Thesis advisor: Clare O'Connor / As organisms get older, the proteins in their cells also age, and as this happens, the amino acids that make up these proteins may become chemically modified and begin to lose their integrity. One example of an age-related modification occurs when the amino acid residue methionine is oxidized by a reactive oxygen species to methionine sulfoxide. Methionine sulfoxide reductase is an enzyme that repairs this damage to the protein by catalyzing a reaction that reduces methionine sulfoxide back to methionine. The fission yeast Schizosachharomyces pombe was used as the experimental model to study methionine sulfoxide reductase in vivo, taking advantage of the variety of tools available with which to study the organism. In S. pombe there are two genes encoding methionine reductase activities, msrA and msrB. The first goal of this project was to construct yeast strains in which the endogenous msrA and msrB genes had been inactivated. This was accomplished via homologous recombination reactions in which the msr genes were replaced with a selectable marker for biosynthesis of uracil (ura4+). After the construction and verification of the two knockout strains, the sensitivities of the strains to reactive oxygen species were tested. Both strains showed reduced resistance to oxidative stress. Future experiments will include more detailed analyses of the abilities of the strains to survive oxidative stress. Finally, the two knockout strains of yeast will be mated with one another in order to produce a double msr knockout, in order to examine the effects of a complete lack of methionine sulfoxide reductase activity on the organism. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

The detrimental effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) have been acknowledged as major factors in aging, senescence and several neurodegenerative diseases and conditions such as Parkinson’s disease and stroke (ischemia/reperfusion). Mammalian models are extremely susceptible to these stresses that follow the restoration of oxygen after anoxia; however, some organisms including the freshwater turtle Trachemys scripta can withstand several bouts of anoxia and repeated reoxygenation without any apparent pathology. T. scripta thus provides us with an alternate vertebrate model in which we can investigate physiological mechanisms of neuroprotection without the damaging effects that come with oxidative stress. The major objective of this study was to investigate the protective mechanisms in the turtle brain under conditions of anoxia and oxidative stress. Specifically, the focus is on the Methionine Sulfoxide Reductase system (Msr), an antioxidant and cellular repair system, and how it is regulated to protect the brain against such stressors. Previous studies in my lab have demonstrated that Msr mRNA and protein levels are differentially upregulated during anoxia and reoxygenation. To investigate the regulation of Msr, FOXO3a was directly induced by transfecting a human FOXO3a plasmid into turtle brain cell cultures, as FOXO3a has been shown to regulate MsrA levels in other animal models. Pharmacological manipulation of FOXO3a was also performed using the green tea extract Epigallocatechin gallate (EGCG) as it has been shown to increase expression of FOXO3a during oxidative stress conditions in other models. I found that an induction of human FOXO3a increased FOXO3a levels and showed protection against cell death during oxidative stress. Furthermore, treatment of cells with EGCG increased expression of FOXO3a only when the cells were exposed to oxidative stress and decreased cell death. Induction of FOXO3a and EGCG treatment did not increase MsrA levels, however MsrB3 levels were upregulated under both treatments but only in the presence of oxidative stress. These results suggest that MsrA and MsrB3 protect the cells from oxidative stress damage through different molecular pathways and that EGCG may be a therapeutic target to treat diseases related to damage by oxidative stress. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 113-119).

Universiẗat, Diss.--Köln, 2006.

Methionine sulfoxide reductase (MSR) is an important antioxidant to help mitigate oxidative stress that contributes to age-associated neurodegenerative diseases, such as Alzheimer’s Disease and Parkinson’s Disease. In MSR deficient Drosophila melanogaster (fruit flies), larvae show a developmental delay like that seen when wild-type larvae are reared on nutrient deficit culture medium. These investigators further showed that serotonin levels were depressed in these nutrient deficient larvae. The overarching aim of this study was to better understand the role of serotonin in MSR regulated physiology. Supplementing food with serotonin partially rescued the slower mouth hook movements (MHM) observed in the MSR-deficient flies. However, supplementation with serotonin altering drugs that cross the blood brain barrier (5-hydroxytryptophan, fluoxetine, or paravi chlorophenylalanine) did not rescue MHM and caused impairments to the growth of larvae during development. This study indicates that serotonin regulates feeding behavior partially through the regulation of MSR production but acts independently to regulate development. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Ergothioneine, a histidine-derived thiol, protects cells against reactive oxygen species and is emerging as a longevity vitamin. Ovothiol, another histidine-derived thiol, is also a potent antioxidant with therapeutic potential due to its anti-inflammatory and anti-proliferative activities. Despite these promising health benefits, the production of ergothioneine is limited by the underlying challenges of its only industrial synthetic method, while ovothiol is not commercially available. Due to these issues, the production of these thiols through metabolic engineering/synthetic biology approaches is appealing. The central steps in the ergothioneine and ovothiol biosynthetic pathways are the oxidative coupling C-S bond formation reaction mediated by non-heme iron sulfoxide synthases, and the pyridoxal-5'-phosphate (PLP)-dependent C-S lyases. This sulfur transfer strategy differs from all other pathways reported. Therefore, these trans-sulfuration reactions in ergothioneine and ovothiol biosyntheses are significant from both basic and translational research perspectives, hence, they were selected as my thesis project. This thesis comprises of five chapters. Sulfur metabolism and the biosynthesis of sulfur-containing natural products are presented in Chapter 1. The computational-guided protein engineering of a thermophilic sulfoxide synthase (EgtB) from Chloracidobacterium thermophiluim is covered in Chapter 2. Chapter 3 describes the mechanistic studies of the reductive C-S lyase (Egt2 from the Neurospora crassa’s ergothioneine biosynthesis), which revealed the involvement of a sulfenic acid intermediate in this reaction. In addition to reconstituting the ergothioneine biosynthetic pathway in vitro presented in Chapter 3, I fully reconstituted the in vitro ovothiol A biosynthetic pathway from Erwinia tasmaniensis, which is described in Chapter 4. In Chapter 5, the mechanistic studies of the ovothiol sulfoxide synthase OvoA using unnatural amino acid incorporation via amber-codon suppression are discussed. The success of this thesis work paves the way for the industrial production of ergothioneine and ovothiol through metabolic engineering/synthetic biology approaches. This study has also laid the foundation for future in-depth mechanistic characterization of these novel enzymes.

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Drosophila melanogaster tolerates several hours of anoxia (the absence of oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to critical cellular molecules, which contribute to aging and development of certain agerelated conditions. The amino acid, methionine, is susceptible to oxidation, although this damage can be reversed by methionine sulfoxide reductases (Msr). This project investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout the lifespan of the animal. The data show that the time for recovery from the protective comma as well as the survival of the animals lacking any Msr activity depends on how quickly the coma is induced by the anoxic conditions. Insight into the roles(s) of Msr genes under anoxic stress can lead us to a path of designing therapeutic drugs around these genes in relation to stroke. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection