title: The repair mechanism of double-stranded breaks in post-mitotic neuronal cells

authors: Coffin, Grace

abstract: A neuron is a fundamental unit of the nervous system. These cells are differentiated and are not readily replaced. They are extremely active and display high metabolic rates. Because of their high activity they are subjected to a large amount of DNA damage throughout their lives. The most genotoxic type of DNA damage is a break in both strands of DNA and there can be as many as 10 to 50 double-stranded breaks (DSBs) in every cell per day. Because of this, a high-fidelity DNA repair mechanism would seem critical for ensuring neuronal longevity.

There are two main mechanisms in which DNA DSBs are repaired: homologous recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is said to occur in proliferating cells, whereas NHEJ is said to occur in post-mitotic cells. HRR is a high-fidelity process that uses a homologous copy of damaged DNA, usually from a sister chromatid, as a template to achieve error-free repair. In contrast, NHEJ is an error-prone system that simply uses a resection/re-ligation method to join the two ends of DNA back together. Because neurons are post-mitotic, they are thought to be repaired by NHEJ. However, given their longevity and high levels of activity, it would seem essential that neurons repair DNA DSBs by an error-free system. 

The goal of this study was to determine whether HRR occurs in post-mitotic neuronal cells. The cell line Neuro-2a (N2a) is a mouse derived neuroblastoma cell line. The first objective of this project was to determine a differentiation protocol for this cell line. It was found, through cell counting and EdU incorporation assays, that cells treated with media containing 1% foetal bovine serum (FBS) and 10 μM retinoic acid (RA), over the course of seven days, showed the greatest number of N2a cells entering a non-proliferative state. 

The plasmids DR-GFP and I-SceI were amplified and purified by bacterial transformation and miniprep. Once the plasmids were purified, DR-GFP was transfected into N2a cells by lipofection. The DR-GFP plasmid is a vector that has been engineered to contain a non-functional full-length green fluorescent protein (GFP) gene with a 5’-premature stop codon/I-SceI recognition sequence. These transfected cells were then treated with puromycin, as the DR-GFP plasmid  contains a puromycin resistance gene, to generate a stable N2a-DRGFP cell line. 

Once stable integration of DR-GFP was achieved, the N2a-DRGFP cells were then transfected with the I-SceI plasmid. I-SceI restriction endonuclease encoded by the I-Scel plasmid, has no recognition sequence in the mouse genome, so the expression of I-SceI will induce a single DSB at the DR-GFP 5’-premature stop codon/I-SceI recognition site. The DR-GFP vector contains a portion of the wild-type GFP sequence, so if HRR occurs after I-SceI expression, the GFP 5’- premature stop codon will be removed and a functional GFP protein will be detected by immunofluorescence. 

No fluorescence was observed after I-SceI transfection in proliferating cells. PCR amplification of a DR-GFP sequence from N2a-DRGFP cells showed that DR-GFP had not been stably integrated into N2a cells. Similarly, co-transfection of I-SceI and DR-GFP into N2a cells also did not show any fluorescence. To determine possible causes for this result, plasmids were sequenced, and it was found that the DR-GFP sequence was incorrect. Because of this, no further experimentation of HRR in post-mitotic cells could occur until the correct DR-GFP plasmid is obtained.

text: The repair mechanism of double-stranded breaks in post-mitotic neuronal cells A neuron is a fundamental unit of the nervous system. These cells are differentiated and are not readily replaced. They are extremely active and display high metabolic rates. Because of their high activity they are subjected to a large amount of DNA damage throughout their lives. The most genotoxic type of DNA damage is a break in both strands of DNA and there can be as many as 10 to 50 double-stranded breaks (DSBs) in every cell per day. Because of this, a high-fidelity DNA repair mechanism would seem critical for ensuring neuronal longevity.

There are two main mechanisms in which DNA DSBs are repaired: homologous recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is said to occur in proliferating cells, whereas NHEJ is said to occur in post-mitotic cells. HRR is a high-fidelity process that uses a homologous copy of damaged DNA, usually from a sister chromatid, as a template to achieve error-free repair. In contrast, NHEJ is an error-prone system that simply uses a resection/re-ligation method to join the two ends of DNA back together. Because neurons are post-mitotic, they are thought to be repaired by NHEJ. However, given their longevity and high levels of activity, it would seem essential that neurons repair DNA DSBs by an error-free system. 

