title: Decoupling enzyme catalysis from thermal denaturation

authors: Easter, Ashley Davys

subjects: equilibrium model

abstract: The equilibrium model (EM) (Daniel et al., 2001) postulates two forms of a folded enzyme, one catalytically active (Eact) and the other inactive (Einact), which interconvert via a fast thermal equilibrium (Keq) (Figure A). This model for enzyme catalysis accounts for experimentally observed time and temperature profiles of enzyme/substrate systems more accurately than the classically derived, single folded-species, model (Figure B). In both models, the denatured species (X) is formed via the kinact process, which is temperature and time-dependent.
(FIgure A) (Figure B) Comparison between the equilibrium model and classical model for enzyme catalysis. For both, the vertical axis is catalytic rate (M s-1), the left-right axis is increasing temperature (K) and the back-front axis is assay duration (s).
The physical basis for the Eact/Einact equilibrium is unknown. To study the equilibrium, the temperature midpoint of the Eact/Einact transition (Teq) has to be separated from the thermostability of the enzyme (Tm) to allow the Einact species to exist in measurable concentrations without exhibiting denaturation.
Mutations were made in a well-studied and NMR-accessible ribonuclease, barnase, to alter the thermostability and/or the Teq of the enzyme activity. The stability properties of each mutant were measured and the activity against two substrates assayed. New models were derived and fitted against wild-type barnase, and an ideal data set, to give insight into alternative irreversible and reversible denaturation pathways. Simulations of these models were developed to benchmark potential dynamics work and explain the movements of species within each model's framework.
Assay data fits to the EM and alternative models show a preference for irreversible denaturation pathways via the Einact species. A mathematically simplified model was also found that accounts for data and could provide an alternative method for determining EM parameters. Although fits of barnase to the EM were statistically good, the denaturation properties could not be reconciled with the literature or experimentally determined values for stability and unfolding. Simulations illustrating how the Eact, Einact and denatured (X) species interact also corroborate this finding.
Despite this discrepancy (in fitted parameters to the EM), it is hypothesised that the Teq and Tm of a disulphide-bridged mutant of barnase have been successfully decoupled. This mutant has been 15N- labelled for future NMR dynamics measurements. New approaches to the EM model are proposed where the separate determination of enzyme thermodynamic properties (e.g., rate and free energy of denaturation) would allow other EM parameters to be fitted independently to each data set.

text: Decoupling enzyme catalysis from thermal denaturation The equilibrium model (EM) (Daniel et al., 2001) postulates two forms of a folded enzyme, one catalytically active (Eact) and the other inactive (Einact), which interconvert via a fast thermal equilibrium (Keq) (Figure A). This model for enzyme catalysis accounts for experimentally observed time and temperature profiles of enzyme/substrate systems more accurately than the classically derived, single folded-species, model (Figure B). In both models, the denatured species (X) is formed via the kinact process, which is temperature and time-dependent.
(FIgure A) (Figure B) Comparison between the equilibrium model and classical model for enzyme catalysis. For both, the vertical axis is catalytic rate (M s-1), the left-right axis is increasing temperature (K) and the back-front axis is assay duration (s).
The physical basis for the Eact/Einact equilibrium is unknown. To study the equilibrium, the temperature midpoint of the Eact/Einact transition (Teq) has to be separated from the thermostability of the enzyme (Tm) to allow the Einact species to exist in measurable concentrations without exhibiting denaturation.
Mutations were made in a well-studied and NMR-accessible ribonuclease, barnase, to alter the thermostability and/or the Teq of the enzyme activity. The stability properties of each mutant were measured and the activity against two substrates assayed. New models were derived and fitted against wild-type barnase, and an ideal data set, to give insight into alternative irreversible and reversible denaturation pathways. Simulations of these models were developed to benchmark potential dynamics work and explain the movements of species within each model's framework.
Assay data fits to the EM and alternative models show a preference for irreversible denaturation pathways via the Einact species. A mathematically simplified model was also found that accounts for data and could provide an alternative method for determining EM parameters. Although fits of barnase to the EM were statistically good, the denaturation properties could not be reconciled with the literature or experimentally determined values for stability and unfolding. Simulations illustrating how the Eact, Einact and denatured (X) species interact also corroborate this finding.
Despite this discrepancy (in fitted parameters to the EM), it is hypothesised that the Teq and Tm of a disulphide-bridged mutant of barnase have been successfully decoupled. This mutant has been 15N- labelled for future NMR dynamics measurements. New approaches to the EM model are proposed where the separate determination of enzyme thermodynamic properties (e.g., rate and free energy of denaturation) would allow other EM parameters to be fitted independently to each data set.

