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: Coastal and sediment dynamics in a high-energy, rocky environment

authors: McComb, Peter

abstract: A study of the wave dynamics, hydrodynamics and sediment dynamics was conducted at an energetic open-coast site at New Plymouth, New Zealand. The location features large, shore-normal reefs of volcanic agglomerate extending from an irregular shoreline, with “black” volcanic sands intermittently overlying a rocky bed. The breakwaters of an artificial harbour (Port Taranaki) interrupt the longshore littoral transport, and dredging and offshore dumping causes “downstream” erosion along the city foreshore. The key study objectives were to investigate the local coastal dynamics, and to examine ways to mitigate the effects of the harbour on the adjacent coast. 

Two large experimental field programmes were conducted involving: i) wave, current and suspended sediment measurements, ii) side-scan sonar and hydrographic survey, iii) sediment tracing, and iv) experimental dumping of 47,000 m³ of dredged sand in a rocky nearshore zone (6-10 m depth). Sediment trap calibration experiments were also performed. 

Analysis of the measured wave data has identified wave-induced motion as a significant source of velocity error in p, u, v meters on taut-wire moorings, and wave directions resolved from the cross-spectra of pressure and velocity have frequency dependent errors due to the phase difference between the pressure and velocity data. Accordingly, the spectra of wave direction should be resolved solely from velocity data. Calibration of sediment traps against an automated water sampler has provided the first strong evidence that traps are effective in deriving the time-averaged suspended sediment concentration SSC in a wave-dominated coastal environment. Rigid cylindrical traps of high Aspect Ratio have a 75% sampling efficiency. 

Wave transformations across the complex nearshore bathymetry are dominated by seabed friction and refraction. This process was simulated using the WBEND wave refraction model, applying a constant friction coefficient for sandy beds, and a Nikuradse roughness for rocky beds. Wind vector was found to influence the spatial distribution of wave energy, with whitecapping conditions under onshore-directed winds inducing a longshore “smoothing” in the wave energy, which appears to act in opposition to wave height reinforcement from refraction. 

Sediment tracing using an artificial fluorescent material proved to be an effective method for observing the transport of littoral sediments over sandy and rocky beds, over distances of up to 5 km. The tracer exhibited a horizontal diffusivity of the order 0.1 m²s⁻¹, of which approximately 5% was expressed in the cross-shore direction. 
The time-averaged entrainment (C₀) of “black” volcanic sands under high-energy conditions may be effectively predicted using the excess non-dimensional skin friction (θ₂.₅) raised to the power of 1.5, with the mean grain size used in the sediment mobility term (Ψ) and the median grain size in the friction factor (fw). The scale of turbulent mixing (lₛ) increases linearly with elevation above the bed, with a gradient of 0.46. Suspended sediments show a systematic (and linear) reduction in mean grain size with increasing elevation above bed. 

SSCs over rocky beds are supply-limited, and inversely proportional to the distance from contiguous sandy beds. Sediments are principally transported in the suspended form, and a system of irregular shore-normal reefs does not significantly obstruct the longshore flux. Rocky beds appear to be resilient to sediment inundation and the sand/rock sediment facies have remained positionally stable over 16 years. Dumping sand in a rocky reef environment does not necessarily result in adjacent beds becoming inundated with sand. In this environment, a nearshore dump mound did not migrate as a contiguous body, but slowly dispersed as suspended sediments. 

Net sediment transport at New Plymouth is directed alongshore to the northeast, although the sediments respond to local and temporal perturbations, which give rise to reversing and circulating fluxes. Port Taranaki traps 174,000 m³ of coastal sediments per year, of which 142,000 m³ accumulates at the breakwater tip. The latter are derived from the shallow zone seaward of the breakwater wall (i.e. within ∼50 m). Sediments in depths > 6 m (i.e. > 100 m from the breakwater wall) bypass the harbour and traverse the rocky reefs toward the eastern beaches. The historical offshore dumping practice removes sediments from the nearshore littoral budget, while the orientation of the main port breakwater causes the longshore sediment flux to be directed away from the adjacent (Kawaroa) coast, further reducing the supply of littoral sediments to that region. 

A new dumping ground has been identified for the breakwater-tip sediments, which is projected to retain the dredged sediments within the nearshore littoral system, feeding sand to the central and eastern city foreshore. Sedimentation of the coast immediately adjacent to the port is not expected to result from the long-term use of this ground.

text: Coastal and sediment dynamics in a high-energy, rocky environment A study of the wave dynamics, hydrodynamics and sediment dynamics was conducted at an energetic open-coast site at New Plymouth, New Zealand. The location features large, shore-normal reefs of volcanic agglomerate extending from an irregular shoreline, with “black” volcanic sands intermittently overlying a rocky bed. The breakwaters of an artificial harbour (Port Taranaki) interrupt the longshore littoral transport, and dredging and offshore dumping causes “downstream” erosion along the city foreshore. The key study objectives were to investigate the local coastal dynamics, and to examine ways to mitigate the effects of the harbour on the adjacent coast. 

