title: A Model In Vitro System to Study Heat Shock Protein 60 (HSP60) Expression in Response to Mitochondrial Impairment in Human Cells

authors: Hall, Luke

subjects: HSP60

abstract: Heat Shock Proteins (HSPs) are a class of ubiquitously expressed and functionally related proteins found in all living organisms from humans to bacteria. Their expression is increased in response to various cellular stressors in what is referred to as the heat shock response. The induction of one particular HSP, HSP60, has been found to be correlated with mitochondria specific cell stress. Recently HSP60 has been found to be secreted and expressed extracellularly, after first being thought to be strictly intracellular. However, the mechanisms of these mechanisms of translocation and secretion have not been clearly identified. 
The aim of this study was to develop a model in vitro system for HSP60 expression in human cells so that treatments which resulted in mitochondrial impairment could be investigated. 
This study looked at several aspects of high glucose, hydrogen peroxide (H₂O₂), and sodium azide treatment on human HeLa cells. At low levels, each treatment had a hormetic effect on HeLa cell growth; however at high concentrations growth was significantly inhibited. Additionally, high treatment concentrations resulted in increased cell lysis as determined by LDH assays. Treatment with concentrations of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide were the only treatment concentrations that did not result in significantly different levels of cell lysis when compared to a control sample. Mitochondrial dehydrogenase activity was also found to be significantly decreased at high treatment concentrations as determined by MTT assays. Thus, 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were identified as optimal conditions for mitochondrial targeted cell stress, as each of these treatments impaired cell growth and inhibited mitochondrial activity while having no significant effect on the degree of cell lysis. 
The 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were then investigated for the intracellular reactive oxygen species (ROS) activity they induced over treatment periods of 24 hours, 3 and 7 days. After 7 days of treatment, the 200μM H₂O₂ and 50μM sodium azide treatments resulted in an approximate three-fold increase in ROS activity, while the 100mM glucose treatment resulted in almost twice as much ROS compared to a control sample.
Finally, the effects of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments on HSP60 and HSP70 expression was also examined over 3 and 7 day time periods. After the 7 day treatment period, the 100mM glucose treatment had induced a 2.43 fold increase in HSP60 expression and a 2.75 fold increase in HSP70 expression, the 200μM H₂O₂ induced a 3.48 fold increase in HSP60 expression and a 3.98 fold increase in HSP70 expression, and finally the 50μM sodium azide induced a 4.74 fold increase in HSP60 expression and a 5.08 fold increase in HSP70 expression. 
It can therefore be concluded that 100mM glucose, 200μM H₂O₂, and 50μM sodium azide results in the upregulation of HSP60 and HSP70 in human HeLa cells. Therefore this model may be used to investigate further aspects of HSP60 induction, such as its translocation and secretion.

text: A Model In Vitro System to Study Heat Shock Protein 60 (HSP60) Expression in Response to Mitochondrial Impairment in Human Cells Heat Shock Proteins (HSPs) are a class of ubiquitously expressed and functionally related proteins found in all living organisms from humans to bacteria. Their expression is increased in response to various cellular stressors in what is referred to as the heat shock response. The induction of one particular HSP, HSP60, has been found to be correlated with mitochondria specific cell stress. Recently HSP60 has been found to be secreted and expressed extracellularly, after first being thought to be strictly intracellular. However, the mechanisms of these mechanisms of translocation and secretion have not been clearly identified. 
The aim of this study was to develop a model in vitro system for HSP60 expression in human cells so that treatments which resulted in mitochondrial impairment could be investigated. 
This study looked at several aspects of high glucose, hydrogen peroxide (H₂O₂), and sodium azide treatment on human HeLa cells. At low levels, each treatment had a hormetic effect on HeLa cell growth; however at high concentrations growth was significantly inhibited. Additionally, high treatment concentrations resulted in increased cell lysis as determined by LDH assays. Treatment with concentrations of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide were the only treatment concentrations that did not result in significantly different levels of cell lysis when compared to a control sample. Mitochondrial dehydrogenase activity was also found to be significantly decreased at high treatment concentrations as determined by MTT assays. Thus, 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were identified as optimal conditions for mitochondrial targeted cell stress, as each of these treatments impaired cell growth and inhibited mitochondrial activity while having no significant effect on the degree of cell lysis. 
The 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were then investigated for the intracellular reactive oxygen species (ROS) activity they induced over treatment periods of 24 hours, 3 and 7 days. After 7 days of treatment, the 200μM H₂O₂ and 50μM sodium azide treatments resulted in an approximate three-fold increase in ROS activity, while the 100mM glucose treatment resulted in almost twice as much ROS compared to a control sample.
Finally, the effects of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments on HSP60 and HSP70 expression was also examined over 3 and 7 day time periods. After the 7 day treatment period, the 100mM glucose treatment had induced a 2.43 fold increase in HSP60 expression and a 2.75 fold increase in HSP70 expression, the 200μM H₂O₂ induced a 3.48 fold increase in HSP60 expression and a 3.98 fold increase in HSP70 expression, and finally the 50μM sodium azide induced a 4.74 fold increase in HSP60 expression and a 5.08 fold increase in HSP70 expression. 
It can therefore be concluded that 100mM glucose, 200μM H₂O₂, and 50μM sodium azide results in the upregulation of HSP60 and HSP70 in human HeLa cells. Therefore this model may be used to investigate further aspects of HSP60 induction, such as its translocation and secretion.