year: 2010

title: International entrepreneurship: A critical analysis of studies in the past two decades and future directions for research

authors: Peiris, Indujeeva K.

subjects: Born global

abstract: International entrepreneurship (IE) is an emerging field with a rapidly growing body of knowledge. This paper examines gaps, issues and trends of the IE in the last two decades. First, it suggests an integrative framework based on international business, entrepreneurship, strategic management, social network and marketing theories. The suggested model highlights the significant role played by the entrepreneur/team, firm and network resources that act as antecedents to international opportunity development and value innovation. Second, it suggests four typologies of firms (born global, enduring global, early exporter and mature exporter) that can be studied under the IE theme. Finally, we discuss future research directions.

text: International entrepreneurship: A critical analysis of studies in the past two decades and future directions for research International entrepreneurship (IE) is an emerging field with a rapidly growing body of knowledge. This paper examines gaps, issues and trends of the IE in the last two decades. First, it suggests an integrative framework based on international business, entrepreneurship, strategic management, social network and marketing theories. The suggested model highlights the significant role played by the entrepreneur/team, firm and network resources that act as antecedents to international opportunity development and value innovation. Second, it suggests four typologies of firms (born global, enduring global, early exporter and mature exporter) that can be studied under the IE theme. Finally, we discuss future research directions.

year: 2012

title: “You’re not coming out – you’ve been there all along and just no one’s looked”: Māori LGBT+ youth and identity exploration

authors: Laurence, Rebekah

subjects: Māori

abstract: This research is a Masters-level project on Māori LGBT-plus (lesbian, gay, bisexual, transgender, and other) youths’ experience of exploring their gender and sexual identities, highlighting how whānau (loosely, ‘extended family’) can provide mana (authority, power) such that youth can exercise mana rangatiratanga (a Maniapoto description of ‘autonomy, self-determination’) regarding their identities. This is a novel, exploratory study.
Adapted elements from a Mana Wāhine (Māori feminist) perspective of Kaupapa Māori theory comprised the theoretical framework for this project. These elements were Mana rangatiratanga (adapted from Tino rangatiratanga), Te reo Māori me ōna tikanga (Māori language and its culture), and Whanaungatanga (relationships). The three elements were adapted to fit a Ngāti Maniapoto worldview. 
Qualitative research methods were used for this study. There were five participants aged between 17 and 24 years, who were Māori and had diverse gender or sexual identities. Semi-structured, face-to-face interviews were conducted with participants, which allowed them to share their experiences of exploring their gender and sexual identities and of whānau support. Interviews were analysed using a process of inductive thematic analysis, which identified and analysed patterns across the dataset.
Eight themes were derived from participants’ experiences. These themes were Ko Tūhura, which described the process of discovery that participants have experienced; Ko Matatapu, which described participants’ experience of authenticity and concealment of their gender and sexual identities; Ko Tautoko, which described how whānau have empowered participants to explore their identities, and how participants have been denied empowerment; Ko Mata-ngaro, which described participants’ early lack of exposure to diverse identities, and their having their identities unseen by others; Ko te Ahunga-ki-te-aunoa, which described how diverse identities have been accepted within participants’ whānau; Ko Tauawhi, which described how participants have been embraced for their diverse identities; Ko Mata-pū-take, which describes participants’ experience of finding somewhere to belong; and Ko Kiritau, which describes participants’ becoming settled in their expression of their gender and sexual identities.
The findings of the thesis describe roles that whānau play in participants’ exploration of their gender and sexual identities and some of the effects that colonisation has had within participants’ whānau regarding their gender and sexual identities. Nonetheless, participants actively resisted these aspects of colonisations and found ways to give voice to their gender and sexual identities.
Given that participants shared that there was a dearth of information regarding gender and sexual identities available to them within their whānau and school environments, organisations within psychology and related fields may work to proactively provide all young Māori people and their whānau with information regarding diverse gender and sexual identities. Moreover, to address the dearth of resources designed for Māori LGBT-plus youth, New Zealand-based researchers may collate information specifically regarding Māori LGBT-plus youth experience of identity exploration and Māori knowledges regarding gender and sexual identities. This information may be collated in community-based resources that are designed specifically for members of this group and their whānau, and professionals who work for them.

