title: Doxycycline-inducible overexpression of NANOG in bovine fibroblasts and nuclear transfer embryos

authors: Chibnall, Alice Margaret

subjects: Embryonic stem cells

abstract: Naïve pluripotent embryonic stem cells (ES cells) have the ability to give rise to all cell types including functional gametes. These cells have been well established in mice and rats. The potential for biomedical and agricultural applications of pluripotent stem cells in livestock are vast. Having this resource will make in vitro techniques more achievable for research purposes and commercial use. Their use for chimera formation will increase the production of high quality embryos, resulting in a faster inclusion of desirable traits in breeding lines. 

The aim of this study was to investigate the molecular characteristics of pluripotency in bovine embryos. This is an incremental step to aid our understanding of the greater system which will encourage the derivation of ES cells within the bovine species. An initial target was the key pluripotency transcription factor NANOG. Nanog has thousands of targets and orchestrates the regulation of the naïve pluripotent molecular environment. This thesis will investigate the overexpression of NANOG with a doxycycline-inducible system in bovine female fibroblasts and nuclear transfer (NT) embryos derived from these. It was hypothesised that the overexpression of NANOG would stimulate the pluripotency network, increasing pluripotency. It has previously been recognised that the re-activation of the X-chromosome in females is an indicator of naïve pluripotency in mouse. Notably, the gene Xist which is important for the inactivation of the X-chromosome is directly repressed by Nanog. It was hypothesised that this would hold true in the bovine system. 

Both the cell line and embryos were analysed for mRNA and protein expression of ectopic NANOG using quantitative PCR (qPCR) and immunocytochemistry. Eight pluripotency-related genes (endogenous NANOG, OCT4, SOX2, KLF4, PDGFRα, SOX17, SOCS3 and FGF4) were quantified using qPCR to investigate the impact of increased NANOG in the NT embryo at day 8.  Expression of XIST mRNA was analysed using qPCR and the inactive X-chromosome was identified using immunocytochemistry to understand the role of NANOG in the re activation of the X-chromosome in the bovine system. 
It was found that the inducible system significantly increased the expression of ectopic NANOG mRNA and protein within the fibroblasts used for NT. A two-fold significant increase of total NANOG mRNA was achieved in the NT embryo, although no additional NANOG protein was identified. The increased NANOG had no effect on the eight pluripotency-related genes investigated. The increased NANOG had no effect on the expression of XIST in the bovine system. No inactive X-chromosomes were identified via immunocytochemistry during this project. Subsequent to this research, details of an alternative overexpression technique using CRISPR technology were published. Use of this system will make research in this field achievable to a higher quality in a shorter timeframe.

text: Doxycycline-inducible overexpression of NANOG in bovine fibroblasts and nuclear transfer embryos Naïve pluripotent embryonic stem cells (ES cells) have the ability to give rise to all cell types including functional gametes. These cells have been well established in mice and rats. The potential for biomedical and agricultural applications of pluripotent stem cells in livestock are vast. Having this resource will make in vitro techniques more achievable for research purposes and commercial use. Their use for chimera formation will increase the production of high quality embryos, resulting in a faster inclusion of desirable traits in breeding lines. 

The aim of this study was to investigate the molecular characteristics of pluripotency in bovine embryos. This is an incremental step to aid our understanding of the greater system which will encourage the derivation of ES cells within the bovine species. An initial target was the key pluripotency transcription factor NANOG. Nanog has thousands of targets and orchestrates the regulation of the naïve pluripotent molecular environment. This thesis will investigate the overexpression of NANOG with a doxycycline-inducible system in bovine female fibroblasts and nuclear transfer (NT) embryos derived from these. It was hypothesised that the overexpression of NANOG would stimulate the pluripotency network, increasing pluripotency. It has previously been recognised that the re-activation of the X-chromosome in females is an indicator of naïve pluripotency in mouse. Notably, the gene Xist which is important for the inactivation of the X-chromosome is directly repressed by Nanog. It was hypothesised that this would hold true in the bovine system. 

Both the cell line and embryos were analysed for mRNA and protein expression of ectopic NANOG using quantitative PCR (qPCR) and immunocytochemistry. Eight pluripotency-related genes (endogenous NANOG, OCT4, SOX2, KLF4, PDGFRα, SOX17, SOCS3 and FGF4) were quantified using qPCR to investigate the impact of increased NANOG in the NT embryo at day 8.  Expression of XIST mRNA was analysed using qPCR and the inactive X-chromosome was identified using immunocytochemistry to understand the role of NANOG in the re activation of the X-chromosome in the bovine system. 
It was found that the inducible system significantly increased the expression of ectopic NANOG mRNA and protein within the fibroblasts used for NT. A two-fold significant increase of total NANOG mRNA was achieved in the NT embryo, although no additional NANOG protein was identified. The increased NANOG had no effect on the eight pluripotency-related genes investigated. The increased NANOG had no effect on the expression of XIST in the bovine system. No inactive X-chromosomes were identified via immunocytochemistry during this project. Subsequent to this research, details of an alternative overexpression technique using CRISPR technology were published. Use of this system will make research in this field achievable to a higher quality in a shorter timeframe.

