both chromosomal rearrangement and chromosomal instability. Both of these broad changes can be induced by exposure to carcinogens. During mitosis, cells can exhibit early and late lagging chromosomes, multipolar spindles or anaphase bridges, all of which contribute to genomic rearrantement. We have studied the link between exposure to carcinogen and prevalence of mitotic defect in both chromosomally stable...
Show moreTumor cells are characterized by an increase in genomic instability, brought about by both chromosomal rearrangement and chromosomal instability. Both of these broad changes can be induced by exposure to carcinogens. During mitosis, cells can exhibit early and late lagging chromosomes, multipolar spindles or anaphase bridges, all of which contribute to genomic rearrantement. We have studied the link between exposure to carcinogen and prevalence of mitotic defect in both chromosomally stable and unstable cell lines as well as ecamined the restorative effects of antioxidants in preventing mitotic defects. We have exposed MES-SA uterine cancer cells to vinyl chloride followed by exposure to an antioxidant : ascorbic acid, B-carotene, or lycopene. Treated cells were then scored for the prevalence of mitotic defects within the population and compared to controls. We have also investigated whether pre-treatment with the antioxidants will weaken the effects of carcinogen exposure in these cell lines.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3359304
Subject Headings: Cellular signal transduction, Cell differentiation, Medical genetics, Cancer, Genetic aspects, Antioxidants, Therapeutic use, Cancer, Chemoprevention, Apoptosis, Molecular aspects, Genetic regulation
Format: Document (PDF)

Title: Adopting the orphan: determining the role of the motor protein KIF9 during the cell cycle.
Creator: Rivera Rios, Miguel E., Harriet L. Wilkes Honors College
Abstract/Description: The kinesin superfamily of microtubule motor proteins is subdivided into families based upon structure and function. KIF9 is the founding member of the Kinesin-9 family, which is a largely uncharacterized group of kinesins. It was originally identified by sequence homology to other kinesins. Subsequent studies have shown that KIF9 interacts with proteins involved in cell shape remodeling, cell migration and proper centrosomal positioning. We have examined KIF9 function in mammalian cells...
Show moreThe kinesin superfamily of microtubule motor proteins is subdivided into families based upon structure and function. KIF9 is the founding member of the Kinesin-9 family, which is a largely uncharacterized group of kinesins. It was originally identified by sequence homology to other kinesins. Subsequent studies have shown that KIF9 interacts with proteins involved in cell shape remodeling, cell migration and proper centrosomal positioning. We have examined KIF9 function in mammalian cells using shRNA-mediated knockdown and GFP-plasmid overexpression. By knocking dow KIF9 expression in these cells, we have seen several effects on normal cell cycle progression. Using various cell cycle markers, we have observed a decrease in the number of cells in late S phase. In addition, there is a marked increase in the number of cells in early mitosis in unexpected time intervals. We propose that KIF9 is required for proper cell progression, via a potentially novel checkpoint mechanism.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3359321, http://purl.flvc.org/fau/fd/FADT3359321
Subject Headings: Cell organelles, Formation, Cellular signal transduction, Protoplasmic streaming, Cells, Motility, Cell division, Research
Format: Document (PDF)

Title: Approaches for raising the level of FOXO3a in animal cells.
Creator: Navarro, Diana., Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description: The turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide...
Show moreThe turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide reductases (Msrs), primarily MsrA and MsrB. The Msr system is capable of reversing oxidation of methionines in proteins and Msr subtypes have been implicated in protecting tissues against oxidative stress, as well as, enhancing the longevity of organisms from yeast to mammals. Preliminary data, unpublished results, indicate that MsrA protein and transcripts are elevated by anoxia. A recent study on Caenorhabditis elegans demonstrated that FOXO is involved in activation of the MsrA promoter. Using the turtle MsrA promoter sequence we worked to determine which regions in the promoter are necessary for activation by anoxia. The results of the present study were 1) to prepare a TAT-FOXO3a fusion protein which could penetrate animal cells and 2) to construct a FOXO3a expression vector for transcription studies on MsrA expression.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3342243
Subject Headings: Cellular signal transduction, Cell proliferation, Oxidative stress, Prevention, Adaptation (Physiology)
Format: Document (PDF)

