title: Clonal cell proliferation in the mammary gland

authors: Jacobson, Gregory M.

abstract: The primary aim of this thesis was to classify the nature of the clonal expansion of cells that gives rise to bovine and murine mammary epithelia. It is generally assumed that the mammary gland is derived from the outgrowth of mammary-determined stem cell progeny, and that this expansion produces a mixture of cellular clones throughout the gland. Molecular techniques that enable the 'visualization' of the size and distribution of these clones will lead to a better appreciation of mammary gland development and architecture. 
In female mammals, the simplest 'mark' of clonality is provided by the inactivation of one or the other of the two X chromosomes. This event occurs early in embryogenesis and gives rise to stem cells that propagate this mark in progeny cells throughout the life of the animal. Two approaches, that utilized X-inactivation as a mark of clonality, were investigated, 1) naturally occurring X-linked gene polymorphisms in cattle, and 2) artificially introduced X-linked transgenes in mice. 
The first part of this thesis deals with the isolation and utilization of polymorphic markers on the bovine X-chromosome as markers of clonality. Such expressed genetic markers are essential if one is to distinguish which of the two X-chromosomes is active in a given cell. Two X-linked bovine genes, glucose-6-phosphate dehydrogenase (g6pd) and the X-inactive specific transcript (Xist) were selected for sequencing and SNP detection. As no specific sequence data was available for these genes, a bovine lambda genomic DNA library was screened. The G6PD clone selected from a lambda library was shown to be a pseudogene and therefore not useful for the present study. Another clone, that hybridized with an Xist gene probe, was shown to actually represent a bovine Huntingtin Associated Protein 1 like (Hap1-like) pseudogene, making it of no utility in this study. However, screening of regions of exon 1 of the Xist gene by direct PCR and sequencing, led to the discovery of an informative G↔T single nucleotide polymorphism (SNP). The variation, which was found in frequencies of 0.8 and 0.2 for 'G' and 'T' variants respectively (32% of females are heterozygous), was used in a number of different ways in order to assess the clonal profile of small 'blocks' of the gland (≤30 mm3). In these preliminary experiments, RT-PCR analysis of Xist expression patterns within each of the small blocks (each comprising around 3 X 107 cells) showed general heterogeneity but the experiments were not conclusive. Limitations of the experimental methods used are discussed and suggestions of alternative approaches, that will offer higher resolution, are presented. 
A second experimental approach, involving mice with a reporter transgene construct introduced into the X-chromosome (H253), was also used. The transgene consisted of 14 tandem repeats of a lacZ nuclear-localized reporter gene coupled with a mouse 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgcr) promoter (Tam and Tan, 1992). A number of researchers had already utilized this transgenic model, and had confirmed that the cellular patterns of lacZ expression reflected actual X-inactivation patterns in a number of tissues. While carrying out initial investigations to ascertain the utility of this reporter system in investigations of the mammary gland it was discovered that the usual description of hmgcr as a 'house-keeping' gene is somewhat of a misnomer, as some tissues in the mouse show very strong patterns of lacZ expression while others gave minimal, or no, detectable expression. Furthermore, transgene expression was observed to increase during pregnancy and it was concluded that this effect was almost certainly a response to increased estrogen levels at this time. 
Whole mount analysis of five mouse mammary glands from transgenic animals revealed heterogeneous patterns of lacZ staining, indicating that mammary epithelia is generally polyclonal. In total, 42% of terminal buds and alveoli displayed a single lacZ staining pattern. Analysis of these staining patterns induct termini indicated that the majority of individual alveoli are derived from two or three division competent (stem) cells.