The goal of this study was to determine whether HRR occurs in post-mitotic neuronal cells. The cell line Neuro-2a (N2a) is a mouse derived neuroblastoma cell line. The first objective of this project was to determine a differentiation protocol for this cell line. It was found, through cell counting and EdU incorporation assays, that cells treated with media containing 1% foetal bovine serum (FBS) and 10 μM retinoic acid (RA), over the course of seven days, showed the greatest number of N2a cells entering a non-proliferative state. 

The plasmids DR-GFP and I-SceI were amplified and purified by bacterial transformation and miniprep. Once the plasmids were purified, DR-GFP was transfected into N2a cells by lipofection. The DR-GFP plasmid is a vector that has been engineered to contain a non-functional full-length green fluorescent protein (GFP) gene with a 5’-premature stop codon/I-SceI recognition sequence. These transfected cells were then treated with puromycin, as the DR-GFP plasmid  contains a puromycin resistance gene, to generate a stable N2a-DRGFP cell line. 

Once stable integration of DR-GFP was achieved, the N2a-DRGFP cells were then transfected with the I-SceI plasmid. I-SceI restriction endonuclease encoded by the I-Scel plasmid, has no recognition sequence in the mouse genome, so the expression of I-SceI will induce a single DSB at the DR-GFP 5’-premature stop codon/I-SceI recognition site. The DR-GFP vector contains a portion of the wild-type GFP sequence, so if HRR occurs after I-SceI expression, the GFP 5’- premature stop codon will be removed and a functional GFP protein will be detected by immunofluorescence. 

No fluorescence was observed after I-SceI transfection in proliferating cells. PCR amplification of a DR-GFP sequence from N2a-DRGFP cells showed that DR-GFP had not been stably integrated into N2a cells. Similarly, co-transfection of I-SceI and DR-GFP into N2a cells also did not show any fluorescence. To determine possible causes for this result, plasmids were sequenced, and it was found that the DR-GFP sequence was incorrect. Because of this, no further experimentation of HRR in post-mitotic cells could occur until the correct DR-GFP plasmid is obtained.

year: 2020

title: Organisational identities and rationalities: A rhetorical and discourse analysis of organisational communication about genetic modification in the New Zealand kiwifruit and dairy industries

authors: Henderson, Alison

abstract: This thesis examines how two of New Zealand's largest primary producer industries negotiated their relationship with the highly controversial practice of genetic modification. This was in an effort to maintain their dominance in export markets, at a time when genetically modified foods were on the one hand regarded as a possible liability in the marketplace, and on the other hand offered the apparent potential for innovation and economic gain. 

The two case studies featured in this thesis-the dairy industry and the kiwifruit industry-both earn significant export incomes and are seen as national icons as well as major institutions for New Zealand. However, these two industries take differing positions in the debate about genetic modification. The kiwifruit industry has urged extreme caution while the dairy industry has argued for rapid involvement in genetic modification. 

The research takes a critical-interpretive perspective on the two cases. The study is both issue- and case-driven, and uses a combination of rhetorical criticism and discourse analysis to examine the social construction of meanings about genetic modification. The research focus is on the intersection of public relations and organisational communication, including issues management, and especially on the ways in which rhetoric is used as a means of managing multiple organisational identities. 

The research findings indicate that the positioning of these two industries on genetic modification is largely market-driven. The differences in their policies result from contrasting industry products, industry markets, and industry cultures. The values and values-related tensions, expressed explicitly and implicitly in the research data, indicate that there is a dynamic interplay between the rationalities used by the industries to justify their positioning on genetic modification and the multiple identities that need to be managed by each industry. In the kiwifruit industry, environmental concerns are highlighted by an emphasis on 'soft' pest management and organic production, and kiwifruit become a luxury raw product sold mainly to markets in Europe and Japan. In the dairy industry, commodity milk products compete on the basis of low-cost, efficient production, and the major markets are in South East Asia and Latin America. 