year: 2013

title: Adsorption of Colour from Pulp and Paper Mill Wastewaters onto Diatomaceous Earths

authors: Carter, Shane Basil

subjects: Adsorption of colour

abstract: Abstract

Production of pulp and paper from raw wood involves the washing of substantial quantities of highly coloured material from the wood pulp. Although most of this colour is trapped within the mill and the mills wastewater treatment system, discharge of coloured wastewater remains a problem of public concern.

Lake Rotorua, New Zealand is filled with sediment consisting almost exclusively of diatoms. The most abundant species, Aulacoseira, has been shown to have very small pores, less than 200 nm, that could be very useful for the capture and holding of large organic molecules. Lake Rotorua is situated close to the two largest pulp and paper mills in New Zealand and may have to be dredged to solve a problem of eutrophication. Wastewaters generated at the Kinleith (Tokoroa) and Tasman (Kawerau) mills in the North island of New Zealand have been used to assess the characteristics of colour removal by Lake Rotorua diatoms.

Vacuum filtration through the diatoms succeeded in removing colour but proved to be impractically slow as an industrial process. Addition of diatoms to wastewater streams in stirred experiments showed that significant adsorption took place. The majority of the colour was removed within 30 seconds of mixing. The efficiency of colour removal was found to vary between wastewater streams within the mills. The pH dependency of this removal was tested and found to be most effective in a pH range of 3.6 to 5.

Attempts to reuse the diatoms showed that chemical backwash regimes were more effective than calcination.

This study concluded that surface silanols were likely to be the principle agent in forming coagulants with the colour material and were subsequently trapped on the diatom surfaces, but more importantly within the pores.

text: Adsorption of Colour from Pulp and Paper Mill Wastewaters onto Diatomaceous Earths Abstract

Production of pulp and paper from raw wood involves the washing of substantial quantities of highly coloured material from the wood pulp. Although most of this colour is trapped within the mill and the mills wastewater treatment system, discharge of coloured wastewater remains a problem of public concern.

Lake Rotorua, New Zealand is filled with sediment consisting almost exclusively of diatoms. The most abundant species, Aulacoseira, has been shown to have very small pores, less than 200 nm, that could be very useful for the capture and holding of large organic molecules. Lake Rotorua is situated close to the two largest pulp and paper mills in New Zealand and may have to be dredged to solve a problem of eutrophication. Wastewaters generated at the Kinleith (Tokoroa) and Tasman (Kawerau) mills in the North island of New Zealand have been used to assess the characteristics of colour removal by Lake Rotorua diatoms.

Vacuum filtration through the diatoms succeeded in removing colour but proved to be impractically slow as an industrial process. Addition of diatoms to wastewater streams in stirred experiments showed that significant adsorption took place. The majority of the colour was removed within 30 seconds of mixing. The efficiency of colour removal was found to vary between wastewater streams within the mills. The pH dependency of this removal was tested and found to be most effective in a pH range of 3.6 to 5.

Attempts to reuse the diatoms showed that chemical backwash regimes were more effective than calcination.

This study concluded that surface silanols were likely to be the principle agent in forming coagulants with the colour material and were subsequently trapped on the diatom surfaces, but more importantly within the pores.

year: 2007

title: Bilingual/Immersion education: Indicators of good practice

authors: May, Stephen

subjects: Maori

abstract: Based on a review of national and international research on bilingualism and bilingual/immersion education, this report explores effective approaches for bilingual education. While the focus was on Māori-medium education, the indicators of good practice can also be applied to other bilingual contexts in Aotearoa/New Zealand, such as Pasifika bilingual education.

text: Bilingual/Immersion education: Indicators of good practice Based on a review of national and international research on bilingualism and bilingual/immersion education, this report explores effective approaches for bilingual education. While the focus was on Māori-medium education, the indicators of good practice can also be applied to other bilingual contexts in Aotearoa/New Zealand, such as Pasifika bilingual education.

year: 2004