Two large experimental field programmes were conducted involving: i) wave, current and suspended sediment measurements, ii) side-scan sonar and hydrographic survey, iii) sediment tracing, and iv) experimental dumping of 47,000 m³ of dredged sand in a rocky nearshore zone (6-10 m depth). Sediment trap calibration experiments were also performed. 

Analysis of the measured wave data has identified wave-induced motion as a significant source of velocity error in p, u, v meters on taut-wire moorings, and wave directions resolved from the cross-spectra of pressure and velocity have frequency dependent errors due to the phase difference between the pressure and velocity data. Accordingly, the spectra of wave direction should be resolved solely from velocity data. Calibration of sediment traps against an automated water sampler has provided the first strong evidence that traps are effective in deriving the time-averaged suspended sediment concentration SSC in a wave-dominated coastal environment. Rigid cylindrical traps of high Aspect Ratio have a 75% sampling efficiency. 

Wave transformations across the complex nearshore bathymetry are dominated by seabed friction and refraction. This process was simulated using the WBEND wave refraction model, applying a constant friction coefficient for sandy beds, and a Nikuradse roughness for rocky beds. Wind vector was found to influence the spatial distribution of wave energy, with whitecapping conditions under onshore-directed winds inducing a longshore “smoothing” in the wave energy, which appears to act in opposition to wave height reinforcement from refraction. 

Sediment tracing using an artificial fluorescent material proved to be an effective method for observing the transport of littoral sediments over sandy and rocky beds, over distances of up to 5 km. The tracer exhibited a horizontal diffusivity of the order 0.1 m²s⁻¹, of which approximately 5% was expressed in the cross-shore direction. 
The time-averaged entrainment (C₀) of “black” volcanic sands under high-energy conditions may be effectively predicted using the excess non-dimensional skin friction (θ₂.₅) raised to the power of 1.5, with the mean grain size used in the sediment mobility term (Ψ) and the median grain size in the friction factor (fw). The scale of turbulent mixing (lₛ) increases linearly with elevation above the bed, with a gradient of 0.46. Suspended sediments show a systematic (and linear) reduction in mean grain size with increasing elevation above bed. 

SSCs over rocky beds are supply-limited, and inversely proportional to the distance from contiguous sandy beds. Sediments are principally transported in the suspended form, and a system of irregular shore-normal reefs does not significantly obstruct the longshore flux. Rocky beds appear to be resilient to sediment inundation and the sand/rock sediment facies have remained positionally stable over 16 years. Dumping sand in a rocky reef environment does not necessarily result in adjacent beds becoming inundated with sand. In this environment, a nearshore dump mound did not migrate as a contiguous body, but slowly dispersed as suspended sediments. 

Net sediment transport at New Plymouth is directed alongshore to the northeast, although the sediments respond to local and temporal perturbations, which give rise to reversing and circulating fluxes. Port Taranaki traps 174,000 m³ of coastal sediments per year, of which 142,000 m³ accumulates at the breakwater tip. The latter are derived from the shallow zone seaward of the breakwater wall (i.e. within ∼50 m). Sediments in depths > 6 m (i.e. > 100 m from the breakwater wall) bypass the harbour and traverse the rocky reefs toward the eastern beaches. The historical offshore dumping practice removes sediments from the nearshore littoral budget, while the orientation of the main port breakwater causes the longshore sediment flux to be directed away from the adjacent (Kawaroa) coast, further reducing the supply of littoral sediments to that region. 

A new dumping ground has been identified for the breakwater-tip sediments, which is projected to retain the dredged sediments within the nearshore littoral system, feeding sand to the central and eastern city foreshore. Sedimentation of the coast immediately adjacent to the port is not expected to result from the long-term use of this ground.

year: 2001

title: A Communitarian Approach to Accountability for the Common Good: A Case Study of Community Participation in Planning and Policy Making for Lake Taupo

authors: Arunachalam, Murugesh

subjects: accountability

abstract: This thesis seeks to gain a theoretical and empirical understanding of the meaning of a communitarian approach to accountability for the common good (CAACG). The hermeneutic analysis adopted in this study starts by explaining the researcher's pre-understandings which includes contemporary ideas on accountability, communitarianism and sustainability. A theoretical communitarian model was designed by synthesising these ideas. Using the theoretical model the researcher attempted to advance the meaning of CAACG in multiple contexts which include: the global context on sustainability; the New Zealand context on local governance; and more specifically in the context of planning and policy making for the sustainable development of the Taupo District. The use of such multiple contexts is crucial for this study. This is because communitarian ideology in New Zealand has historical roots in Local Government which, in recent years has been influenced by the global discourse on sustainable development. The adoption of multiple contexts is aimed at providing a holistic and historical understanding of planning and policy making processes in the Taupo District and the manifestation of CAACG in the processes.

