title: Do PhoH2 proteins regulate SigF in mycobacteria?

authors: Rzoska-Smith, Elizabeth

abstract: There is little known about the role of the PhoH2 proteins in biology. PhoH2 consists of two domains, an N-terminal PIN domain with RNase activity and a C-terminal PhoH domain shown to have ATP induced RNA helicase activity. PhoH2 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis show ATP and magnesium dependent, sequence-specific RNA unwinding and cleavage activity. It is hypothesised based on RNAseq data that PhoH2 from M. smegmatis may be acting as a potential regulator of sigma factor SigF, through its targeted RNA helicase and RNase activity. Regulation may occur directly on the sigF mRNA transcript, or indirectly through regulation of anti-sigma factor antagonists. This thesis used purified PhoH2 proteins to examine the activity of PhoH2 against mRNA substrates designed to span regions of the sigF gene and the genes co-expressed in the sigF operon; rsbW and chaB. Results show that PhoH2 co-purifies with E. coli nucleic acid and that despite this co-purification, activity was observed on sigF mRNA, that was improved with increasing amounts of ATP. qPCR experiments were designed to test expression levels of 12 genes, that showed significant up or down regulation expression in M. smegmatis mc²155 ΔphoH2 compared to wild-type M. smegmatis mc²155. qPCR analysis revealed a significant upregulation of the genes MSMEG_1804 (sigF) and MSMEG_0064 (PPE) in M. smegmatis mc²155 ΔphoH2 compared to M. smegmatis mc²155. These results differ from the gene expression results obtained from RNAseq data, yet results still point towards regulation of SigF by PhoH2. Further experiments using qPCR and activity assays with PhoH2 aim to verify the precise role of PhoH2 in the regulation of SigF.

text: Do PhoH2 proteins regulate SigF in mycobacteria? There is little known about the role of the PhoH2 proteins in biology. PhoH2 consists of two domains, an N-terminal PIN domain with RNase activity and a C-terminal PhoH domain shown to have ATP induced RNA helicase activity. PhoH2 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis show ATP and magnesium dependent, sequence-specific RNA unwinding and cleavage activity. It is hypothesised based on RNAseq data that PhoH2 from M. smegmatis may be acting as a potential regulator of sigma factor SigF, through its targeted RNA helicase and RNase activity. Regulation may occur directly on the sigF mRNA transcript, or indirectly through regulation of anti-sigma factor antagonists. This thesis used purified PhoH2 proteins to examine the activity of PhoH2 against mRNA substrates designed to span regions of the sigF gene and the genes co-expressed in the sigF operon; rsbW and chaB. Results show that PhoH2 co-purifies with E. coli nucleic acid and that despite this co-purification, activity was observed on sigF mRNA, that was improved with increasing amounts of ATP. qPCR experiments were designed to test expression levels of 12 genes, that showed significant up or down regulation expression in M. smegmatis mc²155 ΔphoH2 compared to wild-type M. smegmatis mc²155. qPCR analysis revealed a significant upregulation of the genes MSMEG_1804 (sigF) and MSMEG_0064 (PPE) in M. smegmatis mc²155 ΔphoH2 compared to M. smegmatis mc²155. These results differ from the gene expression results obtained from RNAseq data, yet results still point towards regulation of SigF by PhoH2. Further experiments using qPCR and activity assays with PhoH2 aim to verify the precise role of PhoH2 in the regulation of SigF.

year: 2018

title: Benefits and issues: Visitor sessions with pre-intermediate learners of japanese

authors: Nishimura, Fumiko

abstract: In recent years, the number of Japanese language students in tertiary institutions in New Zealand has gradually decreased (Ogino, 2015), and Japanese departments have experienced budget and staff cuts. As a consequence of these losses, tertiary educators are required to effectively and efficiently conduct the same duties (if not more), but with fewer resources. Accordingly, demand for teaching-learning strategies that require minimal preparation has increased. Visitor sessions (VS) could be a creative answer to this demand, while also having significant benefits for the language learners. This study demonstrates how VS work in our institution, and may give insightful suggestions to other language educators in New Zealand who wish to incorporate VS into their curricula.

text: Benefits and issues: Visitor sessions with pre-intermediate learners of japanese In recent years, the number of Japanese language students in tertiary institutions in New Zealand has gradually decreased (Ogino, 2015), and Japanese departments have experienced budget and staff cuts. As a consequence of these losses, tertiary educators are required to effectively and efficiently conduct the same duties (if not more), but with fewer resources. Accordingly, demand for teaching-learning strategies that require minimal preparation has increased. Visitor sessions (VS) could be a creative answer to this demand, while also having significant benefits for the language learners. This study demonstrates how VS work in our institution, and may give insightful suggestions to other language educators in New Zealand who wish to incorporate VS into their curricula.

year: 2016

title: The Long Bay Restaurant site, R10/1374: Volume 1, the archaeology (HNZPTA authorities 2015/19 and 2016/81): Report to Heritage New Zealand Pouhere Taonga and Auckland Council

authors: Campbell, M

abstract: Auckland Council had proposed refurbishing the Long Bay Restaurant at Long Bay Regional Park (Lot 2 DP 54616), amounting to a near full rebuild of the structure apart from the in situ foundations. Council began the project in mid-2013 with the partial demolition of the existing structure and sawing of the concrete floor slab to install new utilities. Following the discovery of human remains beneath the floor slab on 2 July 2013, work was halted. The remains were inspected by the New Zealand Police who confirmed that they were pre-European kōiwi tangata. An assessment of the site was then undertaken by Beatrice Hudson of ArchOs Archaeology (Hudson 2013) and the find was recorded as site R10/1374 in the New Zealand Archaeological Association (NZAA) Site Recording Scheme (SRS) (this site number applies to any other archaeological features associated with the wider area). Auckland Council applied to the New Zealand Historic Places Trust (now Heritage New Zealand Pouhere Taonga, HNZPT) for an archaeological authority under section 18 of the Historic Places Act 1993 to undertake archaeological test excavations to investigate the nature, extent and condition of the site, and how it would be affected by the proposed development project. Authority 2014/506 was granted on 28 November 2013 and the investigation was carried out on the 20th and 21st of January 2014 (Campbell et al. 2014) (the 2014 excavation is summarised in Chapter 3).

