Finding novel compounds that affect neuronal or muscular function is of great interest, as they can serve as potential pharmacological agents for a variety of neurological disorders. For instance, conopeptides have been developed into powerful drugs like the painkiller PrialtTM. Most conopeptides, however, have yet to be characterized, revealing the need for a rapid and straightforward screening method. We have designed a novel bioassay, which allows for unbiased screening of biological activity of compounds in vivo against numerous molecular targets on a wide variety of neurons and muscles in a rapid and straightforward manner. For this, we paired nanoinjection of compounds with electrophysiological recordings from the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. / by Monica Mejia. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated. The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein. A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines. Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD. / Arts, Faculty of / Philosophy, Department of / Graduate

Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The transcription efficiency of transfer RNA genes is modulated by sequences contained in their 5'-flanking region. For a tRNA val₄ gene a pentanucleotide with the sequence TCGCT was identified between positions -33 and -38. I have previously proposed that this sequence may be involved in specifically determining the rate of transcription of this gene. A general form of this sequence, TNNCT was found associated with other Drosophila tRNA genes which showed high ill vitro transcription efficiency. To further elucidate the role of TCGCT in tRNA transcription, single and double base-pair changes were created in the sequence TCGCT using site-specific mutagenesis. Mutations in the nucleotides -38T, -35C and -34T showed decreased levels of transcription whereas nucleotide changes at the nucleotides -37C and -36G did not reduce template activity. Therefore the sequence which modulates transcription of the tRNAVal₄ gene does have the general form TNNCT. Competition experiments between the Val₄ mutant -38G.-35A and a tRNASer₇ gene showed the TNNCT mutant to be a better competitor for transcription than the wild type template. Experiments analyzing the time-course of transcription, the effects of temperature and the effects of ionic strength indicated that TNNCT was not involved in determining the efficiency of stable complex formation. It is proposed that the pentanucleotide is probably responsible for influencing the rate of initiation of transcription. A sequence TGCCT contained in the anticodon stem/loop region of the Val₄ gene was also mutagenized and shown to be involved in complex stability or the elongation of Val₄ tRNAs. Using deletion analysis of the 5'-flanking sequences of a tRNASer₇ gene, a second positive transcription regulatory element was delimited. This sequence was also found in the 5'-flanks of the tRNAVal₄ and a tRNAArg gene. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed. / by Kori Mulholland. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

The inevitable aging process can be partially attributed to the accumulation of oxidative damage that results from the action of free radicals. Methionine sulfoxide reductases (Msr) are a class of enzymes that repair oxidized methionine residues. The two known forms of Msr are MsrA and MsrB which reduce the R- and S- enantiomers of methionine sulfoxide, respectively. Our lab has created the first genetic animal model that is fully deficient for any Msr activity. Previously our lab showed that these animals exhibit a 20 hour delay in development of the third instar larvae (unpublished data). My studies have further shown that the prolonged third-instar stage is due to a reduced growth rate associated with slower food intake and a markedly slower motility. These Msr-deficient animals also exhibit decreased egg-laying that can be attributed to a lack of female receptivity to mating. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

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