title: Expression of TNF- α at mRNA level in Thp-1 cells exposed to LPS and HSP60: Possible impact on diabetic vascular inflammation

authors: Azfar, Afreen

subjects: mRNA

abstract: Diabetes mellitus is a chronic disease characterized by hyperglycaemia. It occurs when the b cells of the pancreas produce either low levels of insulin or no insulin at all. The chronic hyperglycaemic condition of diabetes is associated with the dysfunction, long-term damage and failure of organs such as the heart and blood vessels. Diabetes is closely linked to the prevalence of cardiovascular diseases, which is one of the highest causes of death worldwide. In particular, the hyperglycaemic and chronic inflammatory state of diabetes has been linked with atherogenesis, which is when fatty deposits build up in the arteries and they become inflamed. It still remains unknown how diabetes contributes to atherosclerosis.
Heat Shock Protein 60 (HSP60) is an important endogenous inflammatory mediator elevated in diabetic patients. HSP60 can be released from various cell types, including monocyte cells, which are the most common cell type found in atherosclerotic plaques. The mechanism behind how HSP60 triggers endothelial cell inflammation remains unknown, but has been hypothesised to be similar to that of a Toll like receptor 4 ligand, lipopolysaccharide (LPS). Understanding the molecular mechanism of HSP60 could give insights into the pathological, cellular and molecular basis of vascular inflammation in diabetes as well as other vascular diseases such as cardiovascular disease.
It is hypothesized that monocyte-derived HSP60 has a role in causing inflammation in the diabetic vasculature through the expression of pro- inflammatory cytokines such as TNF-α, which could contribute to atherosclerosis. In this study a human monocyte cell line (Thp-1) was used to mimic monocyte behaviour. First a growth curve was established, which indicated when the cells were in their exponential phase. This enabled RNA to be extracted during the period when cells were growing the fastest.
Thp-1 cells were then exposed to LPS for 6, 12, and 24 hours to determine the expression of TNF-α. After conducting RT-PCR, a 12 hour time point was selected. Since inconsistent results were obtained using this semi- quantitative analysis, qPCR was carried out for the rest of the project.
Thp-1 cells were then exposed to 50ng/mL and 100ng/mL of LPS for 12 hours. After conducting and analysing qPCR using the delta-delta Ct method and the Pfaffl method, a ~2 fold increase was seen in the expression of a pro-inflammatory cytokine, TNF-α. These results however were not significant (determined by a Student’s T test).
In order to obtain a higher level of TNF-α expression, this experiment was repeated with 10ng/mL and 50ng/mL of LPS for 4 hours, based on previous published research. These results were analysed using the delta-delta Ct method and the Pfaffl method. The results were not what was expected; only an insignificant <1 fold increase was seen in the samples.
Thp-1 cells were also exposed to 500ng/mL and 1500ng/mL of HSP60 for 4 hours in order to see a TNF-α induction. HSP60 has been hypothesised to work through the same mechanism as LPS to trigger TNF-α , leading to an inflammatory response. However again, no significant increase in expression was seen (as determined by a Student’s T test).
The inability to detect a significant up-regulation of the expression of TNF-α at mRNA level with LPS (positive control) and HSP60 in Thp-1 cells was surprising as this is well documented in the literature. This could possibly have been due to the high passage number of the Thp-1 cell cultures used in this study; mycoplasma contamination or stability of the LPS used. The study therefore needs to be repeated with cells of a lower passage number and cells checked for mycoplasma contamination to establish appropriate time frames of exposure and concentrations of LPS and HSP60 required to see a TNF-α induction. This could then be followed by experiments using receptor antagonists, such as TAK-242, (a TLR4 antagonist) to determine if TNF-α expression levels would give an indication of whether or not HSP60 was using the same pathway as LPS, and if it indeed played a part in causing diabetic vascular inflammation.

