title: Prolactin-induced proteins and dependence on protein synthesis for β-casein expression in mouse mammary cells

authors: Beaton, Angela

abstract: The regulation of milk protein gene expression in mammary epithelial cells occurs in response to multiple hormonal signals, plus cell-matrix and cell-cell interactions. A number of signalling molecules have been implicated in this process, but the exact molecular mechanisms regulating milk protein gene expression remain to be established. The project described in this thesis focuses on investigating the de novo synthesis of proteins in response to prolactin in mammary epithelial cells, to determine the role of de novo synthesis in lactogenic signalling, and to identify proteins which are induced by prolactin that could act as mediators of lactogenic signals. Two main approaches were taken:

(1) The results of proteome analyses using two-dimensional electrophoresis revealed a number of prolactin-responsive proteins in cells from the mouse mammary epithelial cell line, COMMA-D, and also lactation-associated proteins in freshly isolated mouse mammary epithelial cells. Two prolactin-induced and lactation-associated proteins, p77 and p63, were identified as glucose regulated protein 78 (grp78) and protein disulphide isomerase (PDI), and assessed as potential regulators of lactation. These proteins are reticuloplasmins and probably function in mammary epithelial cells in the processing of milk proteins for secretion. The results of this investigation have shown that grp78 and PDI were not rapidly induced in response to prolactin, thus it is unlikely that these proteins are involved in mediating lactogenic signals in response to prolactin.

(2) The requirement for de novo protein synthesis for maximal prolactin-stimulated β-casein gene expression was evaluated in COMMA-D cells using the protein synthesis inhibitors cycloheximide (CHX) and anisomycin. It appears that protein synthesis is required at the level of transcription for maximal induction and maintenance of β-casein gene expression (over the first 8 h of prolactin treatment). Furthermore, this effect was shown to be very rapid and independent of the lactogenic hormones, insulin and hydrocortisone, and the extracellular matrix. To determine the molecular basis for the effect of CHX on β-casein mRNA levels, STAT5 protein levels and DNA-binding activity were analysed in COMMA-D cells cultured in the presence or absence of prolactin and/or CHX. The results of Western analyses showed that STAT5 protein levels were not significantly altered in  the presence or absence of (prolactin and/ or) CHX. A DNA probe, denoted STAT5(30), derived from the rat β-casein promoter sequence (-104 to -75), which contains a high affinity STAT5 consensus sequence was used for EMSA. CHX treatment stimulated the induction of a DNA-binding activity of distinct mobility to STAT5. This protein-DNA complex was shown to contain the p50 subunit of NF-κB. Detailed analysis revealed that CHX-stimulated NF-κB bound to a putative κB half-site on the STAT5(30) probe (GGAATT), which overlaps the high-affinity STAT5 DNA-binding site (TTCTTGGAA). Therefore, the effect of CHX appears to be mediated by inhibition of IκB synthesis, leading to the activation of NF-κB. Since the κB site overlaps the STAT5 binding site, it is possible that NF-κB competes for binding to the β-casein promoter and sterically hinders STAT5 binding and transactivation of β-casein transcription. It is conceivable that competition between NF-κB and STAT5 is functionally significant if NF-κB is activated in response to physiological signals in the mammary gland. This remains to be demonstrated.

text: Prolactin-induced proteins and dependence on protein synthesis for β-casein expression in mouse mammary cells The regulation of milk protein gene expression in mammary epithelial cells occurs in response to multiple hormonal signals, plus cell-matrix and cell-cell interactions. A number of signalling molecules have been implicated in this process, but the exact molecular mechanisms regulating milk protein gene expression remain to be established. The project described in this thesis focuses on investigating the de novo synthesis of proteins in response to prolactin in mammary epithelial cells, to determine the role of de novo synthesis in lactogenic signalling, and to identify proteins which are induced by prolactin that could act as mediators of lactogenic signals. Two main approaches were taken:

(1) The results of proteome analyses using two-dimensional electrophoresis revealed a number of prolactin-responsive proteins in cells from the mouse mammary epithelial cell line, COMMA-D, and also lactation-associated proteins in freshly isolated mouse mammary epithelial cells. Two prolactin-induced and lactation-associated proteins, p77 and p63, were identified as glucose regulated protein 78 (grp78) and protein disulphide isomerase (PDI), and assessed as potential regulators of lactation. These proteins are reticuloplasmins and probably function in mammary epithelial cells in the processing of milk proteins for secretion. The results of this investigation have shown that grp78 and PDI were not rapidly induced in response to prolactin, thus it is unlikely that these proteins are involved in mediating lactogenic signals in response to prolactin.

