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- Creator:
- Chen, Sylvia
- Description:
- Worldwide, there are 65 million people that have epilepsy and approximately 200,000 additional individuals are diagnosed with epilepsy annually. There is no cure and currently available anticonvulsants, which are medications to treat seizures, are not universally effective. Additional studies are needed to generate better treatment options. Drosophila melanogaster provides a valuable and efficient model to study seizure disorders due to the low cost of maintenance, genetic tractability, and short lifespan, which allows for observation at various life stages. D. melanogaster also possesses similarities to humans on cellular and subcellular levels. These advantages allow the development of a model for the high throughput study of epilepsy. This study aims to induce single and multiple seizures and monitor locomotion, learning, and memory. The results from the negative geotaxis assay, which takes advantage of D. melanogaster’s innate climbing behavior, suggest that single seizure events have minimal impact, whereas multiple seizures result in deficits in visuo-motor/motor behavior. Additionally, a visual place learning assay was used to study the cognitive abilities and results suggest a single seizure event impacts learning and memory. Lastly, whole brain slides were made and determined lesions in brain tissue result from repeated seizure events. The significant findings from this research may contribute to improve therapeutics for epilepsy by providing a method of high throughput screening.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Gonzalez, Dalia Banesa
- Description:
- This study involved Drosophila melanogaster, the common fruit fly, as the model organism to study the behavioral differences seen in flies overexpressing the human tau protein throughout their adult life span. The tau protein (MAPT – microtubule associated protein tau) is linked to Alzheimer’s Disease (AD). It is seen in both humans and Drosophila and has been noted to decrease learning and cognitive abilities in both. This study aimed to look at the cognitive and behavioral difference seen as adult flies age. The adult flies were tested at three different ages (young, middle, elderly) in order to look for any progression declines in behavior as the flies aged and expressed the human tau for longer periods of time. The human tau was driven into two different areas of the brain (ellipsoid body, mushroom body) that have been shown to be important regions for learning and memory in insects. The flies were tested in two different behavioral assays, a visual place learning assay and a negative geotaxis assay, in order to quantify spatial learning and memory. Additionally, fluorescence microscopy was used to analyze the amount of tau present in the brain at different times of the adult’s life, which confirmed the increased expression of tau as flies aged. As tau-expressing flies aged, both visual place learning and memory were impacted. These flies also showed a decline in planning ability. The behavioral declines seen in this study can be correlated to patients with AD who also overexpress this abnormal tau protein. This study aims to create a better Drosophila melanogaster model for AD. Moreover, the findings of this study can contribute to a better model organism for AD, which allow us to test interventions prior to the onset of dementia.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Montiel, Christian M.
- Description:
- The Escherichia coli cAMP receptor protein (CRP) requires both DNA binding and RNA polymerase recruitment for its transcriptional activation function. The DNA recognition of CRP is performed by the F-helix (residues 180-185) and several F-helix residues (Arg180, Glu181, Gly184, Arg185) are well-characterized as to their importance in DNA binding. However, no apparent function has been known for the other two residues (Thr182 and Val183). In this work, I show that the position-183 amino acid (Val 183) is important for the transcriptional activity of CRP. First, Ala substitution at position 183 led to an increase in transcriptional activation activity. Second, CRP mutants altered at position 183 were created to show an inverse correlation between amino acid size and transcriptional activation activity (V183A > V183G > wild type CRP > V183I > V183M > V183F = no activity). Third, the loss of the activity in V183F could be restored by a smaller amino acid substitution (IleGly) at position 172, implying that it is the combined size of positions 172 and 183 which is important. A structural analysis of CRP reveals that the position-183 amino acid faces the opposite of target DNA and is in close proximity to the position-172 amino acid. The requirement of a small residue at position 183 is hypothesized to avoid steric hindrance to the position-172 residue as steric hindrance would misalign the nearby AR1 residues, leading to poor RNA polymerase recruitment and transcriptional activation.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Thomas, Jason Michael