In this interpretive study the term  text  is defined as the empirical data which consists of public documents, website material, minutes of community meetings, field notes and transcriptions of interviews. The empirical data is about processes and outcomes of collaboration between the community groups, public authorities and private entities in formulating strategies and policies for sustainable development of the Taupo District. Interpretation of the empirical data involved understanding the  text  from the vantage point of the pre-understandings of the interpreter. The interpretation of the  text  is is aimed at explaining the manifestation of CAACG in the Taupo District. The methodological orientations of the thesis are predominantly consistent with the hermeneutic tradition of Gadamer (1975). However, although the interpreter started with the intention of strictly confining to the subjectivist approach of Gadamer (1975), at times the hermeneutic methodology adopted by the researcher encroached into the methodological orientations of objectivist hermeneutics. The use of objectivist hermeneutic was inevitable as it was necessary to understand the authorial intention in the text before the interpreter understands the text from the perspective of his pre-understandings. Hence, the researcher rejects the assumption of objective-subjective dichotomy and subscribes more to the philosophical arguments advanced by contemporary scholars (such as Boland Jr, 1989 and Ricoeur, 1981) who find both the subjective and objective philosophies as necessary for interpreting texts. 

The hermeneutic analysis undertaken in this study suggests that the meaning of CAACG appears in the context of communal processes (including planning and policy making processes) and the strategies and policies formulated by the Taupo community. The meaning of CAACG may not be obvious to any reader of the  text . It arises from the interpretation of the  text  from the perspective of the interpreter's pre-understandings on a communitarian approach to accountability. The interpretation suggests the existence of a community of interests, community values and concerns, and communal processes in the Taupo District.  Community values and concerns are associated with Lake Taupo. The primary concern of the community is the pollution of Lake Taupo caused by animal farming in the land surrounding the lake and the impact the pollution has on the environmental, economic and cultural values of the community. The communal processes involved collaboration between the community and public authorities in planning and policy making for the protection of Lake Taupo. The interpretation also indicates the manifestation of several dimensions of accountability in the communal processes; joint accountability or 360 degree accountability in the Taupo District; and the holistic meaning of environmental and social accounting.

In the Taupo District the operation of the CAACG can be affected by several factors. Symmetry of power can be affected in terms of the preferential treatment given to the indigenous community and its segregation from the rest of the community. The strong influence of local authorities in the planning and decision making processes makes the community appear helpless without the local authorities. Communitarian processes intended to empower the community may actually strengthen the position of local authorities. Power has actually shifted from Central Government to local authorities and may not necessarily have shifted to communities. However, the endeavours to engage the community in the Taupo District can be seen as attempts to build the capacity of the community to participate in the processes and time will tell how effective the CAACG will prove to be.

This thesis is the pioneer in advancing the theorization of the CAACG and has added a substantial contribution to accountability literature. It suggests a new way of looking at environmental and social accounting in which the emphasis is on community involvement through reporting and deliberation (dialectical dimension of accountability) on the impacts of human activities on the natural environment. The CAACG is premised on the centrality of community and the assumption of a 360 degree accountability in which everyone in the community has mutual responsibility to protect the common good and can be subject to critical enquiry for the adverse impacts of their activities on the common good. The CAACG does not accord private corporations the privilege status of  reporting entities  but considers environmental and social accounting as the agenda of the community. 

The concept of CAACG is not utopian as it may sound to some readers of this thesis. There are ample evidence of communities throughout the world demanding a voice in statutory planning and decision making for sustainable development. The increasing focus on environmental sustainability and community participation in the planning and decision making processes arises from greater awareness of the detrimental impacts of environmental pollution and the realisation that the decision on the common good cannot be left to free market forces and private corporations. The natural environment belongs to a community of interests which wants to participate in joint responsibility and collective planning and decision making. No individual or group has the right to make decisions about the natural environment without consulting the community.