(2) The requirement for de novo protein synthesis for maximal prolactin-stimulated β-casein gene expression was evaluated in COMMA-D cells using the protein synthesis inhibitors cycloheximide (CHX) and anisomycin. It appears that protein synthesis is required at the level of transcription for maximal induction and maintenance of β-casein gene expression (over the first 8 h of prolactin treatment). Furthermore, this effect was shown to be very rapid and independent of the lactogenic hormones, insulin and hydrocortisone, and the extracellular matrix. To determine the molecular basis for the effect of CHX on β-casein mRNA levels, STAT5 protein levels and DNA-binding activity were analysed in COMMA-D cells cultured in the presence or absence of prolactin and/or CHX. The results of Western analyses showed that STAT5 protein levels were not significantly altered in  the presence or absence of (prolactin and/ or) CHX. A DNA probe, denoted STAT5(30), derived from the rat β-casein promoter sequence (-104 to -75), which contains a high affinity STAT5 consensus sequence was used for EMSA. CHX treatment stimulated the induction of a DNA-binding activity of distinct mobility to STAT5. This protein-DNA complex was shown to contain the p50 subunit of NF-κB. Detailed analysis revealed that CHX-stimulated NF-κB bound to a putative κB half-site on the STAT5(30) probe (GGAATT), which overlaps the high-affinity STAT5 DNA-binding site (TTCTTGGAA). Therefore, the effect of CHX appears to be mediated by inhibition of IκB synthesis, leading to the activation of NF-κB. Since the κB site overlaps the STAT5 binding site, it is possible that NF-κB competes for binding to the β-casein promoter and sterically hinders STAT5 binding and transactivation of β-casein transcription. It is conceivable that competition between NF-κB and STAT5 is functionally significant if NF-κB is activated in response to physiological signals in the mammary gland. This remains to be demonstrated.

year: 1998

title: Media studies in New Zealand schools and universities: a research study

authors: Lealand, Geoff

abstract: This report describes the outcome of research conducted between January and December 2008. It draws on research done in 2006, which sought to investigate media teaching developments in the secondary sector (NCEA Media Studies, in particular), and the possible consequences for media teaching (Media Studies, in particular) in the tertiary sector. The 2008 research exercise was designed to extend and expand the information gathered in the 2006 research exercise, by focusing more closely on what is happening in New Zealand classrooms, in respect of NCEA Media Studies. The emphasis in the 2008 research is on the content and delivery of Media Studies Achievement Studies, and the consequences for students, with such prior knowledge, when they encounter first year Media Studies courses in New Zealand tertiary institutions.

text: Media studies in New Zealand schools and universities: a research study This report describes the outcome of research conducted between January and December 2008. It draws on research done in 2006, which sought to investigate media teaching developments in the secondary sector (NCEA Media Studies, in particular), and the possible consequences for media teaching (Media Studies, in particular) in the tertiary sector. The 2008 research exercise was designed to extend and expand the information gathered in the 2006 research exercise, by focusing more closely on what is happening in New Zealand classrooms, in respect of NCEA Media Studies. The emphasis in the 2008 research is on the content and delivery of Media Studies Achievement Studies, and the consequences for students, with such prior knowledge, when they encounter first year Media Studies courses in New Zealand tertiary institutions.

year: 2009

title: Demystifying a relationship between voluntary work and Māori

authors: Te Momo, Oliver Helena Fiona

abstract: The purpose of this thesis is to provide evidence that critically reviews the relationship between Māori and voluntary work in social service organisations. Since colonisation very little research has been initiated that investigated the involvement of Māori in voluntary activity yet recent statistics show that Māori are the highest participants in this type of work. This thesis provides an analysis of the relationship of Māori to voluntary work and its evolution in a Colonial State, Liberal State, Welfare State, and Neo-Liberal State in New Zealand. Three perspectives of Māori voluntary work provide the foundation for the analysis of this relationship. The first perspective describes voluntary work from a personal experience as an insider. The second perspective explores literature that records Māori involvement in voluntary activity. The third perspective documents life experiences from Māori voluntary workers in New Zealand communities. The three perspectives provide an empirical foundation for the type of relationship that has developed between Māori and voluntary work; Māori and the State; Māori and their place in New Zealand society. My interest in Te Ao Māori (the Māori world) as a valid and thriving ontological position and my academic interest in critical theory provide the theoretical lens for my analysis. 