- Description:
- Bacteria must be able to withstand a myriad of stresses from both their environment, and in the case of pathogens, from their host. Low molecular weight thiols serve as redox buffers to maintain a reducing environment in the cell and participate in various protective mechanisms, including detoxification of reactive oxygen and nitrogen species, resistance against electrophiles and antibiotics, and protection against environmental stressors. In this study, I analyze the transcriptome of two heterotrophic clinically relevant bacteria, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus SAUSA300 and transposon mutants disrupted in thiol biosynthesis. The transposon mutants are disrupted in P. aeruginosa glutathione biosynthesis gene, gshA, and S. aureus bacillithiol biosynthesis gene, bshC. I also analyze the transcriptome of the autotrophic cyanobacterium, Synechocystis PCC 6803 as well as a knock-out mutant disrupted in egtB, which is involved in ergothioneine biosynthesis. P. aeruginosa gshA mutant is downregulated in type II and type VI secretion systems and upregulated in type III secretion systems while the S. aureus bshA mutant is downregulated in type VII secretion suggesting that thiols play a role in secretion of effectors. Strikingly, both P. aeruginosa and S. aureus switch to anerobic respiration with nitrate as the electron acceptor in the thiol mutants. A number of other virulence factors are also differentially expressed in these mutants agreeing with the attenuation of the mutants in disease models. In contrast, genes related to high light resistance, acid stress tolerance, and osmotic stress tolerance are downregulated in Synechocystis PCC 6803. This suggests that ergothioneine is involved in abiotic stress tolerance in the autotrophic bacteria. Overall, we demonstrate that LMW thiols are involved in a myriad of functions have not been previously identified.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Ajanel Gomez, Jehiel Abigail
- Description:
- Discovered in 2004, tunneling nanotubes (TNTs) are actin-based, bridge-like structures that allow the intercellular transport of cellular components, electrical signals, and pathogens. Thus, TNTs have been implicated in viral propagation, neurodegenerative diseases, and cancer. Most research has focused on examining the molecules within cells that aid in TNT formation and function, but little has been documented on the role that exogenous molecules have in TNT formation. Past research has determined that hydrogen peroxide (H2O2) induces TNTs in various cell lines; however, the amount of H2O2 used was above was high (100μM). The aim of this study was to establish if TNT formation can be induced by pathophysiological H2O2 concentrations. In addition, we also investigated glutamate, an inducer of actin-based filopodia in astrocytes, as a possible TNT inducer. The data obtained using fluorescence microcopy suggest that low concentrations of H2O2 and glutamate are able to increase TNTs in both HeLa and CAD cell lines. Furthermore, cells treated with low concentrations of H2O2 or glutamate showed an increase in the expression of Myo10, a protein that has been linked to TNT formation and function. Lastly, the data obtained with a microscope capillary single cell sorting system suggest that H2O2 and glutamate can induce the formation as well as the elongation of cellular protrusions in distances ranging from 14-96 μm. Altogether, these results provide useful insights regarding TNT formation pathways in cells as well as the exogenous molecules involved in the formation of TNTs.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Trujillo, Andreina
- Description:
- Tunneling nanotubes (TNTs) have been recently discovered as an essential form of cell-cell communication in multicellular organisms. Although TNTs appear to be an important cell-cell signaling pathway, its mechanism of formation and functions are still not fully understood. A central issue is determining whether mitochondria play a vital role in the formation of TNTs. Previous studies have shown that exposure to a low concentration of ethidium bromide (EtBr) in mammalian cells caused a complete loss of mitochondrial DNA (mtDNA) leading to non-functional mitochondria (Rho0 cells); therefore, this technique will be applied to produce a HeLa Rho0 cell line to study the effects of non-functional mitochondria on TNT. Here we will determine if Rho0 cells were