text: Expression of TNF- α at mRNA level in Thp-1 cells exposed to LPS and HSP60: Possible impact on diabetic vascular inflammation Diabetes mellitus is a chronic disease characterized by hyperglycaemia. It occurs when the b cells of the pancreas produce either low levels of insulin or no insulin at all. The chronic hyperglycaemic condition of diabetes is associated with the dysfunction, long-term damage and failure of organs such as the heart and blood vessels. Diabetes is closely linked to the prevalence of cardiovascular diseases, which is one of the highest causes of death worldwide. In particular, the hyperglycaemic and chronic inflammatory state of diabetes has been linked with atherogenesis, which is when fatty deposits build up in the arteries and they become inflamed. It still remains unknown how diabetes contributes to atherosclerosis.
Heat Shock Protein 60 (HSP60) is an important endogenous inflammatory mediator elevated in diabetic patients. HSP60 can be released from various cell types, including monocyte cells, which are the most common cell type found in atherosclerotic plaques. The mechanism behind how HSP60 triggers endothelial cell inflammation remains unknown, but has been hypothesised to be similar to that of a Toll like receptor 4 ligand, lipopolysaccharide (LPS). Understanding the molecular mechanism of HSP60 could give insights into the pathological, cellular and molecular basis of vascular inflammation in diabetes as well as other vascular diseases such as cardiovascular disease.
It is hypothesized that monocyte-derived HSP60 has a role in causing inflammation in the diabetic vasculature through the expression of pro- inflammatory cytokines such as TNF-α, which could contribute to atherosclerosis. In this study a human monocyte cell line (Thp-1) was used to mimic monocyte behaviour. First a growth curve was established, which indicated when the cells were in their exponential phase. This enabled RNA to be extracted during the period when cells were growing the fastest.
Thp-1 cells were then exposed to LPS for 6, 12, and 24 hours to determine the expression of TNF-α. After conducting RT-PCR, a 12 hour time point was selected. Since inconsistent results were obtained using this semi- quantitative analysis, qPCR was carried out for the rest of the project.
Thp-1 cells were then exposed to 50ng/mL and 100ng/mL of LPS for 12 hours. After conducting and analysing qPCR using the delta-delta Ct method and the Pfaffl method, a ~2 fold increase was seen in the expression of a pro-inflammatory cytokine, TNF-α. These results however were not significant (determined by a Student’s T test).
In order to obtain a higher level of TNF-α expression, this experiment was repeated with 10ng/mL and 50ng/mL of LPS for 4 hours, based on previous published research. These results were analysed using the delta-delta Ct method and the Pfaffl method. The results were not what was expected; only an insignificant <1 fold increase was seen in the samples.
Thp-1 cells were also exposed to 500ng/mL and 1500ng/mL of HSP60 for 4 hours in order to see a TNF-α induction. HSP60 has been hypothesised to work through the same mechanism as LPS to trigger TNF-α , leading to an inflammatory response. However again, no significant increase in expression was seen (as determined by a Student’s T test).
The inability to detect a significant up-regulation of the expression of TNF-α at mRNA level with LPS (positive control) and HSP60 in Thp-1 cells was surprising as this is well documented in the literature. This could possibly have been due to the high passage number of the Thp-1 cell cultures used in this study; mycoplasma contamination or stability of the LPS used. The study therefore needs to be repeated with cells of a lower passage number and cells checked for mycoplasma contamination to establish appropriate time frames of exposure and concentrations of LPS and HSP60 required to see a TNF-α induction. This could then be followed by experiments using receptor antagonists, such as TAK-242, (a TLR4 antagonist) to determine if TNF-α expression levels would give an indication of whether or not HSP60 was using the same pathway as LPS, and if it indeed played a part in causing diabetic vascular inflammation.