Title: An investigation of membrane transporter proteins in the distal vertebrate retina: excitatory amino acid transporters and sodium potassium chloride cotransporters.
Creator: Purpura, Lauren Angeline, Shen, Wen, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Neurons are able to maintain membrane potential and synaptic integrity by an intricate equilibrium of membrane transporter proteins and ion channels. Two membrane proteins of particular importance in the vertebrate retina are the excitatory amino acid transporters (EAATs) which are responsible for the reuptake of glutamate into both glial and neuronal cells and the sodium potassium chloride cotransporters (NKCCs) that are responsible for the uptake of chloride ions into the cell. NKCCs are...
Show moreNeurons are able to maintain membrane potential and synaptic integrity by an intricate equilibrium of membrane transporter proteins and ion channels. Two membrane proteins of particular importance in the vertebrate retina are the excitatory amino acid transporters (EAATs) which are responsible for the reuptake of glutamate into both glial and neuronal cells and the sodium potassium chloride cotransporters (NKCCs) that are responsible for the uptake of chloride ions into the cell. NKCCs are electro-neutral with the uptake of 2 Cl- coupled to an exchange of a potassium and Na+ ion into the cells. Therefore, there is little change of cell membrane potential in the action of NKCCs. In this study the localization and function of EAATs in the distal retina is investigated. Whole cell patch clamp recordings in lower vertebrate retina have demonstrated that EAAT2 is the main synaptic EAATs in rod photoreceptors and it is localized to the axon terminals. Furthermore, the action of the transporter seems to be modified by intracellular calcium concentration. There is also evidence that EAAT2 might be regulated by feedback from the neuron network by glycinergic and GABAergic mechanisms. The second half of this study investigates expression of NKCCs in the retina by western blot analysis and quantitative polymerase chain reaction. There are two forms of NKCCs, NKCC1 and NKCC2. NKCC1 is mostly expressed in the central nervous system and NKCC2 was thought to only be expressed in the kidneys. NKCC1 is responsible for the majority of chloride uptake into neuronal and epithelial cells and NKCC1 is expressed in the distal retina where photoreceptors synapse on second order horizontal and bipolar cells. This study found the expression of NKCC1 in the distal retina to be regulated by temporal light and dark adaptation. Light adaptation increased phosphorylated NKCC1 expression (the active form of the cotransporter). The increase in NKCC1 expression during light adaptation was modulated by dopamine. Specifically, a D1 receptor agonist increased phosphorylated NKCC1 expression. Dopamine is an essential chemical and receptor known for initiating light adaptation in retina. Finally, an NKCC1 knockout mouse model was examined and it revealed that both forms of NKCC are expressed in the vertebrate retina.
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Date Issued: 2014
PURL: http://purl.flvc.org/fau/fd/FA00004224, http://purl.flvc.org/fau/fd/FA00004224
Subject Headings: Biological transport, Carrier proteins, Cellular signal transduction, Neural receptors, Retina -- Cytology
Format: Document (PDF)

Title: Characterization of the MHC II B of the bald eagle.
Creator: Smith, Andrew., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: The Major Histocompatibility Complex class II B (MHC II B) gene encodes a protein that is part of the adaptive immune system and critical for the non-self recognition ability of immune cells. This gene has been characterized in the Bald Eagle, ten unique alleles were found in two subpopulations at the geographic extremes of the range margins. Geographic genetic variation is suggested by the presence of population specific alleles. The results showed considerable divergence of groups of Bald...
Show moreThe Major Histocompatibility Complex class II B (MHC II B) gene encodes a protein that is part of the adaptive immune system and critical for the non-self recognition ability of immune cells. This gene has been characterized in the Bald Eagle, ten unique alleles were found in two subpopulations at the geographic extremes of the range margins. Geographic genetic variation is suggested by the presence of population specific alleles. The results showed considerable divergence of groups of Bald Eagle alleles when compared to alleles from other birds of prey. Particular codons within the exon II show signs of balancing selection driving the evolution of the MHC II B. Transcription data showed statistically significant differential expression of alleles. This can be interpreted as meaning a particular locus is being preferentially expressed in blood. The analysis of the polymorphism of this adaptive marker may aid managers of wildlife during this age of global climate change and the biodiversity crisis.
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Date Issued: 2010
PURL: http://purl.flvc.org/FAU/2979375
Subject Headings: Major histocompatibility complex, Cellular signal transduction, Immunogenetics, Genetic polymorphisms
Format: Document (PDF)

Title: Characterization of SNAG-zinc finger protein (ZFP) transcription factors.
Creator: Chiang, Cindy Chung-Yue., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional...
Show moreTranscriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/186676
Subject Headings: Zinc-finger proteins, Synthesis, Metalloproteins, Synthesis, Genetic transcription, Regulation, Cellular signal transduction, Gene expression
Format: Document (PDF)

