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Characterisation of conserved amino acids from the low-oxygen expressed D1 protein in the model organism Synechocystis sp. PCC 6803
No full textOxygenic photosynthesis exploits light, water and carbon dioxide to transform light energy into chemical energy. This process is fundamental in shaping the current oxygenic atmosphere and supports most life on earth. The light-dependent reaction of oxygenic photosynthesis uses light energy and converts it into ATP and NADPH, this process involves four major membrane-bound protein complexes; these include Photosystem II, Photosystem I, cytochrome b6f and ATP synthase. Photosystem II is a multi-protein subunit embedded into the thylakoid membrane of plants, algae and cyanobacteria; it is a highly conserved structure with four core protein complexes, including two homologous proteins D1 and D2, and chlorophyll- binding proteins 43 and 47. The D1 protein is encoded by a psbA gene and is highly susceptible to light damage. Cyanobacteria can have multiple copies of this gene and phylogenetic analysis has shown several variants of the D1 protein. The different D1 isoforms can be divided into different groups; however, distribution is not linked to any distinct traits—the cyanobacterium Synechocystis sp. PCC 6803 carries three copies of the psbA gene; psbA2 and psbA3 encode one type of D1, expressed under normal growth and most stress conditions. The psbA1 gene encodes the D1' protein, an early form of D1 expressed under micro-aerobic conditions. Conserved amino acid substitutions in the D1' protein could affect Photosystem II function, altering photosynthesis. The aim of this study was to investigate if the conserved amino acid changes in the low-oxygen expressed D1 protein altered the function and activity of Photosystem II reaction centres, affecting photosynthetic performance. Eight amino acid residues were characterised by introducing these different residues into D1 protein in Synechocystis sp. PCC 6803: these included Gly80, Phe158, Phe186, Ile192, Thr286, Thr292, Met293 and Ala336. Most of the amino acid residues are located either between or in proximity to the chlorophyll molecule P680 and electron carrier Yz, or close to the oxygen-evolving complex of Photosystem II. Overall, 16 mutant strains were generated by Quickchange II
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polymerase chain reactions; these included a combination of single, double and triple strains. The mutant strains were analysed by measuring the photoautotrophic growth rate, Photosystem II activity, assembly, and electron transfer activity. All strains grew photoautotrophically; however, the growth rate was impaired in the F158L, F186L, T286A and A336V mutant strains. Photosystem II activity in more than half of the mutant strains were altered; F158L, F186L and G80A:F158L strains displayed the most significant reductions. The levels of assembled active Photosystem II reaction centres reduced in all the mutant strains except G80A, I192V and A203T:A336V. The electron transfer activity between QA and QB in most mutant strains was impaired, and back reaction by charge recombination was altered; all mutant strains displayed a donor side effect in Photosystem II, particularly strains with mutations at Gly80, Phe158, Phe186 and Thr286. The results presented demonstrate the importance of the studied residues in Photosystem II’s function, supporting the view that these conserved amino acid residues play a crucial role, either directly or indirectly, in electron transfer activity within the Photosystem II reaction centres. The accumulation of the various amino acid substitutions could provide a D1' that is functionally distinct from normally expressed D1 and confer a selective advantage under specific environmental conditions
Ināianei, i Mua, ā Muri Ake – Now, then, next: A whakapapa analysis of engagement approaches to tangata, whenua and wai: A community case study in Te Tai Tokerau (Northland)
Get PDFThrough case study research located in Tautoro, Northland, Aotearoa New Zealand, this thesis investigates the challenges hapū (kin-community groups) and their marae (ancestral centers of tribal identity, meeting houses) face in their traditional role as kaitiaki (guardians) of land and water.
Harnessing connection and whakapapa (genealogy) to gather ancestral narratives while living and working within the community at the center of the case study, coupled with examinations of legal documents, historical land transfers and western scientific studies, the past and present relationship between hapū, Pākehā (New Zealand Europeans) and the local environment is explored.
Ināianei, i Mua, ā Muri Ake – Now, Then, Next: A Whakapapa Analysis of Engagement Approaches to Tangata, Whenua and Wai: A Case Study in Te Tai Tokerau (Northland) finds that for the hapū of Tautoro, water and land are viewed in a contrasting way to Eurocentric capitalist models. It establishes clearly that ancestral rights exist, and that consultation with hapū over proposed sale and development of land and related water take has historically been performed poorly, resulting in harm to local environments and thus harm to the people of the land.
We see through the case study how a kanohi-ki-te-kanohi (face-to-face), marae-based negotiation with hapū, acknowledging water as a taonga (treasure) with its own inherent mauri (life force) and the kaitiaki role of local hapū, can result in improved outcomes for all parties, and for the local environment.