successfully engineered by labeling the mtDNA with Quant-it PicoGreen Reagent and labeling of the plasma membrane with WGA Rhodamine to determine the effects on TNT formation. We will also determine the expression levels of Myosin-X (Myo10), a known TNT inducer, in Rho0 cells versus control cells. Overall results revealed that mitochondria do play a role in TNT formation. As cells were in the process of becoming Rho0 cells, TNT formation increased peaking at week 4. As Rho0 cells stabilized, TNT formation significantly decreased. In addition, cells were seen to expel their mitochondria into the extracellular medium in week 6, a process known as mitochondrial extrusion. Lastly, Myo10 protein levels significantly increased during the production of a stable Rho0 cell line at week 4. The increase in Myo10 protein levels correlated with an increase in TNT formation.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Chand, Shristi
- Description:
- Bacterial second messenger, c-di-GMP, regulates important functions such as biofilm formation, motility, and virulence. C-di-GMP also has an immunostimulatory property, so can be used as a vaccine adjuvant. In this project, DgcZ (a diguanylate cyclase) was engineered to create hyperactive mutants in c-di-GMP production. For this, two protein sites were targeted: substrate (GTP) binding site and product (c-di-GMP) inhibition site. At the GTP binding site, three residues (L134, D182 and R204) were tested through codon randomization and in vivo c-di-GMP production assay, but none of the positions provided high activity DgcZ mutants superior to wild type DgcZ. In contrast, the engineering at the product inhibition site was fruitful as superior mutants could be made at both R197 and E200 positions. So far, R197T, E200A and E200G were identified to be better than wild type DgcZ in in vivo c-di-GMP production based on both Clp toxicity-masking assay and c-di-GMP toxicity assay. These mutants were toxic to the host cell when highly produced. The toxicity problem was circumvented by tagging one of the mutants (E200A) with PelB, a signal sequence to the periplasmic space, at the N- terminus and the fused PelB-E200A was not toxic to the cells. In the future, the resultant PelB-E200A needs to be tested thoroughly through protein expression, purification and in vitro c-di-GMP production assay for mass production of c-di-GMP.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Ziser, Brian.
- Description:
- Clearcut logging of numerous small stands of trees over the last fifteen years has provided suitable areas for the study of early secondary plant succession on the western slope of the central Sierra Nevada mountains. Familiarity with early successional patterns in these areas is essential in the development of appropriate silvicultural practices for the maintenance of viable, productive forests, as well as aiding in effective management of wildlife resources. Clearcut logging has been widely practiced and researched in the coastal Redwood forests of Northern California, and in the Douglas Fir dominated forests of Oregon and Washington. However, as indicated by the available literature, no significant research on secondary succession following clearcutting has been conducted in the Sierra Nevada mountains. This project was undertaken to provide information about early regrowth patterns on these clearcut areas.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Yoshimura, Glenn.
- Description:
- Six species of Simulium larvae found in the Kings River near Piedra, Fresno County, California, are described. The black fly larvae and pupae were reared in the laboratory for taxonomic association of the immature instars, the pupae and the adults. The differential morphology of S. argus Williston, S . aureum Fries, S. canadense Hearle, S. virgatum Coquillett, and S. vittatum Zetterstedt is presented in greater detail than ever before, with the S. arcticum Malloch larva being described for the first time.
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology
- Creator:
- Willson, Ruth.
- Description:
- Nitidulid beetles have been recognized as major pests of figs In California since 1900 (Simmons et al., 1931). Early workers were concerned primarily with Carpophllus hemlpterus (Linnaeus), the dried-fruit beetle, as the principal nitidulid pest (Smith et al., I927). More recent studies have shown four other pestiferous nitidulids: Carpophllus freemanl Dobson, Carpophllus mutllatus Erichson, Urophorus humeralls (Fabrlcius), the pineapple beetle, and Haptoncus luteolus Erichson. Of these species, C. mutllatus was found in 56^ the Calimyrna figs in one sample from Madera County, California (Smilanick and Ehler, 1975)*
- Resource Type:
- Masters Thesis
- Campus Tesim:
- Fresno
- Department:
- Biology