Fluorescent Detection of Reactive Oxygen Species in Saccharomyces Cerevisiae Applied to Chronological Lifespan

During the course of normal aerobic metabolism, cells are exposed to a wide range of reactive oxygen species such as the superoxide anion, hydrogen peroxide, and the hydroxyl radical. These reactive oxygen species (ROS) are highly reactive metabolites of oxygen and can damage a wide range of macromolecules in the cell, including nucleic acids, proteins, and lipids, and can even, in some severe cases, lead to cell death. Normally, molecular oxygen is relatively unreactive and harmless in its ground state; however, it can undergo partial reduction via electrons that are leaked from the electron transport chain to form both the superoxide anion and hydrogen peroxide, both of which can react further to form the dangerously reactive hydroxyl radical. In order to combat the toxic and potentially deadly effects of ROS, cells are equipped with various antioxidant defense mechanisms, which include enzymes like superoxide dismutase 1 (Sod1p). Our objective is to observe these various reactive oxygen species using yeast (Saccharomyces cerevisiae) as a model organism and explore different biochemical staining assays such as Amplex Red (AR) and Dihydroethidium (DHE). These stains can both be used to track live cells and quantify ROS levels. This will allow us to study how ROS changes during chronological yeast lifespan. Although there are many types of reactive oxygen species that exist in various parts of the cell, our work thus far has aimed to track extracellular hydrogen peroxide via AR and superoxide generation in the mitochondria via DHE. Our initial results indicate that we are able to track superoxide production using DHE in wild type cell and sod1∆ yeast strains spectroscopically. Ultimately, we will use both fluorescence spectroscopy and live cell imaging via fluorescence microscopy to assess superoxide levels in multiple yeast strains. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

Fluorescent Detection of Reactive Oxygen Species in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Applied to Chronological Lifespan

During the course of normal aerobic metabolism, cells are exposed to a wide range of reactive oxygen species such as the superoxide anion, hydrogen peroxide, and the hydroxyl radical. These reactive oxygen species (ROS) are highly reactive metabolites of oxygen and can damage a wide range of macromolecules in the cell, including nucleic acids, proteins, and lipids, and can even, in some severe cases, lead to cell death. Normally, molecular oxygen is relatively unreactive and harmless in its ground state; however, it can undergo partial reduction via electrons that are leaked from the electron transport chain to form both the superoxide anion and hydrogen peroxide, both of which can react further to form the dangerously reactive hydroxyl radical. In order to combat the toxic and potentially deadly effects of ROS, cells are equipped with various antioxidant defense mechanisms, which include enzymes like superoxide dismutase 1 (∆sod1). Our objective is to observe these various reactive oxygen species using yeast (Saccharomyces cerevisiae) as a model organism and explore different biochemical staining assays such as Amplex Red (AR) and Dihydroethidium (DHE). These stains can both be used to track live cells and quantify ROS levels. This will allow us to study how ROS changes during chronological yeast lifespan. Although there are many types of reactive oxygen species that exist in various parts of the cell, our work thus far has aimed to track extracellular hydrogen peroxide via AR and superoxide generation in the mitochondria via DHE. Our initial results indicate that we are able to track superoxide production using DHE in wild type cell and ∆sod1 yeast strains spectroscopically. Ultimately, we will use both fluorescence spectroscopy and live cell imaging via fluorescence microscopy to assess superoxide levels in multiple yeast strains. Our results will provide insight into the role of ROS in aging as we quantify levels during yeast lifespan

Career Aspirations of Older Workers: An Australian Study

Global challenges associated with the ageing workforce include lower levels of education and negative attitudes of older workers towards learning and covert age discrimination in the workplace. This report discusses initial findings from a survey of older workers employed in regional areas in Australia. The older workers surveyed were predominantly blue collar with low levels of formal education. Contrary to the stereotypical views, there were few attitude differences between older (>40 years) and younger workers (≤40 years). However, gender, education level and job type had a greater impact on attitudes in the older workers when compared to their younger colleagues

Making-Up Conditions Of Employment: The Unequal Burdens Test as a Flawed Mode of Analysis in Jespersen v. Harrah\u27s Operating Co.

Part I of this Note reviews Title VII and foundational caselaw, including cases regarding sex discrimination and appearance standards. Part II examines the Ninth Circuit\u27s Jespersen opinion. Part III compares the Supreme Court decision in Price Waterhouse v. Hopkins, which expanded Title VII protection to include gender stereotyping, with the Jespersen holding. Part III also explores a Seventh Circuit case, Carroll v. Talman Federal Savings and Loan Association of Chicago, and Judge Thomas\u27s dissent in Jespersen, which both argue for inclusion of less tangible factors such as gender stereotyping in the unequal burdens test. Part III finally contends that the unequal burdens test should consider a job-relatedness element in the initial weighing of burdens based on the intent of Title VII. Finally, this Note concludes that by incorporating intangible considerations such as gender stereotyping and weighing job-relatedness in the plaintiff s initial showing under the unequal burdens test, the Ninth Circuit will better protect employees from discriminatory appearance and grooming standards

Making-Up Conditions Of Employment: The Unequal Burdens Test as a Flawed Mode of Analysis in Jespersen v. Harrah\u27s Operating Co.