text: Clonal cell proliferation in the mammary gland The primary aim of this thesis was to classify the nature of the clonal expansion of cells that gives rise to bovine and murine mammary epithelia. It is generally assumed that the mammary gland is derived from the outgrowth of mammary-determined stem cell progeny, and that this expansion produces a mixture of cellular clones throughout the gland. Molecular techniques that enable the 'visualization' of the size and distribution of these clones will lead to a better appreciation of mammary gland development and architecture. 
In female mammals, the simplest 'mark' of clonality is provided by the inactivation of one or the other of the two X chromosomes. This event occurs early in embryogenesis and gives rise to stem cells that propagate this mark in progeny cells throughout the life of the animal. Two approaches, that utilized X-inactivation as a mark of clonality, were investigated, 1) naturally occurring X-linked gene polymorphisms in cattle, and 2) artificially introduced X-linked transgenes in mice. 
The first part of this thesis deals with the isolation and utilization of polymorphic markers on the bovine X-chromosome as markers of clonality. Such expressed genetic markers are essential if one is to distinguish which of the two X-chromosomes is active in a given cell. Two X-linked bovine genes, glucose-6-phosphate dehydrogenase (g6pd) and the X-inactive specific transcript (Xist) were selected for sequencing and SNP detection. As no specific sequence data was available for these genes, a bovine lambda genomic DNA library was screened. The G6PD clone selected from a lambda library was shown to be a pseudogene and therefore not useful for the present study. Another clone, that hybridized with an Xist gene probe, was shown to actually represent a bovine Huntingtin Associated Protein 1 like (Hap1-like) pseudogene, making it of no utility in this study. However, screening of regions of exon 1 of the Xist gene by direct PCR and sequencing, led to the discovery of an informative G↔T single nucleotide polymorphism (SNP). The variation, which was found in frequencies of 0.8 and 0.2 for 'G' and 'T' variants respectively (32% of females are heterozygous), was used in a number of different ways in order to assess the clonal profile of small 'blocks' of the gland (≤30 mm3). In these preliminary experiments, RT-PCR analysis of Xist expression patterns within each of the small blocks (each comprising around 3 X 107 cells) showed general heterogeneity but the experiments were not conclusive. Limitations of the experimental methods used are discussed and suggestions of alternative approaches, that will offer higher resolution, are presented. 
A second experimental approach, involving mice with a reporter transgene construct introduced into the X-chromosome (H253), was also used. The transgene consisted of 14 tandem repeats of a lacZ nuclear-localized reporter gene coupled with a mouse 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgcr) promoter (Tam and Tan, 1992). A number of researchers had already utilized this transgenic model, and had confirmed that the cellular patterns of lacZ expression reflected actual X-inactivation patterns in a number of tissues. While carrying out initial investigations to ascertain the utility of this reporter system in investigations of the mammary gland it was discovered that the usual description of hmgcr as a 'house-keeping' gene is somewhat of a misnomer, as some tissues in the mouse show very strong patterns of lacZ expression while others gave minimal, or no, detectable expression. Furthermore, transgene expression was observed to increase during pregnancy and it was concluded that this effect was almost certainly a response to increased estrogen levels at this time. 
Whole mount analysis of five mouse mammary glands from transgenic animals revealed heterogeneous patterns of lacZ staining, indicating that mammary epithelia is generally polyclonal. In total, 42% of terminal buds and alveoli displayed a single lacZ staining pattern. Analysis of these staining patterns induct termini indicated that the majority of individual alveoli are derived from two or three division competent (stem) cells.

year: 2004

title: Life on parole: An extended investigation of how pre-release and post-release factors contribute to desistence from crime