This thesis contributes to recent work on corporate identity and organisational communication. Significantly, it demonstrates how the blurring of boundaries between internal and external organisational communication has increased the need for a more complex understanding of how organisations manage multiple organisational identities, particularly where broader socio-political issues, including national identities and international markets, are concerned.

text: Organisational identities and rationalities: A rhetorical and discourse analysis of organisational communication about genetic modification in the New Zealand kiwifruit and dairy industries This thesis examines how two of New Zealand's largest primary producer industries negotiated their relationship with the highly controversial practice of genetic modification. This was in an effort to maintain their dominance in export markets, at a time when genetically modified foods were on the one hand regarded as a possible liability in the marketplace, and on the other hand offered the apparent potential for innovation and economic gain. 

The two case studies featured in this thesis-the dairy industry and the kiwifruit industry-both earn significant export incomes and are seen as national icons as well as major institutions for New Zealand. However, these two industries take differing positions in the debate about genetic modification. The kiwifruit industry has urged extreme caution while the dairy industry has argued for rapid involvement in genetic modification. 

The research takes a critical-interpretive perspective on the two cases. The study is both issue- and case-driven, and uses a combination of rhetorical criticism and discourse analysis to examine the social construction of meanings about genetic modification. The research focus is on the intersection of public relations and organisational communication, including issues management, and especially on the ways in which rhetoric is used as a means of managing multiple organisational identities. 

The research findings indicate that the positioning of these two industries on genetic modification is largely market-driven. The differences in their policies result from contrasting industry products, industry markets, and industry cultures. The values and values-related tensions, expressed explicitly and implicitly in the research data, indicate that there is a dynamic interplay between the rationalities used by the industries to justify their positioning on genetic modification and the multiple identities that need to be managed by each industry. In the kiwifruit industry, environmental concerns are highlighted by an emphasis on 'soft' pest management and organic production, and kiwifruit become a luxury raw product sold mainly to markets in Europe and Japan. In the dairy industry, commodity milk products compete on the basis of low-cost, efficient production, and the major markets are in South East Asia and Latin America. 

This thesis contributes to recent work on corporate identity and organisational communication. Significantly, it demonstrates how the blurring of boundaries between internal and external organisational communication has increased the need for a more complex understanding of how organisations manage multiple organisational identities, particularly where broader socio-political issues, including national identities and international markets, are concerned.

year: 2005

title: Indigenous peoples, data, and the coloniality of surveillance

authors: Cormack, Donna

abstract: In Aotearoa New Zealand (Aotearoa NZ), Māori (the Indigenous peoples of New Zealand) have long been objects of surveillance by state institutions and agents. State representations have centred on constructions of difference and deviance, on understandings of Indigenous peoples as dangerous, and on the management of Indigenous resistance to colonialism. This chapter considers how contemporary state surveillance practices in Aotearoa NZ, enabled by the expanded use of big data and linked government datasets, function to regulate and manage Māori. Through this lens, we explore continuities of current data practices for Indigenous peoples with the racialised logics and social orders set in place as part of global systems of imperialism and colonialism. Recognising that resistance has always been a part of Indigenous responses to colonialism, we also explore how Māori Data Sovereignty (MDSov), as part of broader Indigenous Data Sovereignty (IDS) movements globally, provides opportunities to counter and disrupt prevailing data relations and to imagine alternative futures.

text: Indigenous peoples, data, and the coloniality of surveillance In Aotearoa New Zealand (Aotearoa NZ), Māori (the Indigenous peoples of New Zealand) have long been objects of surveillance by state institutions and agents. State representations have centred on constructions of difference and deviance, on understandings of Indigenous peoples as dangerous, and on the management of Indigenous resistance to colonialism. This chapter considers how contemporary state surveillance practices in Aotearoa NZ, enabled by the expanded use of big data and linked government datasets, function to regulate and manage Māori. Through this lens, we explore continuities of current data practices for Indigenous peoples with the racialised logics and social orders set in place as part of global systems of imperialism and colonialism. Recognising that resistance has always been a part of Indigenous responses to colonialism, we also explore how Māori Data Sovereignty (MDSov), as part of broader Indigenous Data Sovereignty (IDS) movements globally, provides opportunities to counter and disrupt prevailing data relations and to imagine alternative futures.

year: 2022