title: The Effect of Hydration on Enzyme Activity and Dynamics

authors: Lopez, Murielle

subjects: protein hydration

abstract: Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. 
At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site.
Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

text: The Effect of Hydration on Enzyme Activity and Dynamics Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. 
At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site.
Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

year: 2008

title: The amendment to section 59 of the Crimes Act 1961: A case study in legislative change

authors: Austin, Geraldine Rachel

subjects: anti-smacking

abstract: In June of 2007 the New Zealand Government passed the Crimes (Substituted section 59) Amendment Act 2007, legally abolishing the right of parents to physically discipline their children and making New Zealand one of 29 States to have (so far) achieved full prohibition of corporal punishment. However the hypothesis of this thesis is that this process of abolishing corporal punishment can be characterised as a lost opportunity - the opportunity to engage in productive debate about the way in which our country’s children are viewed and raised and to address some of the underlying practices and attitudes which contribute towards our appalling rates of child abuse. Instead it degenerated into one of the most contentious debates in recent political history. Rational debate deteriorated into abuse, misunderstandings and deliberate thwarting of reasoned discussion. As a result in 2010 we are in a position where the boundaries surrounding permissible and impermissible discipline are arguably less clear than they were before. This thesis analyses the process of legislative change with a view to showing how, why and where things went wrong. It argues that this process was stymied by two crucial factors; firstly the influence of right wing lobby groups who garnered support for their religiously motivated viewpoints by capitalising on the public’s misplaced fears and underlying attitudes towards children ; and secondly the failure of the Government to adequately advocate for, and educate the public on, the law which it passed. Ultimately these two factors were heavily influenced by the fact that our collective and individual opinions on physical discipline have arisen as a result of the historically dominant construction of children as inherently evil – a construction which we inherited from Britain and which is intrinsically aligned with the practice of corporal punishment. The thesis then demonstrates that the negative repercussions of the debate process could be partially ameliorated through education and that lessons learned in this process can be utilised in the future

text: The amendment to section 59 of the Crimes Act 1961: A case study in legislative change In June of 2007 the New Zealand Government passed the Crimes (Substituted section 59) Amendment Act 2007, legally abolishing the right of parents to physically discipline their children and making New Zealand one of 29 States to have (so far) achieved full prohibition of corporal punishment. However the hypothesis of this thesis is that this process of abolishing corporal punishment can be characterised as a lost opportunity - the opportunity to engage in productive debate about the way in which our country’s children are viewed and raised and to address some of the underlying practices and attitudes which contribute towards our appalling rates of child abuse. Instead it degenerated into one of the most contentious debates in recent political history. Rational debate deteriorated into abuse, misunderstandings and deliberate thwarting of reasoned discussion. As a result in 2010 we are in a position where the boundaries surrounding permissible and impermissible discipline are arguably less clear than they were before. This thesis analyses the process of legislative change with a view to showing how, why and where things went wrong. It argues that this process was stymied by two crucial factors; firstly the influence of right wing lobby groups who garnered support for their religiously motivated viewpoints by capitalising on the public’s misplaced fears and underlying attitudes towards children ; and secondly the failure of the Government to adequately advocate for, and educate the public on, the law which it passed. Ultimately these two factors were heavily influenced by the fact that our collective and individual opinions on physical discipline have arisen as a result of the historically dominant construction of children as inherently evil – a construction which we inherited from Britain and which is intrinsically aligned with the practice of corporal punishment. The thesis then demonstrates that the negative repercussions of the debate process could be partially ameliorated through education and that lessons learned in this process can be utilised in the future

year: 2010

title: Te Poho o Te Arawa: Te Mana Whenua o Ngāti Whakaue ki Maketū

authors: Potaka, Ngahu

subjects: Ngāti Whakaue

abstract: Maketū is a small coastal town in the Bay of Plenty and is the final resting place of the Te Arawa canoe, the ancestral waka of the Te Arawa people. This is the home of Ngāti Whakaue ki Maketū a coastal hapū of the tribe Ngāti Whakaue from the Te Arawa confederation of tribes. Ngāti Whakaue ki Maketū is actually a collection of smaller hapū who descend from the union of their ancestor Whakaue Kaipapa and his wife Rangiuru, and they trace their overall decent from the celebrated chief Tama te Kapua. It is on their ancestral land in Maketū that Ngāti Whakaue ki Maketū have resided and established their mana whenua (tribal authority) for over 170 years. In that time they have faced many internal and external challenges to their mana whenua including military engagement, political maneuvering, and other social issues.

This thesis traces the historical origins and mana whenua of Ngāti Whakaue ki Maketū and analyses the various key components that combine to ensure Ngāti Whakaue ki Maketū maintain mana whenua across their tribal lands. This thesis also explores how mana whenua is a vehicle for maintaining tribal authority and well being for Ngāti Whakaue ki Maketū. Part of this study is the creation of a mana whenua strategic plan that will help Ngāti Whakaue ki Maketū maintain and secure mana whenua in Maketū for future generations.

It is the proposition of this thesis that mana whenua has, is and will always play an important part in the social identity and fabric of Ngati Whakaue ki Maketū. However as Ngāti Whakaue ki Maketū stand at the dawn of a new era, it is important for this related group to understand how mana whenua operates, and can be nurtured as a means of protection to ensure the survival of Ngāti Whakaue ki Maketū in this ever changing world.

text: Te Poho o Te Arawa: Te Mana Whenua o Ngāti Whakaue ki Maketū Maketū is a small coastal town in the Bay of Plenty and is the final resting place of the Te Arawa canoe, the ancestral waka of the Te Arawa people. This is the home of Ngāti Whakaue ki Maketū a coastal hapū of the tribe Ngāti Whakaue from the Te Arawa confederation of tribes. Ngāti Whakaue ki Maketū is actually a collection of smaller hapū who descend from the union of their ancestor Whakaue Kaipapa and his wife Rangiuru, and they trace their overall decent from the celebrated chief Tama te Kapua. It is on their ancestral land in Maketū that Ngāti Whakaue ki Maketū have resided and established their mana whenua (tribal authority) for over 170 years. In that time they have faced many internal and external challenges to their mana whenua including military engagement, political maneuvering, and other social issues.

This thesis traces the historical origins and mana whenua of Ngāti Whakaue ki Maketū and analyses the various key components that combine to ensure Ngāti Whakaue ki Maketū maintain mana whenua across their tribal lands. This thesis also explores how mana whenua is a vehicle for maintaining tribal authority and well being for Ngāti Whakaue ki Maketū. Part of this study is the creation of a mana whenua strategic plan that will help Ngāti Whakaue ki Maketū maintain and secure mana whenua in Maketū for future generations.

It is the proposition of this thesis that mana whenua has, is and will always play an important part in the social identity and fabric of Ngati Whakaue ki Maketū. However as Ngāti Whakaue ki Maketū stand at the dawn of a new era, it is important for this related group to understand how mana whenua operates, and can be nurtured as a means of protection to ensure the survival of Ngāti Whakaue ki Maketū in this ever changing world.

year: 2014