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: Interfacial modification of hemp fiber reinforced composites using fungal and alkali treatment

authors: Pickering, Kim L.

subjects: natural fibers

abstract: Increasing worldwide environmental awareness is encouraging scientific research into developing cheaper, more sustainable materials. Industrial hemp fiber is one of the strongest and stiffest available natural fibers [K. L. Pickering, M. Priest, T. Watts, G. Beckermann, and S. N. Alam, J. Adv. Mater. 37, 15 (2005)] and therefore has great potential in composite materials. Incorporated into a thermoplastic matrix, it gives a structural material that is cheap, lightweight, and recyclable. However, natural fibers are commonly incompatible with common molding thermoplastics such as polypropylene, which limits the performance of the composites produced. The main objective of the current work was to investigate the use of fungi to treat hemp fiber to create better bonding characteristics in natural fiber reinforced polypropylene composites. X-ray diffraction (XRD), ζ-potential, lignin testing, thermal analysis, and scanning electron microscopy (SEM) were used to characterize the effect of treatment on hemp fibers. A combined alkali and fungi treated fiber composite produced the highest tensile strength of 48.3 MPa, an increase of 32% compared to composites with untreated fiber.

text: Interfacial modification of hemp fiber reinforced composites using fungal and alkali treatment Increasing worldwide environmental awareness is encouraging scientific research into developing cheaper, more sustainable materials. Industrial hemp fiber is one of the strongest and stiffest available natural fibers [K. L. Pickering, M. Priest, T. Watts, G. Beckermann, and S. N. Alam, J. Adv. Mater. 37, 15 (2005)] and therefore has great potential in composite materials. Incorporated into a thermoplastic matrix, it gives a structural material that is cheap, lightweight, and recyclable. However, natural fibers are commonly incompatible with common molding thermoplastics such as polypropylene, which limits the performance of the composites produced. The main objective of the current work was to investigate the use of fungi to treat hemp fiber to create better bonding characteristics in natural fiber reinforced polypropylene composites. X-ray diffraction (XRD), ζ-potential, lignin testing, thermal analysis, and scanning electron microscopy (SEM) were used to characterize the effect of treatment on hemp fibers. A combined alkali and fungi treated fiber composite produced the highest tensile strength of 48.3 MPa, an increase of 32% compared to composites with untreated fiber.

year: 2007

title: Te Papa-o-Rotu Marae Management and Administration at the End of the Twentieth Century: Negotiating Bureaucratisation

authors: Collins, Adelaide

subjects: informal organising

abstract: Te Papa-o-Rotu Marae is a Māori community settlement located in the Waikato region of New Zealand. Its hapu (sub-tribe) community was one of 33 hapu that formed the Tainui confederation claiming compensation from the Crown for land confiscated in the nineteenth century.  The claim was settled in 1995 and it was within this context that research for this study was conducted at the marae from August 1997 to December 1999.  

This ethnographic study examines the way that the community at Te Papa-o-Rotu Marae managed its affairs through its two management bodies, the Marae Committee and the Trustees.  It is argued in this thesis that the marae's mode of management is in transition from an informal to formal mode, and from an inward to outward looking focus.  Bureaucratic administration, it is argued, has been the major catalyst for the transition and has been introduced into marae operations through an accumulation of state legislation affecting Māori land and communities. Furthermore, some aspects of bureaucratic administration have been legitimated and appropriated by the iwi authority, which has passed this on to the Marae Committee.  The community have been complicit in the adoption of bureaucratic administration by accommodating the requirements of both the state and the iwi authority.  However, a persistent question was whether the marae could maintain its own rangatiratanga (authority, self-determination, control) and separate identity in the face of increasing pressure to conform to a bureaucratic management style.

The community managed the marae communally by way of hui (gatherings) and meetings, which were observed using a combined methodological approach of Kaupapa Māori research and ethnography, as described in Chapter 2.  The philosophy of kotahitanga (solidarity) underpinned the social organisation of the Tainui tribal confederation, so understanding the place of the marae in its wider socio-political environment has helped in comprehending the nature of the pressure on the community to increase its scale of operations and is explained in Chapter 3. 

Chapter 4 introduces the accumulation of influences that have brought about a change of managerial style from informal to formal organising.  The practical effect of these influences are demonstrated in the management structure and administrative systems that the community used. These are described phenomenologically in Chapters 5 and 6 respectively.  The management plan, compiled since 1995, had a strong emphasis on management structural review and participation in tribal development initiatives and is discussed in Chapter 7. The implementation of a collaborative development project between the iwi authority and Marae Committee is described in Chapter 8.  The final chapter reflects on the impact of bureaucratic administration on marae management as well as the dynamism of the community and how the rangatiratanga of the marae has thus far been maintained.

text: Te Papa-o-Rotu Marae Management and Administration at the End of the Twentieth Century: Negotiating Bureaucratisation Te Papa-o-Rotu Marae is a Māori community settlement located in the Waikato region of New Zealand. Its hapu (sub-tribe) community was one of 33 hapu that formed the Tainui confederation claiming compensation from the Crown for land confiscated in the nineteenth century.  The claim was settled in 1995 and it was within this context that research for this study was conducted at the marae from August 1997 to December 1999.  

This ethnographic study examines the way that the community at Te Papa-o-Rotu Marae managed its affairs through its two management bodies, the Marae Committee and the Trustees.  It is argued in this thesis that the marae's mode of management is in transition from an informal to formal mode, and from an inward to outward looking focus.  Bureaucratic administration, it is argued, has been the major catalyst for the transition and has been introduced into marae operations through an accumulation of state legislation affecting Māori land and communities. Furthermore, some aspects of bureaucratic administration have been legitimated and appropriated by the iwi authority, which has passed this on to the Marae Committee.  The community have been complicit in the adoption of bureaucratic administration by accommodating the requirements of both the state and the iwi authority.  However, a persistent question was whether the marae could maintain its own rangatiratanga (authority, self-determination, control) and separate identity in the face of increasing pressure to conform to a bureaucratic management style.

The community managed the marae communally by way of hui (gatherings) and meetings, which were observed using a combined methodological approach of Kaupapa Māori research and ethnography, as described in Chapter 2.  The philosophy of kotahitanga (solidarity) underpinned the social organisation of the Tainui tribal confederation, so understanding the place of the marae in its wider socio-political environment has helped in comprehending the nature of the pressure on the community to increase its scale of operations and is explained in Chapter 3. 

Chapter 4 introduces the accumulation of influences that have brought about a change of managerial style from informal to formal organising.  The practical effect of these influences are demonstrated in the management structure and administrative systems that the community used. These are described phenomenologically in Chapters 5 and 6 respectively.  The management plan, compiled since 1995, had a strong emphasis on management structural review and participation in tribal development initiatives and is discussed in Chapter 7. The implementation of a collaborative development project between the iwi authority and Marae Committee is described in Chapter 8.  The final chapter reflects on the impact of bureaucratic administration on marae management as well as the dynamism of the community and how the rangatiratanga of the marae has thus far been maintained.

year: 2005