authors: Brown, Andrew

subjects: prevention of recidivism

abstract: Prevention of recidivism is the universally accepted goal of the New Zealand/ Aotearoa justice system, the nation’s Department of Corrections, politicians and public (“Our vision goal and priorities reducing re-offending”, New Zealand Department of Corrections, 2015). What can be done within prisons prior to release and then in the community post-release to reduce recidivism needs to be better understood. Parolees endure significant challenges when they transition back to their communities. Finding suitable accommodation, confirming stable employment, and maintaining prosocial support networks rate highly among these challenges (Bucklen & Zajac, 2009). While a high proportion of parolees fail to meet these challenges and recidivate soon after release, there are some who do not. Understanding the factors that enable those who avoid further convictions post-release is an important step in designing interventions and processes that might lead to higher a proportion of parolees similarly succeeding (Polaschek & Yesberg, 2016). Of equal importance is the need to understand the factors that increase parolees’ risks of recidivating. Measures have been designed by researchers to understand first, how men prepare for, and conceptualise, their re- entries and second, how they then experience life on parole. The Release Plan Quality (RPQ) measure was designed to analyse prisoners’ release plans and rate their quality on various metrics. Another measure, the Parole Experiences Measure (PEM) rates the quality of parolees’ experiences during re-entry and transition (Wilson, Kilgour & Polaschek, 2013). This thesis examines the PEM and the RPQ in relation to four recidivism outcomes at one- year post-release, namely; conviction for breach of parole conditions, reconviction for new offending (excluding convictions for breach of parole), new convictions for violence offences and convictions leading to reimprisonment. Logistic regression analysis results indicated that PEM outcomes were more predictive of the four recidivism outcomes than the RPQ results. Better RPQ results did, however, predict better PEM results. PEM results (particularly PEM results from the PEM external circumstances subscale) at the two-month mark post-release were found to best predict the four recidivism outcomes. This indicated that environmental issues external to the parolee such as employment and accommodation were more likely to impact risk of recidivism than internal, personal factors such as mental health and personal attitudes. The effectiveness of the PEM two-month results for predicting the four recidivism outcomes remained after controlling for possible confounding variables. This research suggests first, that parolees who have better release plans will have better experiences when on parole and second that parolees who have early positive experiences on parole particularly in relation to external factors such as employment and accommodation are somewhat more likely to remain crime-free post-release.

text: Life on parole: An extended investigation of how pre-release and post-release factors contribute to desistence from crime Prevention of recidivism is the universally accepted goal of the New Zealand/ Aotearoa justice system, the nation’s Department of Corrections, politicians and public (“Our vision goal and priorities reducing re-offending”, New Zealand Department of Corrections, 2015). What can be done within prisons prior to release and then in the community post-release to reduce recidivism needs to be better understood. Parolees endure significant challenges when they transition back to their communities. Finding suitable accommodation, confirming stable employment, and maintaining prosocial support networks rate highly among these challenges (Bucklen & Zajac, 2009). While a high proportion of parolees fail to meet these challenges and recidivate soon after release, there are some who do not. Understanding the factors that enable those who avoid further convictions post-release is an important step in designing interventions and processes that might lead to higher a proportion of parolees similarly succeeding (Polaschek & Yesberg, 2016). Of equal importance is the need to understand the factors that increase parolees’ risks of recidivating. Measures have been designed by researchers to understand first, how men prepare for, and conceptualise, their re- entries and second, how they then experience life on parole. The Release Plan Quality (RPQ) measure was designed to analyse prisoners’ release plans and rate their quality on various metrics. Another measure, the Parole Experiences Measure (PEM) rates the quality of parolees’ experiences during re-entry and transition (Wilson, Kilgour & Polaschek, 2013). This thesis examines the PEM and the RPQ in relation to four recidivism outcomes at one- year post-release, namely; conviction for breach of parole conditions, reconviction for new offending (excluding convictions for breach of parole), new convictions for violence offences and convictions leading to reimprisonment. Logistic regression analysis results indicated that PEM outcomes were more predictive of the four recidivism outcomes than the RPQ results. Better RPQ results did, however, predict better PEM results. PEM results (particularly PEM results from the PEM external circumstances subscale) at the two-month mark post-release were found to best predict the four recidivism outcomes. This indicated that environmental issues external to the parolee such as employment and accommodation were more likely to impact risk of recidivism than internal, personal factors such as mental health and personal attitudes. The effectiveness of the PEM two-month results for predicting the four recidivism outcomes remained after controlling for possible confounding variables. This research suggests first, that parolees who have better release plans will have better experiences when on parole and second that parolees who have early positive experiences on parole particularly in relation to external factors such as employment and accommodation are somewhat more likely to remain crime-free post-release.

year: 2019

title: Māori language interfaces

authors: Keegan, Te Taka Adrian Gregory

abstract: This paper considers the usability of the interface and the ramifications for te reo Māori if the interface language, the messages displayed in those text boxes, is written in te reo Māori.

text: Māori language interfaces This paper considers the usability of the interface and the ramifications for te reo Māori if the interface language, the messages displayed in those text boxes, is written in te reo Māori.

year: 2017