text: A Communitarian Approach to Accountability for the Common Good: A Case Study of Community Participation in Planning and Policy Making for Lake Taupo This thesis seeks to gain a theoretical and empirical understanding of the meaning of a communitarian approach to accountability for the common good (CAACG). The hermeneutic analysis adopted in this study starts by explaining the researcher's pre-understandings which includes contemporary ideas on accountability, communitarianism and sustainability. A theoretical communitarian model was designed by synthesising these ideas. Using the theoretical model the researcher attempted to advance the meaning of CAACG in multiple contexts which include: the global context on sustainability; the New Zealand context on local governance; and more specifically in the context of planning and policy making for the sustainable development of the Taupo District. The use of such multiple contexts is crucial for this study. This is because communitarian ideology in New Zealand has historical roots in Local Government which, in recent years has been influenced by the global discourse on sustainable development. The adoption of multiple contexts is aimed at providing a holistic and historical understanding of planning and policy making processes in the Taupo District and the manifestation of CAACG in the processes.

In this interpretive study the term  text  is defined as the empirical data which consists of public documents, website material, minutes of community meetings, field notes and transcriptions of interviews. The empirical data is about processes and outcomes of collaboration between the community groups, public authorities and private entities in formulating strategies and policies for sustainable development of the Taupo District. Interpretation of the empirical data involved understanding the  text  from the vantage point of the pre-understandings of the interpreter. The interpretation of the  text  is is aimed at explaining the manifestation of CAACG in the Taupo District. The methodological orientations of the thesis are predominantly consistent with the hermeneutic tradition of Gadamer (1975). However, although the interpreter started with the intention of strictly confining to the subjectivist approach of Gadamer (1975), at times the hermeneutic methodology adopted by the researcher encroached into the methodological orientations of objectivist hermeneutics. The use of objectivist hermeneutic was inevitable as it was necessary to understand the authorial intention in the text before the interpreter understands the text from the perspective of his pre-understandings. Hence, the researcher rejects the assumption of objective-subjective dichotomy and subscribes more to the philosophical arguments advanced by contemporary scholars (such as Boland Jr, 1989 and Ricoeur, 1981) who find both the subjective and objective philosophies as necessary for interpreting texts. 

The hermeneutic analysis undertaken in this study suggests that the meaning of CAACG appears in the context of communal processes (including planning and policy making processes) and the strategies and policies formulated by the Taupo community. The meaning of CAACG may not be obvious to any reader of the  text . It arises from the interpretation of the  text  from the perspective of the interpreter's pre-understandings on a communitarian approach to accountability. The interpretation suggests the existence of a community of interests, community values and concerns, and communal processes in the Taupo District.  Community values and concerns are associated with Lake Taupo. The primary concern of the community is the pollution of Lake Taupo caused by animal farming in the land surrounding the lake and the impact the pollution has on the environmental, economic and cultural values of the community. The communal processes involved collaboration between the community and public authorities in planning and policy making for the protection of Lake Taupo. The interpretation also indicates the manifestation of several dimensions of accountability in the communal processes; joint accountability or 360 degree accountability in the Taupo District; and the holistic meaning of environmental and social accounting.

In the Taupo District the operation of the CAACG can be affected by several factors. Symmetry of power can be affected in terms of the preferential treatment given to the indigenous community and its segregation from the rest of the community. The strong influence of local authorities in the planning and decision making processes makes the community appear helpless without the local authorities. Communitarian processes intended to empower the community may actually strengthen the position of local authorities. Power has actually shifted from Central Government to local authorities and may not necessarily have shifted to communities. However, the endeavours to engage the community in the Taupo District can be seen as attempts to build the capacity of the community to participate in the processes and time will tell how effective the CAACG will prove to be.

This thesis is the pioneer in advancing the theorization of the CAACG and has added a substantial contribution to accountability literature. It suggests a new way of looking at environmental and social accounting in which the emphasis is on community involvement through reporting and deliberation (dialectical dimension of accountability) on the impacts of human activities on the natural environment. The CAACG is premised on the centrality of community and the assumption of a 360 degree accountability in which everyone in the community has mutual responsibility to protect the common good and can be subject to critical enquiry for the adverse impacts of their activities on the common good. The CAACG does not accord private corporations the privilege status of  reporting entities  but considers environmental and social accounting as the agenda of the community. 

The concept of CAACG is not utopian as it may sound to some readers of this thesis. There are ample evidence of communities throughout the world demanding a voice in statutory planning and decision making for sustainable development. The increasing focus on environmental sustainability and community participation in the planning and decision making processes arises from greater awareness of the detrimental impacts of environmental pollution and the realisation that the decision on the common good cannot be left to free market forces and private corporations. The natural environment belongs to a community of interests which wants to participate in joint responsibility and collective planning and decision making. No individual or group has the right to make decisions about the natural environment without consulting the community.

year: 2010