text: “You’re not coming out – you’ve been there all along and just no one’s looked”: Māori LGBT+ youth and identity exploration This research is a Masters-level project on Māori LGBT-plus (lesbian, gay, bisexual, transgender, and other) youths’ experience of exploring their gender and sexual identities, highlighting how whānau (loosely, ‘extended family’) can provide mana (authority, power) such that youth can exercise mana rangatiratanga (a Maniapoto description of ‘autonomy, self-determination’) regarding their identities. This is a novel, exploratory study.
Adapted elements from a Mana Wāhine (Māori feminist) perspective of Kaupapa Māori theory comprised the theoretical framework for this project. These elements were Mana rangatiratanga (adapted from Tino rangatiratanga), Te reo Māori me ōna tikanga (Māori language and its culture), and Whanaungatanga (relationships). The three elements were adapted to fit a Ngāti Maniapoto worldview. 
Qualitative research methods were used for this study. There were five participants aged between 17 and 24 years, who were Māori and had diverse gender or sexual identities. Semi-structured, face-to-face interviews were conducted with participants, which allowed them to share their experiences of exploring their gender and sexual identities and of whānau support. Interviews were analysed using a process of inductive thematic analysis, which identified and analysed patterns across the dataset.
Eight themes were derived from participants’ experiences. These themes were Ko Tūhura, which described the process of discovery that participants have experienced; Ko Matatapu, which described participants’ experience of authenticity and concealment of their gender and sexual identities; Ko Tautoko, which described how whānau have empowered participants to explore their identities, and how participants have been denied empowerment; Ko Mata-ngaro, which described participants’ early lack of exposure to diverse identities, and their having their identities unseen by others; Ko te Ahunga-ki-te-aunoa, which described how diverse identities have been accepted within participants’ whānau; Ko Tauawhi, which described how participants have been embraced for their diverse identities; Ko Mata-pū-take, which describes participants’ experience of finding somewhere to belong; and Ko Kiritau, which describes participants’ becoming settled in their expression of their gender and sexual identities.
The findings of the thesis describe roles that whānau play in participants’ exploration of their gender and sexual identities and some of the effects that colonisation has had within participants’ whānau regarding their gender and sexual identities. Nonetheless, participants actively resisted these aspects of colonisations and found ways to give voice to their gender and sexual identities.
Given that participants shared that there was a dearth of information regarding gender and sexual identities available to them within their whānau and school environments, organisations within psychology and related fields may work to proactively provide all young Māori people and their whānau with information regarding diverse gender and sexual identities. Moreover, to address the dearth of resources designed for Māori LGBT-plus youth, New Zealand-based researchers may collate information specifically regarding Māori LGBT-plus youth experience of identity exploration and Māori knowledges regarding gender and sexual identities. This information may be collated in community-based resources that are designed specifically for members of this group and their whānau, and professionals who work for them.

year: 2020