year: 2018

title: Problematising constructions of ‘expert teachers’ and ‘vulnerable children’ in New Zealand Early Childhood

authors: Kamenarac, Olivera

subjects: teacher identities

abstract: Over the last two decades, early childhood education (ECE) has come to the top of the political agenda in New Zealand and internationally. The interest of governments and policy-makers in ECE has been driven by the thinking that an 'investment' in ECE is a key a country's future social and economic development (Heckman 2000). Through an interplay of the existing early childhood discourses and emerging economic discourses, the contexts in which early childhood teachers understand and undertake their work and view themselves as professionals have, thus rapidly changed (Penn 2013; Brown et al. 2018; Pupala, Kascak, and Tesar 2016). This article problematises constructions of 'expert teachers' and 'vulnerable children', which emerged through an interplay of discourses of social intervention, economic investment and vulnerability in New Zealand ECE. It argues that the identity constructions on offer have a powerful potential to reinforce social-interventionist and neoliberal narratives in ECE, and move its purpose away from the idea of collective democracy (Dewey 1976). Keywords: teacher identities; New Zealand early childhood education; early childhood policies and politics; discourses of vulnerability; poststructural perspectives

text: Problematising constructions of ‘expert teachers’ and ‘vulnerable children’ in New Zealand Early Childhood Over the last two decades, early childhood education (ECE) has come to the top of the political agenda in New Zealand and internationally. The interest of governments and policy-makers in ECE has been driven by the thinking that an 'investment' in ECE is a key a country's future social and economic development (Heckman 2000). Through an interplay of the existing early childhood discourses and emerging economic discourses, the contexts in which early childhood teachers understand and undertake their work and view themselves as professionals have, thus rapidly changed (Penn 2013; Brown et al. 2018; Pupala, Kascak, and Tesar 2016). This article problematises constructions of 'expert teachers' and 'vulnerable children', which emerged through an interplay of discourses of social intervention, economic investment and vulnerability in New Zealand ECE. It argues that the identity constructions on offer have a powerful potential to reinforce social-interventionist and neoliberal narratives in ECE, and move its purpose away from the idea of collective democracy (Dewey 1976). Keywords: teacher identities; New Zealand early childhood education; early childhood policies and politics; discourses of vulnerability; poststructural perspectives

year: 2021

title: Ngā Whanaketanga: Minimising contradictions and maximising opportunities for teacher learning.

authors: Hohepa, Margie Kahukura

subjects: Māori medium teacher professional development

abstract: In 2010 the assessment of student progress and achievement against National Standards became mandatory in English medium schooling in Aotearoa New Zealand. In that same year information gathering and revision work was carried out on the then draft Whanaketanga, developed in 2009. The Whanaketanga are deemed to be the equivalent to National Standards for classrooms implementing Te Marautanga o Aotearoa, the curriculum for immersion Māori medium settings. The information gathering and revision work was carried out in readiness for mandatory implementation of the Whanaketanga in 2011. The work also provided important opportunities for Māori medium teacher professional learning and development. The introduction of National Standards has been controversial. The development of Whanaketanga has also been controversial and challenging in nature. The first half of this paper provides an overview of Whanaketanga development and the challenges its developers worked hard to address. The second part discusses implications for Māori medium teacher professional learning and development, drawn from findings from research case studies of the information gathering and revision work.

text: Ngā Whanaketanga: Minimising contradictions and maximising opportunities for teacher learning. In 2010 the assessment of student progress and achievement against National Standards became mandatory in English medium schooling in Aotearoa New Zealand. In that same year information gathering and revision work was carried out on the then draft Whanaketanga, developed in 2009. The Whanaketanga are deemed to be the equivalent to National Standards for classrooms implementing Te Marautanga o Aotearoa, the curriculum for immersion Māori medium settings. The information gathering and revision work was carried out in readiness for mandatory implementation of the Whanaketanga in 2011. The work also provided important opportunities for Māori medium teacher professional learning and development. The introduction of National Standards has been controversial. The development of Whanaketanga has also been controversial and challenging in nature. The first half of this paper provides an overview of Whanaketanga development and the challenges its developers worked hard to address. The second part discusses implications for Māori medium teacher professional learning and development, drawn from findings from research case studies of the information gathering and revision work.

year: 2012