Auckland Council then applied to HNZPT for an authority under section 44 of the Heritage New Zealand Pouhere Taonga Act 2014 to modify or destroy any further material found during the refurbishment project, including associated car parks and footpaths. Authority 2015/19 was granted on 7 August 2014 and works resumed. On 6 November 2014 further kōiwi were found within the project footprint and work once again ceased. 

Following extensive consultation with mana whenua and HNZPT, Auckland Council determined that the refurbishment would continue but it was agreed that the entire footprint of the development would be cleared archaeologically in order to provide assurance that no further kōiwi would remain within the project area. As the scope of the archaeological investigation had changed, a new archaeological authority, superseding authority 2015/19, was required and a full archaeological assessment of efects was also required in support of the authority application (Campbell and Hudson 2015). Authority 2016/81 was granted by HNZPT on 25 August 2015. This report fulfils the conditions of both authority 2015/19 and 2016/81.

Excavation began on 5 October 2015 with site clearance and removal of the concrete pad but not the ground beam foundations, while full excavation commenced on 12 October under the direction of Matthew Campbell (Excavation Director, CFG Heritage) and Beatrice Hudson (Osteologist, ArchOs Archaeology). Excavation continued until 3 November, by which stage kōiwi of a further 10 individuals had been found, bringing the total to 12. Following an onsite hui on 4 November between representatives of mana whenua, HNZPT, Auckland Council and the excavation team, the excavation was halted but mana whenua requested that the 3 possible burials that had been located but not excavated be lifted, and on 9 November the site was shut down. At a second on site hui on 9 December Auckland Council announced that they had resolved to cancel the restaurant project, but mana whenua expressed a desire that the 13 x 12 m excavation area opened up at the time be excavated to a sterile layer to lift all kōiwi. Excavation resumed on 18 January 2016 and continued until 4 March, when all kōiwi and archaeological deposits and features had been excavated.

text: The Long Bay Restaurant site, R10/1374: Volume 1, the archaeology (HNZPTA authorities 2015/19 and 2016/81): Report to Heritage New Zealand Pouhere Taonga and Auckland Council Auckland Council had proposed refurbishing the Long Bay Restaurant at Long Bay Regional Park (Lot 2 DP 54616), amounting to a near full rebuild of the structure apart from the in situ foundations. Council began the project in mid-2013 with the partial demolition of the existing structure and sawing of the concrete floor slab to install new utilities. Following the discovery of human remains beneath the floor slab on 2 July 2013, work was halted. The remains were inspected by the New Zealand Police who confirmed that they were pre-European kōiwi tangata. An assessment of the site was then undertaken by Beatrice Hudson of ArchOs Archaeology (Hudson 2013) and the find was recorded as site R10/1374 in the New Zealand Archaeological Association (NZAA) Site Recording Scheme (SRS) (this site number applies to any other archaeological features associated with the wider area). Auckland Council applied to the New Zealand Historic Places Trust (now Heritage New Zealand Pouhere Taonga, HNZPT) for an archaeological authority under section 18 of the Historic Places Act 1993 to undertake archaeological test excavations to investigate the nature, extent and condition of the site, and how it would be affected by the proposed development project. Authority 2014/506 was granted on 28 November 2013 and the investigation was carried out on the 20th and 21st of January 2014 (Campbell et al. 2014) (the 2014 excavation is summarised in Chapter 3).

Auckland Council then applied to HNZPT for an authority under section 44 of the Heritage New Zealand Pouhere Taonga Act 2014 to modify or destroy any further material found during the refurbishment project, including associated car parks and footpaths. Authority 2015/19 was granted on 7 August 2014 and works resumed. On 6 November 2014 further kōiwi were found within the project footprint and work once again ceased. 

Following extensive consultation with mana whenua and HNZPT, Auckland Council determined that the refurbishment would continue but it was agreed that the entire footprint of the development would be cleared archaeologically in order to provide assurance that no further kōiwi would remain within the project area. As the scope of the archaeological investigation had changed, a new archaeological authority, superseding authority 2015/19, was required and a full archaeological assessment of efects was also required in support of the authority application (Campbell and Hudson 2015). Authority 2016/81 was granted by HNZPT on 25 August 2015. This report fulfils the conditions of both authority 2015/19 and 2016/81.

Excavation began on 5 October 2015 with site clearance and removal of the concrete pad but not the ground beam foundations, while full excavation commenced on 12 October under the direction of Matthew Campbell (Excavation Director, CFG Heritage) and Beatrice Hudson (Osteologist, ArchOs Archaeology). Excavation continued until 3 November, by which stage kōiwi of a further 10 individuals had been found, bringing the total to 12. Following an onsite hui on 4 November between representatives of mana whenua, HNZPT, Auckland Council and the excavation team, the excavation was halted but mana whenua requested that the 3 possible burials that had been located but not excavated be lifted, and on 9 November the site was shut down. At a second on site hui on 9 December Auckland Council announced that they had resolved to cancel the restaurant project, but mana whenua expressed a desire that the 13 x 12 m excavation area opened up at the time be excavated to a sterile layer to lift all kōiwi. Excavation resumed on 18 January 2016 and continued until 4 March, when all kōiwi and archaeological deposits and features had been excavated.

year: 2019