Personal experience as a Māori volunteer opened doors to the communities I entered and added strength to the research, the first perspective. Western research in Māori communities often by non-Māori researchers, was conducted to gather knowledge for Western policy makers. Māori knowledge was not recognised as valid and the people were treated as objects. Knowing this, I searched for an approach that valued Māori participants, valued Māori people, and contained cultural aspects that separated the procedure from Western research. A review of literature, the second perspective, introduced a cultural approach to research termed kaupapa Māori research, generated out of and for Te Ao Māori detailed further in Chapter Three.

Participants’ life experiences coupled with literature provide a wealth of knowledge and a testimony to the type of relationship that exists between Māori and voluntary work. Criticism from Māori communities as to the exploitation of Māori volunteers has swelled in recent years so that the environment volunteers work in is no longer attractive to Māori people. Therefore, documenting the life experiences of Māori who participate in voluntary work as the primary source of information was imperative and produced evidence to describe the relationship between Māori and voluntary work in the thesis. A triangular study: case study, community study, and cross-section study (Chapters Six, Seven, and Eight), provide the ‘grass roots voices’ and represent the third perspective. 

My observation of Māori organisations’ involvement in voluntary work generated in me a deep concern in what appeared to be an involvement in activity that improved neither the lives of the volunteers nor the wider problems to which they attributed their aroha. This involvement activity is a product of historical developments whereby the Western ideology of voluntary work became confused with a Māori ideology of collective participation. This confusion has created an environment where Māori voluntary organisations are working unpaid with insufficient resources in oppressive conditions in the attempt to provide social solutions. I argue that the Māori ideology of collective participation has been co-opted in the emerging conflict of neo-liberalism to provide social services in communities which government agencies exploit. In conclusion, the thesis is a journey through the world of voluntary work for Māori in communal organisations.

text: Demystifying a relationship between voluntary work and Māori The purpose of this thesis is to provide evidence that critically reviews the relationship between Māori and voluntary work in social service organisations. Since colonisation very little research has been initiated that investigated the involvement of Māori in voluntary activity yet recent statistics show that Māori are the highest participants in this type of work. This thesis provides an analysis of the relationship of Māori to voluntary work and its evolution in a Colonial State, Liberal State, Welfare State, and Neo-Liberal State in New Zealand. Three perspectives of Māori voluntary work provide the foundation for the analysis of this relationship. The first perspective describes voluntary work from a personal experience as an insider. The second perspective explores literature that records Māori involvement in voluntary activity. The third perspective documents life experiences from Māori voluntary workers in New Zealand communities. The three perspectives provide an empirical foundation for the type of relationship that has developed between Māori and voluntary work; Māori and the State; Māori and their place in New Zealand society. My interest in Te Ao Māori (the Māori world) as a valid and thriving ontological position and my academic interest in critical theory provide the theoretical lens for my analysis. 

Personal experience as a Māori volunteer opened doors to the communities I entered and added strength to the research, the first perspective. Western research in Māori communities often by non-Māori researchers, was conducted to gather knowledge for Western policy makers. Māori knowledge was not recognised as valid and the people were treated as objects. Knowing this, I searched for an approach that valued Māori participants, valued Māori people, and contained cultural aspects that separated the procedure from Western research. A review of literature, the second perspective, introduced a cultural approach to research termed kaupapa Māori research, generated out of and for Te Ao Māori detailed further in Chapter Three.

Participants’ life experiences coupled with literature provide a wealth of knowledge and a testimony to the type of relationship that exists between Māori and voluntary work. Criticism from Māori communities as to the exploitation of Māori volunteers has swelled in recent years so that the environment volunteers work in is no longer attractive to Māori people. Therefore, documenting the life experiences of Māori who participate in voluntary work as the primary source of information was imperative and produced evidence to describe the relationship between Māori and voluntary work in the thesis. A triangular study: case study, community study, and cross-section study (Chapters Six, Seven, and Eight), provide the ‘grass roots voices’ and represent the third perspective. 

My observation of Māori organisations’ involvement in voluntary work generated in me a deep concern in what appeared to be an involvement in activity that improved neither the lives of the volunteers nor the wider problems to which they attributed their aroha. This involvement activity is a product of historical developments whereby the Western ideology of voluntary work became confused with a Māori ideology of collective participation. This confusion has created an environment where Māori voluntary organisations are working unpaid with insufficient resources in oppressive conditions in the attempt to provide social solutions. I argue that the Māori ideology of collective participation has been co-opted in the emerging conflict of neo-liberalism to provide social services in communities which government agencies exploit. In conclusion, the thesis is a journey through the world of voluntary work for Māori in communal organisations.

year: 2003