title: GFP tagged Hsp60 used to investigate the translocation of Hsp60 in response to mitochondrial stress in HeLa cells

authors: Short, Matthew Edward

subjects: Hsp60

abstract: Heat shock protein 60 (Hsp60) is a mitochondrial stress protein that is elevated in response to mitochondrial impairment. It is involved in protein folding and, more recently, it has been identified as a signalling molecule. It has been found elevated in patients suffering from diabetes, atherosclerosis and cardiomyopathy. In order to investigate the translocation of Hsp60 from mitochondria we employed RG224428, a plasmid housing genes for a human Hsp60 connected to a green fluorescent protein (GFP) bound at the C-terminus. The plasmid was cloned inside DH5α E. coli (DH5α) and purified using an endotoxin-free plasmid extraction kit. Using endotoxin-free DNA we transfected the human cervical cancer cell line HeLa with the Hsp60-GFP DNA. It has been found that endotoxin (or otherwise known as lipopolysaccharide) can affect Hsp60 expression in mammalian cells, so by using endotoxin-free plasmid DNA we could induce controlled stress treatments without the unwanted effects the DH5α endotoxin would exhibit. Propidium iodide staining found the plasmid posed very low-to-no toxicity on HeLa cells. Mitochondrial staining using Mitotracker CMX Ros showed that the Hsp60-GFP was being taken up by mitochondria almost exclusively. High-glucose and sodium azide were used to stress the transfected HeLa cells and the translocation of the Hsp60-GFP signal was recorded using an Olympus FV1000 laser scanning confocal microscope.
We found that the Hsp60-GFP colocalises with mitochondria to a high degree. We also found that high-glucose can be linked to the translocation of Hsp60-GFP out of mitochondria. The results here support published data that suggest higher glucose concentrations may play a role in the translocation of Hsp60 out of mitochondria and into extracellular fluids. We have also found that the use of fluorescent-confocal microscopy may provide a useful way to detect the movement of the biomarker Hsp60 under stress and therapeutic treatments.

text: GFP tagged Hsp60 used to investigate the translocation of Hsp60 in response to mitochondrial stress in HeLa cells Heat shock protein 60 (Hsp60) is a mitochondrial stress protein that is elevated in response to mitochondrial impairment. It is involved in protein folding and, more recently, it has been identified as a signalling molecule. It has been found elevated in patients suffering from diabetes, atherosclerosis and cardiomyopathy. In order to investigate the translocation of Hsp60 from mitochondria we employed RG224428, a plasmid housing genes for a human Hsp60 connected to a green fluorescent protein (GFP) bound at the C-terminus. The plasmid was cloned inside DH5α E. coli (DH5α) and purified using an endotoxin-free plasmid extraction kit. Using endotoxin-free DNA we transfected the human cervical cancer cell line HeLa with the Hsp60-GFP DNA. It has been found that endotoxin (or otherwise known as lipopolysaccharide) can affect Hsp60 expression in mammalian cells, so by using endotoxin-free plasmid DNA we could induce controlled stress treatments without the unwanted effects the DH5α endotoxin would exhibit. Propidium iodide staining found the plasmid posed very low-to-no toxicity on HeLa cells. Mitochondrial staining using Mitotracker CMX Ros showed that the Hsp60-GFP was being taken up by mitochondria almost exclusively. High-glucose and sodium azide were used to stress the transfected HeLa cells and the translocation of the Hsp60-GFP signal was recorded using an Olympus FV1000 laser scanning confocal microscope.
We found that the Hsp60-GFP colocalises with mitochondria to a high degree. We also found that high-glucose can be linked to the translocation of Hsp60-GFP out of mitochondria. The results here support published data that suggest higher glucose concentrations may play a role in the translocation of Hsp60 out of mitochondria and into extracellular fluids. We have also found that the use of fluorescent-confocal microscopy may provide a useful way to detect the movement of the biomarker Hsp60 under stress and therapeutic treatments.

year: 2013

title: Embodying Sexy: Narrative Inquiry into Female Sexuality and Consumer Culture