year: 2015

title: Where firms choose to disclose voluntary environmental information

authors: de Villiers, Charl

abstract: Corporate environmental performance is of increasing importance to investors, public policy makers and the general public. Firms disclose environmental information (mostly) voluntarily in their annual reports and on their websites. These disclosures are important, because they provide environmental performance information and influence capital markets. We compare environmental disclosure in annual reports and on websites with a long-term (bad) and a short-term (crisis) environmental performance measure. We find evidence to support our hypotheses that different levels of environmental disclosure are made in annual reports and on websites under different conditions. More specifically, firms disclose more environmental information on their websites when faced with an environmental crisis and more in their annual reports when they have a bad environmental reputation.

text: Where firms choose to disclose voluntary environmental information Corporate environmental performance is of increasing importance to investors, public policy makers and the general public. Firms disclose environmental information (mostly) voluntarily in their annual reports and on their websites. These disclosures are important, because they provide environmental performance information and influence capital markets. We compare environmental disclosure in annual reports and on websites with a long-term (bad) and a short-term (crisis) environmental performance measure. We find evidence to support our hypotheses that different levels of environmental disclosure are made in annual reports and on websites under different conditions. More specifically, firms disclose more environmental information on their websites when faced with an environmental crisis and more in their annual reports when they have a bad environmental reputation.

year: 2011

title: Iwi interests and the RMA: An evaluation of the quality of first generation council plans

authors: Jefferies, Richard

subjects: New Zealand

abstract: This working paper analyses the processes adopted by councils for involving hapū
/iwi in plan implementation, including the resource consents process. Three topic issues were investigated to assess plan implementation — urban amenity,
storm water, and issues of importance to iwi. Questions were asked about the capacity of hapū/iwi to engage in the resource consent
process, which resource issues were of concern to them, their relationship with
council and consent applicants, and their perception of the consent process. Most resources listed in the questionnaire were of concern to hapū/iwi, with water
quality, wāhi tapu and heritage the most commonly cited. In conclusion, we found a general dissatisfaction on the part of hapū/iwi with
councils’ performance with respect to both Treaty relationships and consent
processing under the RMA. A further contributing factor to the poor relationships
found between hapū/iwi and councils, was the lack of clarity over the role of hapū and
iwi in resource management. In several districts, diverging responses from hapū/iwi
and councils to questions about level of understanding and commitment suggests there
is a need for more effective communication. These problems are compounded by the
generally low capacity of hapū/iwi to participate in resource consent processes. These findings suggest that there is much to be done to improve relationships and
behaviour of these key stakeholder groups in the plan implementation process if key
provisions in the RMA related to hapū/iwi interests are to be fulfilled. The differences
shown in reciprocal perceptions have serious implications for establishing a sound
working partnership between councils and hapū/iwi in their areas. Making clear these
discrepancies is a first step towards taking the measures needed for building a better
partnership. Further, the capacity of hapū/iwi to participate could be better utilised if
there was greater integration between regional and district councils on issues of
significance and processes for iwi involvement.

text: Iwi interests and the RMA: An evaluation of the quality of first generation council plans This working paper analyses the processes adopted by councils for involving hapū
/iwi in plan implementation, including the resource consents process. Three topic issues were investigated to assess plan implementation — urban amenity,
storm water, and issues of importance to iwi. Questions were asked about the capacity of hapū/iwi to engage in the resource consent
process, which resource issues were of concern to them, their relationship with
council and consent applicants, and their perception of the consent process. Most resources listed in the questionnaire were of concern to hapū/iwi, with water
quality, wāhi tapu and heritage the most commonly cited. In conclusion, we found a general dissatisfaction on the part of hapū/iwi with
councils’ performance with respect to both Treaty relationships and consent
processing under the RMA. A further contributing factor to the poor relationships
found between hapū/iwi and councils, was the lack of clarity over the role of hapū and
iwi in resource management. In several districts, diverging responses from hapū/iwi
and councils to questions about level of understanding and commitment suggests there
is a need for more effective communication. These problems are compounded by the
generally low capacity of hapū/iwi to participate in resource consent processes. These findings suggest that there is much to be done to improve relationships and
behaviour of these key stakeholder groups in the plan implementation process if key
provisions in the RMA related to hapū/iwi interests are to be fulfilled. The differences
shown in reciprocal perceptions have serious implications for establishing a sound
working partnership between councils and hapū/iwi in their areas. Making clear these
discrepancies is a first step towards taking the measures needed for building a better
partnership. Further, the capacity of hapū/iwi to participate could be better utilised if
there was greater integration between regional and district councils on issues of
significance and processes for iwi involvement.

year: 2002-10-31