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

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

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

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

This study has investigated the involvement of photosynthetic electron transport chain on the regulation of gene expression of methionine sulfoxide reductase (UfMSR) in the marine macroalga Ulva fasciata Delile.UfMSRA is from copper stress and UfMSRB ir from hypersalinity stress. UfMSRA is similar to Arabidopsis AtMSRA4 and UfMSRB is similar to AtMSRB1. UfMSRA is specific to the MetSO S-enantiomer and UfMSRB catalytically reduces the MetSO R-enantiomer. Both enzymes are required, since in the cell oxidation of Met residues at the sulfur atom results in a racemic mixture of the two stereoisomers. UfMSRA and UfMSRB transcripts were increased by white light, blue light and red light with the maximum at 1 h following a decline, but kept constant in the dark. The magnitude of UfMSRA and UfMSRB transcript increase showed a positive linear correlation to increasing light intensity from 0-1200 u mole¡Pm-2¡Ps-1. The treatment with linear electron transport chain inhibitors, hydroxylamine, 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and stigmatellin, effectively inhibited PS II activity under 300 u mole¡Pm-2¡Ps-1 irradiance. DBMIB and stigmatellin can increase UfMSRA transcript that was reversed by 2,6-dichlorophenolindophenol (DCPIP), a PS I electron donor. It indicates that the block of electron transport of the downstream of cytochrome b6f indeuces UfMSRA gene expression. Hydroxylamine, DCMU and DBMIB decreased UfMSRB transcript that was not reversed by DCPIP while stigmatellin increased UfMSRB mRNA level, reflecting a role of reduced state with Qo site located at cytochrome b6f on the induction of UfMSRB gene expression. The cyclic electron transport chain inhibitors, antimycin A that inhibited photosynthetic electron transport, can inhibit the increase of UfMSRA and UfMSRB transcripts by irradiance. UfMSRA and UfMSRB gene expression were both modulated by cyclic electron transport chain and linear electron transport chain. These results reveal that photosynthetic electron transport chain modulates UfMSRA and UfMSRB gene expression by change its redox state.

Chlamydomonas reinhardtii can utilize CO2 for autotrophic growth (HSM plus 5% CO2) or acetate for mixotrophic growth (TAP). This study was to elucidate the differential regulation of methionine sulfoxide reductase (MSR) gene expression between HSM plus 5% CO2 and TAP cultured cells, and also to determine the difference of gene expression in response to high light (1,000 £gE m-2 s-1). The role of photosynthetic electron transport (PET) in the regulation of MSR gene expression was also examined by the use of PET inhibitors. High light inhibited PSII activity (Fv/Fm and Fv'/Fm') of HSM plus 5% CO2 and TAP cultured cells., while the responses of CrMSR gene expression in mixotrophically grown cells were different from autotrophically grown cells, High light increased the expression of CrMSRA1, CrMSRA2, CrMSRA3, CrMSRA5, CrMSRB1.2, and CrMSRB2.1, but inhibited the expression of CrMSRA4 and CrMSRB2.2 in autotrophically grown cells. The expression of CrMSRA3, CrMSRA5, and CrMSRB2.1 in mixotrophically grown cells was increased by high light but that of CrMSRA1, CrMSRA4, and CrMSRB2.2 was inhbited. The number of MSR isoform that was up-regulated by high light was greater in autotrophically grown cell than that in mixotrophically grown cells. Using the PET inhibitors (3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)), most of the CrMSRA expression was regulated by reduced QA for autotrophically grown cells while reduced PQ was the main site for mixotrophically grown cells by high light. The expression of CrMSRB in autotrophically grown cells was mainy modulated by QA (-) or Cytb6f (-), while that was not affected by PET, except a role of Cytb6f (-) on the high light-induced CrMSRB2.2 expression. We fouind that CrMSRB gene expression in autotrophically grown cells was highly affected by PET but not for micotrophically grtown cells. The present result that H2O2 did not accumulate in autotrophically and mixotrophically grown cells suggests that H2O2 may be not involved in the regulation of high light regulation of CrMSR gene expression. The present study shows that the mRNA expression of CrMSR isoforms in Chlamydomonas was diffrerentially regulated between autotrophically and mixttrophically grown cells. The relationship between the utilization of different C source and CrMSR gene expression will be discussed.

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Doctor of Philosophy(PhD) / Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.