authors: Travis, Courtney

subjects: Female Sexuality

abstract: This research investigates female sexuality and consumer culture. It focuses on sexy as a form of expressing sexuality. The main objectives of this research were to understand what sexy meant to a group of young women and how they were socialised into their meaning through the consumer culture they live in. A narrative inquiry is used to understand these women’s embodied experience using collective biography and digital storytelling methods. A group of seven women participated in three group meetings to collectively research sexy. Collective biography and digital storytelling were layered upon one another to understand these women’s experiences of sexy and to enable collaborative analysis. Through the meetings the women progressed through the meetings in their comprehension of their own and the collective’s meaning of sexy. They did so through the creation of memory and digital stories. The research highlighted sexy as complex, contradictory, consequential, and dynamic for these women. Sexy was complex because it was embodied as multisensory. It was also contradictory because the images of sexy presented in consumer culture are not consistent with these women’s beliefs. The contrary nature of sexy resulted in envy, low self-esteem, and objectification. Sexy was also dynamic because it changed as the women matured. Sexy is embodied. It requires more than simply achieving just the look. It is experienced as multisensory. These women’s experiences and images of sexy differ from those presented in consumer culture. The research challenges the assumption that adult women can and do act on their knowledge that adverts are fantasy. It provides a new insight by highlighting transformation through the collaborative research process. These women were able to understand how they had been socialised into their meaning of sexy and make better informed choices. The research also highlighted the fine line these women walk between slutty and sexy due to the virgin/whore dichotomy. This research contributes in two ways: by suggesting future directions for the sexual education of adolescent girls and more research on young adult women, their experiences of sexuality, and the influences from consumer culture.

text: Embodying Sexy: Narrative Inquiry into Female Sexuality and Consumer Culture This research investigates female sexuality and consumer culture. It focuses on sexy as a form of expressing sexuality. The main objectives of this research were to understand what sexy meant to a group of young women and how they were socialised into their meaning through the consumer culture they live in. A narrative inquiry is used to understand these women’s embodied experience using collective biography and digital storytelling methods. A group of seven women participated in three group meetings to collectively research sexy. Collective biography and digital storytelling were layered upon one another to understand these women’s experiences of sexy and to enable collaborative analysis. Through the meetings the women progressed through the meetings in their comprehension of their own and the collective’s meaning of sexy. They did so through the creation of memory and digital stories. The research highlighted sexy as complex, contradictory, consequential, and dynamic for these women. Sexy was complex because it was embodied as multisensory. It was also contradictory because the images of sexy presented in consumer culture are not consistent with these women’s beliefs. The contrary nature of sexy resulted in envy, low self-esteem, and objectification. Sexy was also dynamic because it changed as the women matured. Sexy is embodied. It requires more than simply achieving just the look. It is experienced as multisensory. These women’s experiences and images of sexy differ from those presented in consumer culture. The research challenges the assumption that adult women can and do act on their knowledge that adverts are fantasy. It provides a new insight by highlighting transformation through the collaborative research process. These women were able to understand how they had been socialised into their meaning of sexy and make better informed choices. The research also highlighted the fine line these women walk between slutty and sexy due to the virgin/whore dichotomy. This research contributes in two ways: by suggesting future directions for the sexual education of adolescent girls and more research on young adult women, their experiences of sexuality, and the influences from consumer culture.

year: 2011

title: Indigenous cancer research: Reflections on roles and responsibilities

authors: Scott, Nina

abstract: When asked for a commentary on the roles and responsibilities of nonindigenous researchers in indigenous cancer research, several potential nonindigenous coauthors were contacted. The majority wanted to “run for the hills,” signifying that many researchers feel uncomfortable, out of their depth, and fearful regarding methodologies and principles for safe and effective research with and for indigenous peoples.

Most health research is relevant for indigenous peoples, and most researchers are nonindigenous. Thus, if researchers don’t engage with indigenous research principles, health research can and does result in significant harm for indigenous peoples.1,2 Nonengagement may also prevent potential positive outcomes for indigenous communities (eg, health gain, workforce and community development) and impinge on indigenous rights to health and development.3

In this commentary, we outline our reflections and observations on the roles and responsibilities of nonindigenous cancer researchers and identify some of the principles that guide researchers in Aotearoa (Aotearoa is the name given by the indigenous Māori for New Zealand). We do not attempt to provide an in-depth analysis or comprehensive best practice summary, but hope to encourage more discussion in this critical and evolving area.

text: Indigenous cancer research: Reflections on roles and responsibilities When asked for a commentary on the roles and responsibilities of nonindigenous researchers in indigenous cancer research, several potential nonindigenous coauthors were contacted. The majority wanted to “run for the hills,” signifying that many researchers feel uncomfortable, out of their depth, and fearful regarding methodologies and principles for safe and effective research with and for indigenous peoples.

Most health research is relevant for indigenous peoples, and most researchers are nonindigenous. Thus, if researchers don’t engage with indigenous research principles, health research can and does result in significant harm for indigenous peoples.1,2 Nonengagement may also prevent potential positive outcomes for indigenous communities (eg, health gain, workforce and community development) and impinge on indigenous rights to health and development.3

In this commentary, we outline our reflections and observations on the roles and responsibilities of nonindigenous cancer researchers and identify some of the principles that guide researchers in Aotearoa (Aotearoa is the name given by the indigenous Māori for New Zealand). We do not attempt to provide an in-depth analysis or comprehensive best practice summary, but hope to encourage more discussion in this critical and evolving area.

year: 2020