Title: Cleavage of brain glutamic acid decarboxylase 65 by calpain under pathological conditions.
Creator: Buddhala, Chandana, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on...
Show moreBrain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica, d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3342053
Subject Headings: Glutamic acids, Antagonists, Proteolytic enzymes, Research, Cellular signal transduction, Calpain, Glutamic acid, Metabolism
Format: Document (PDF)

Title: Comprehensive study of the ZAD family of zinc finger transcription factors in Drosophila melanogaster.
Creator: Krystel, Joseph., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: The zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S...
Show moreThe zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S-transferase (GST)-ZAD domain chimeric proteins for use in pull down protein binding assays,and GST-Zinc finger (ZnF) array domain chimera for electrophoretic mobility shift assays (EMSA). Protein binding assays indicated two putative conserved interactors, similar to the analogous KRAB system in mammals. ... Competitive bindings were carried out to show a specificity of binding conferred by the identified conserved positions. While the consensus binding sites show relatively few similarities, the predicted target genes identified by the consensus binding sites show significant overlap. The nature of this overlap conforms to the known characteristics of the ZAD family but points to a more positive selection to maintain conservation of function.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3355627
Subject Headings: Cellular signal transduction, Drosophila melanogaster, Cytogenetics, Transcription factors, Zinc-finger proteins, Synthesis, Genetic transcription, Regulation, Gene expression
Format: Document (PDF)

Title: Elucidation of the features of the zinc finger associated domain (ZAD) family of transportation factors in Drosophila melanogaster.
Creator: Krystel, Joseph., Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible...
Show moreThe zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/186768
Subject Headings: Cellular signal transduction, Drosophila melanogaster, Cytogenetics, Transcription factors, Zinc-finger proteins, Synthesis, Genetic transcription, Regulation, Gene expression
Format: Document (PDF)

Title: DNAJC25 Pro90Leu J-domain mutation demonstrates decreased chaperone activity in vitro.
Creator: Chauss, Daniel C., Charles E. Schmidt College of Medicine, Department of Biomedical Science
Abstract/Description: Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently,...
Show moreMolecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25.
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Date Issued: 2012
PURL: http://purl.flvc.org/FAU/3342040
Subject Headings: Cell physiology, Methodology, Molecular chaperones, Physiological effect, Cellular signal transduction, Proteolytic enzymes
Format: Document (PDF)

Title: Chronic variable stress affects hippocampal neurotrophic factor gene expression in the novelty-seeking phenotype: epigenetic regulation.
Creator: Oztan, Ozge., Charles E. Schmidt College of Medicine
Abstract/Description: Experimentally naive rats exhibit varying degrees of novelty exploration. Some rats display high rates of locomotor reactivity to novelty (high responders; HR), and others display low rates (low responders; LR). The novelty-seeking phenotype (LRHR) is introduced as a model of stress responsiveness. In this thesis I examined effects of chronic variable physical and social stress or control handling on the levels of various neurotrophins in the hippocampus, and changes in mossy fibre terminal...
Show moreExperimentally naive rats exhibit varying degrees of novelty exploration. Some rats display high rates of locomotor reactivity to novelty (high responders; HR), and others display low rates (low responders; LR). The novelty-seeking phenotype (LRHR) is introduced as a model of stress responsiveness. In this thesis I examined effects of chronic variable physical and social stress or control handling on the levels of various neurotrophins in the hippocampus, and changes in mossy fibre terminal fields in LRHR rats. A positive correlation is seen between histone deacetylase 2 and brain-derived neurotrophic factor (BDNF) levels both of which are oppositely regulated in LRHR CA3 fields in response to chronic social stress. Increase in BDNF levels in CA3 field accompanied increase in supra-pyramidal mossy fibre terminal field size (SP-MF) in HRs, and decrease in BDNF levels accompanied decrease in SP-MF volume in LRs. Epigenetic regulation of neurotrophic support underlying these structural changes is discussed.
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Date Issued: 2009
PURL: http://purl.flvc.org/FAU/215290
Subject Headings: Rats as laboratory animals, Cellular signal transduction, Gene expression, Hippocampus (Brain), Physiology, Neural transmission, Genetic regulation
Format: Document (PDF)

Title: Developmental delays in methionine sulfoxide reductase mutants in Drosophila Melanogaster.
Creator: Hausman, William, Binninger, David, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biological Sciences
Abstract/Description: Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster...
Show moreAging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass.
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Date Issued: 2014
PURL: http://purl.flvc.org/fau/fd/FA00004200, http://purl.flvc.org/fau/fd/FA00004200
Subject Headings: Aging -- Molecular aspects, Cellular signal transduction, Drosophila melanogaster -- Genetics, Mitochondrial pathology, Mutation (Biology), Oxidative stress
Format: Document (PDF)

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