Part I of this Note reviews Title VII and foundational caselaw, including cases regarding sex discrimination and appearance standards. Part II examines the Ninth Circuit\u27s Jespersen opinion. Part III compares the Supreme Court decision in Price Waterhouse v. Hopkins, which expanded Title VII protection to include gender stereotyping, with the Jespersen holding. Part III also explores a Seventh Circuit case, Carroll v. Talman Federal Savings and Loan Association of Chicago, and Judge Thomas\u27s dissent in Jespersen, which both argue for inclusion of less tangible factors such as gender stereotyping in the unequal burdens test. Part III finally contends that the unequal burdens test should consider a job-relatedness element in the initial weighing of burdens based on the intent of Title VII. Finally, this Note concludes that by incorporating intangible considerations such as gender stereotyping and weighing job-relatedness in the plaintiff s initial showing under the unequal burdens test, the Ninth Circuit will better protect employees from discriminatory appearance and grooming standards

A Study of Written Communication: Showing Your Steps

In this action research study of my teaching of sixth grade mathematics, I investigated the importance of showing work on daily assignments. I wanted to find out what happens when I ask students to show their work, specifically, whether it would improve students’ grades or not and whether I could help the students to understand the importance of showing their work. I discovered that students need to be shown the proper way to show their work, how to look at a problem and then how to show all of their steps to get to the answer. They need to be encouraged and be held accountable for showing their work when asked. Once they were able to show work, they could start to see the value in showing their work and they tended to show their work more often. Students became more confident in themselves as mathematics students and, in some cases, their grades improved. As a result of this research, I plan to teach and explain to my future classes about how showing their work can benefit them in a variety of ways. They will be able to use the knowledge that they gain in my classroom in their future math classes in middle and high school

Fumarate Inhibits TNF-α Release

Vertebrates utilize two forms of immunity to combat pathogens. Innate immunity is considered the first line of defense that utilizes immediate action and three barriers. Innate immunity responses typically occur within minutes of pathogen exposure resulting in cellular receptor activation and acute pro-inflammatory cytokine release. Inflammatory macrophages engage bacterial endotoxins, including Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA). And respond by releasing tumor necrosis factor-α (TNF-α). While beneficial for neutralizing acute pathogen exposure, prolonged TNF-α release results in chronic inflammation and tissue damage. In the present study, we examined two methylated derivatives of a citric acid cycle intermediate, fumarate, as potential mediators of endotoxin-induced TNF-α release. We determined that 10µM dimethyl fumarate (DMF) significantly inhibited LPS-mediated soluble TNF-α released from the mouse monocyte cell line RAW 264.7, with no effect on cell viability or reactive oxygen species (ROS) generation. Monomethyl fumarate (MMF) and DMF showed minimal effects on cell viability or ROS generation. We found that DMF at 10µM is anti-inflammatory with minimal monocyte cytotoxicity. The cellular mechanism of DMF action remains unknown

Experimental studies of adult longevity of the blowfly, Calliphora stygia (Diptera: Calliphoridae)

There are two commonly used environmental manipulations that have been shown to extend longevity across a range of animal species: temperature (in ectotherms) and dietary restriction (in endotherms and ectotherms). Using these two manipulations in model organisms, a number of correlations between levels of oxidative damage and longevity have been found, which are largely consistent with the „oxidative stress hypothesis of ageing‟. However, before mechanisms identified in a few model organisms can be accepted as being general features of the ageing process, it is essential that these manipulations are shown to extend lifespan in a broader group of animals. Ageing is measured through both demographic measures of mortality and by determination of changes in physiological functionality at cellular and molecular levels. Rodent models are typically used, due to their physiological similarity to humans and because their body size enables thorough biochemical analysis. The lifespans of rodents, however, require experiments lasting several years to obtain demographic measurements. By contrast, invertebrate models have the demographic advantage of short lifespans and relative ease of husbandry and experimental manipulation, but their small size often limits biochemical measures, which are based on pooled samples from many individuals. The ageing process is not however, constant across tissue-types, therefore, pooling individuals can lead to both underestimating individual variation in the ageing process and missing age-related tissue specific effects. This thesis examines longevity and membrane fatty acid composition in a new model organism, the blowfly, Calliphora stygia. This species has the advantage of a short lifespan but a relatively large body size among flies, that allows for individualbased measurements. This is advantageous not only for biochemical measures, but also for implementation of treatments such as dietary restriction, where food consumption of populations can be measured over the entire lifespan. I examined the effects of temperature and diet energy content on longevity and demographic and cellular senescence. Temperature effects were examined both after exposure to constant temperatures, and after transfer to lower or higher temperatures. Diet energy content was varied by either altering the sugar content of the diet, or by varying the yeast content of the diet, to examine potential differences between diet composition and energy consumption