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: The structure and evolution of industrial clusters: Transactions, technology and knowledge spillovers

authors: Iammarino, Simona

subjects: industrial clusters

abstract: In this paper, we investigate the relationship between location patterns, innovation processes and industrial clusters. In order to do this we extend a transactions costs-based classification into a knowledge-based taxonomy of clusters, along the lines suggested by a critical revision of the main assumptions underlying most of the existing literature on spatial clusters. Our arguments show that the transactions costs approach and the innovation and technological regimes framework are broadly consistent, and that real insights into the microfoundations, nature, and evolution of clusters can be provided by these classification systems.

text: The structure and evolution of industrial clusters: Transactions, technology and knowledge spillovers In this paper, we investigate the relationship between location patterns, innovation processes and industrial clusters. In order to do this we extend a transactions costs-based classification into a knowledge-based taxonomy of clusters, along the lines suggested by a critical revision of the main assumptions underlying most of the existing literature on spatial clusters. Our arguments show that the transactions costs approach and the innovation and technological regimes framework are broadly consistent, and that real insights into the microfoundations, nature, and evolution of clusters can be provided by these classification systems.

year: 2006

title: Investigating the Social Supports of Successful Māori Undergraduate Appellants at the University of Waikato

authors: Diamond, Danielle Jade

subjects: Social Supports

abstract: Educational disparities between Māori and non-Māori tertiary students are evident in regards to participation, retention, and completion rates at secondary and tertiary levels. Instead of focusing on negative statistics, it was of interest to investigate Māori students who had failed sufficient papers to initially be denied re-entry to university study, yet continued to attain their degrees, as a result of a successful appeal. Māori undergraduate students, who had successfully appealed from 2003-2012, were recruited for my research. The availability and utilisation of social supports were investigated, along with the decision to make an appeal, the role of cultural expectations, and barriers experienced by participants. A mixed method approach was utilised in this research through use of an adapted overarching Kaupapa Māori framework, statistical analysis, and 13 interviews. My research found that social supports were beneficial for participants throughout their appeal, as they provided: emotional, instrumental, appraisal, and informational support. Although the social supports utilised by participants were satisfactory, barriers to accessing formal supports were also found, which included: perceived stigma having failed their papers, pride, as well as being whakamā. The appeal process was a beneficial intervention from the University of Waikato, which provided participants another opportunity to overcome the factors that contributed to their initial failure. Post appeal, participants implemented strategies to address and reduce their factors in order to become academically engaged. Participants were deemed to be resilient from their continued pursuit of degree attainment, implementation of their strategies, and their use of perceived social supports.

text: Investigating the Social Supports of Successful Māori Undergraduate Appellants at the University of Waikato Educational disparities between Māori and non-Māori tertiary students are evident in regards to participation, retention, and completion rates at secondary and tertiary levels. Instead of focusing on negative statistics, it was of interest to investigate Māori students who had failed sufficient papers to initially be denied re-entry to university study, yet continued to attain their degrees, as a result of a successful appeal. Māori undergraduate students, who had successfully appealed from 2003-2012, were recruited for my research. The availability and utilisation of social supports were investigated, along with the decision to make an appeal, the role of cultural expectations, and barriers experienced by participants. A mixed method approach was utilised in this research through use of an adapted overarching Kaupapa Māori framework, statistical analysis, and 13 interviews. My research found that social supports were beneficial for participants throughout their appeal, as they provided: emotional, instrumental, appraisal, and informational support. Although the social supports utilised by participants were satisfactory, barriers to accessing formal supports were also found, which included: perceived stigma having failed their papers, pride, as well as being whakamā. The appeal process was a beneficial intervention from the University of Waikato, which provided participants another opportunity to overcome the factors that contributed to their initial failure. Post appeal, participants implemented strategies to address and reduce their factors in order to become academically engaged. Participants were deemed to be resilient from their continued pursuit of degree attainment, implementation of their strategies, and their use of perceived social supports.

year: 2013