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: Fabrication of advanced heat sink materials by powder metallurgy approach

authors: Chang, Yaqi

abstract: To guarantee a safe work environment for high-end chips, electronic packaging materials should have high thermal conductivity, matched coefficient of thermal expansion to semiconductor, low density, good impact toughness, and tensile strength. Diamond/graphite flakes-reinforced composites are considered as the new generation of heat sink material, however, the poor chemical affinity between copper and diamond and between copper and graphite flakes hinder the composites from achieving high thermal conductivity. The interface between copper matrix and diamond reinforcement or graphite flakes reinforcement is the key factor for improving heat transfer of the composites. 

This study aims to investigate the mechanisms of how the interface improves the thermal conductivity of the diamond/graphite flakes-reinforced composites. The primary research includes: (1) study the effect of vacuum sintering process on the formation of carbide layer; (2) investigate the effect of the volume fraction of diamond particles on relative density and thermal conductivity of the copper/diamond composites, and (3) explore the feasibility to fabricate graphite flakes reinforced copper composites by powder forging and investigate the effect of graphite flakes’ volume fraction on the thermal conductivity of the fabricated composites.

Tungsten carbide (WC) can be formed during the vacuum sintering process, however, the formed WC were broken during the subsequent 1050 ℃ hot forging process. The WC can be formed during the vacuum sintering process above 550 ℃ for 30mins, on both diamond -{100}facet and -{111}facet. Cu-55WDia-1050HF has a thermal conductivity of 230.5W/(mK) which is higher than Cu-55WDia-VS800C2h-1050 of 195.1W/(mK). With the volume fraction of diamond from 45vol% to 65vol%, the thermal conductivity reduced from 250.8 to 65.1W/(mK). For copper/graphite flakes composites, the addition of carbide forming element Ti helped to improve the thermal conductivity. Because of the excessive copper fluidity in Cu-Ti-50GFs-1050HF, Cu-Ti-50GFs-1050HF has the lower thermal conductivity of 202.7 W/(mK) than thermal conductivity of Cu-Ti-50GFs-950HF which is 375.0 W/(mK). With the volume fraction of the graphite flakes from 30% to 70%, the relative density changed from 100% to 83.3%.

text: Fabrication of advanced heat sink materials by powder metallurgy approach To guarantee a safe work environment for high-end chips, electronic packaging materials should have high thermal conductivity, matched coefficient of thermal expansion to semiconductor, low density, good impact toughness, and tensile strength. Diamond/graphite flakes-reinforced composites are considered as the new generation of heat sink material, however, the poor chemical affinity between copper and diamond and between copper and graphite flakes hinder the composites from achieving high thermal conductivity. The interface between copper matrix and diamond reinforcement or graphite flakes reinforcement is the key factor for improving heat transfer of the composites. 

This study aims to investigate the mechanisms of how the interface improves the thermal conductivity of the diamond/graphite flakes-reinforced composites. The primary research includes: (1) study the effect of vacuum sintering process on the formation of carbide layer; (2) investigate the effect of the volume fraction of diamond particles on relative density and thermal conductivity of the copper/diamond composites, and (3) explore the feasibility to fabricate graphite flakes reinforced copper composites by powder forging and investigate the effect of graphite flakes’ volume fraction on the thermal conductivity of the fabricated composites.

Tungsten carbide (WC) can be formed during the vacuum sintering process, however, the formed WC were broken during the subsequent 1050 ℃ hot forging process. The WC can be formed during the vacuum sintering process above 550 ℃ for 30mins, on both diamond -{100}facet and -{111}facet. Cu-55WDia-1050HF has a thermal conductivity of 230.5W/(mK) which is higher than Cu-55WDia-VS800C2h-1050 of 195.1W/(mK). With the volume fraction of diamond from 45vol% to 65vol%, the thermal conductivity reduced from 250.8 to 65.1W/(mK). For copper/graphite flakes composites, the addition of carbide forming element Ti helped to improve the thermal conductivity. Because of the excessive copper fluidity in Cu-Ti-50GFs-1050HF, Cu-Ti-50GFs-1050HF has the lower thermal conductivity of 202.7 W/(mK) than thermal conductivity of Cu-Ti-50GFs-950HF which is 375.0 W/(mK). With the volume fraction of the graphite flakes from 30% to 70%, the relative density changed from 100% to 83.3%.

year: 2021

title: Ati Ue: Te Aitanga a Tuoro

authors: James, Steven (Tipene) Robert

subjects: Ngati Whakaue

abstract: This thesis is based on the manuscripts of my tupuna Tuoro Akapita Pango that are held in a treasured whānau box. The manuscripts contain history and kōrero of Te Arawa written by multiple generations of the whānau Pango. Within this thesis I explore the value of inter-generational knowledge through considering what lies within these whānau manuscripts and taking the opportunity to discover more history about Ngāti Whakaue through a whānau lens. Researching these manuscripts enables reconnection of the past and the present; the reconnection between forgotten knowledge and whānau. 
Specifically, this thesis proposes a Ngāti Whakaue approach to engaging historical materials and research. It centres the voices of the people, such as the many family members involved within the process, as well as the box of writings and the many gems that hide within. Also, it asks what the manuscripts mean for the current descendants involved in the research. 
The research has been a means of reconnecting with the past to give relevance and context to the present. The objective is demonstrating the value of iwi-specific research methods so that whānau feel encouraged to embark on their own journey.

text: Ati Ue: Te Aitanga a Tuoro This thesis is based on the manuscripts of my tupuna Tuoro Akapita Pango that are held in a treasured whānau box. The manuscripts contain history and kōrero of Te Arawa written by multiple generations of the whānau Pango. Within this thesis I explore the value of inter-generational knowledge through considering what lies within these whānau manuscripts and taking the opportunity to discover more history about Ngāti Whakaue through a whānau lens. Researching these manuscripts enables reconnection of the past and the present; the reconnection between forgotten knowledge and whānau. 
Specifically, this thesis proposes a Ngāti Whakaue approach to engaging historical materials and research. It centres the voices of the people, such as the many family members involved within the process, as well as the box of writings and the many gems that hide within. Also, it asks what the manuscripts mean for the current descendants involved in the research. 
The research has been a means of reconnecting with the past to give relevance and context to the present. The objective is demonstrating the value of iwi-specific research methods so that whānau feel encouraged to embark on their own journey.

year: 2021