The purpose of this thesis is to gather narratives for future descendants of the hapū of Tautoro, inform policy at local and central government level in relation to collaboration with hapū, and provide a template for capitalist enterprises whose activities on the land threaten the kaitiakitanga (guardianship) of local hapū. In a climate-change environment, this is important not only to Aotearoa New Zealand’s hapū groups, but also to indigenous communities elsewhere in the world facing similar crises
RCT study comparing the effectiveness of Mixed Reality, Three‐dimensional Monoscopy, and Textbook Style Learning Tools for Anatomical Education in pre‐clinical Medicine
Get PDFThis study, to our knowledge, was the first of its kind which compared the learning tools of textbook style, 3DM, and MR learning tools qualitatively and quantitatively in the short-term anatomy acquisition and retention over a month. It was found that the text-only group and 3DM group on average performed better than the MR group in the short-term only in nominal type questions. While none of the learning tools were superior or inferior for performance in spatial type and mixed type questions in the short-term. The MR group retained their performance in nominal and spatial type knowledge after a month, while the text-only group retained their performance only in spatial type knowledge and 3DM group was unable to retain performance in nominal or spatial type knowledge after a month. The ability to focus using the MR learning tool on average was less than the 3DM group. Despite this, the open responses from participants emphasised the use of the MR learning tool compared to the textbook style and 3DM learning tools had improved engagement, immersion, excitement, enjoyment, and was easier to understand the text and 2D images. Furthermore, exploring the EEG BIS data, the participants in the MR group also averaged a higher awakeness level during the learning session compared to the text-only and 3DM groups
Carbazole complexes as catalysts for hydrogen evolution and CO2 reduction
No full textIn chapter 1, after an introduction to Schiff bases, a literature survey of synthetic routes to the key diformyl-carbazole head units, required for all ligands made in this thesis, is discussed. An overview of the previously reported Schiff base ligands and complexes derived from 1,8-diformyl-3,6-ditertbutylcarbazole HUtBu follows. Then an overview of homogeneous molecular catalysts for hydrogen evolution (HER) and CO2 reduction (CO2RR) is provided. Finally, the systems targeted in this thesis are presented.
In chapter 2 HUtBu and a back-to-back ethylene-linked diformyl-carbazole (HU2Et) are prepared via the established routes and are characterised. The low solubility of HU2Et would make further work with it challenging, but the focus in this thesis is entirely on systems derived from HUtBu.
In chapter 3 a new non-cyclic pentadentate N5-donor Schiff-base ligand, HL2Etpyr, prepared from HUtBu and two equivalents of 2-(2-pyridyl)ethylamine is presented. Four tetrafluoroborate monometallic complexes, [MIIL2Etpyr](BF4), where M = Co, Ni, Cu, and Zn, along with two non-tetrafluoroborate cobalt(II) complexes, [CoIIL2EtPyr]2[CoII(NCS)4], and [CoIIL2Etpyr]2[CoIICl4], were isolated as solvates and structurally characterised. The cations in all six complexes are isostructural and feature the metal(II) centre in a trigonal bipyramidal N5-donor environment. Only the Zn(II) complex is fluorescent. Cyclic voltammograms of all four of the tetrafluoroborate complexes in MeCN show one reversible redox process at positive potentials and the Co(II) complex displays a second reversible process. Finally, the [MIIL2Etpyr](BF4), where M = Co, Ni and Cu, complexes are tested as HER electrocatalysts. Controlled potential electrolysis carried out at -1.6 V in MeCN in the presence of 80 mM acetic acid shows electrocatalytic HER performance in the order Ni(II) > Cu(II) > Co(II), but disappointingly, the control Ni(II) tetrafluoroborate salt is more active than all three catalysts.
In chapter 4 the macrocyclic ligand, HLPr, prepared from HUtBu and one equivalent of bis(3-aminopropyl)amine, previously reported by Dr Malthus in his thesis, is remade due to our interest in the 3d tetrafluoroborate complexes. Reversible redox processes are seen for [MIILpr](BF4), where M = Cu, Ni and Zn. Furthermore, the M = Cu and Ni complexes are investigated as electrocatalysts for HER, and the results indicate that NiLPr performed better than CuLPr but the control, Ni(BF4)2, performed far better than both catalysts.
In chapter 5 the two new N5-donor macrocycles, HLEt-Pyr and HLPrPyr, were prepared from HLEt and HLPr by alkylation with one equivalent of 2-(bromomethyl)pyridine. Five monometallic [MLR](BF4) complexes, where M = Co, Ni, and Cu; (R = Et-Pyr or Pr-Pyr) and one dinuclear [Ni2IIL2(Et-Pyr)(CH3OH)](BF4)2 complexes are prepared and characterised. The CVs of the [NiIILEt-Pyr](BF4), [CuIILEt-Pyr](BF4), and [CuIILPr Pyr](BF4)2 complexes revealed reversible redox processes at positive and negative potentials. Selected complexes are tested as HER electrocatalysts.
In chapter 6 the open-armed complexes which are reported in chapter 3 are tested under photocatalytic conditions as catalysts for HER and CO2RR. The performance under HER conditions is not encouraging as the controls show the simple salts are more active than the complexes in generating hydrogen. The results indicate that CO2 is reduced to CO and CH4, although the CH4 obtained is sub-stoichiometric. HER, a competitor during CO2RR, overshadowed CO and CH4 formation. Possible formation of other products such as formate still needs to be checked by our collaborator in Montreal.
Finally, in chapter 7 a summary of the key findings is provided
Rethinking Hazard Management in New Zealand
Get PDFNew Zealand is characterised by a high susceptibility to natural hazards. The last decade has been characterised by the Christchurch 2010 earthquake and devastating aftershock sequence that included the infamous ‘2011 Christchurch earthquake’ alongside the 2016 Kaikōura earthquake have catalysed discussions around the state of the civil defence emergency management framework. In New Zealand, civil defence emergency management are managed under a framework that is fed by a range of different statutory legislation, including the Civil Defence Emergency Management Act (CDEMA) 2002 and the Resource Management Act (RMA) 1991. Combined with other non-statutory documents such as the newly developed National Disaster Resilience Strategy 2019 and international commitments to hazard and risk reduction such as the Sendai Framework for Disaster Risk Resilience 2015 – 2030. The combination of the legislative devices named above, and others have combined to create the hazard and emergency management framework based on the ‘4R’s’ within the CDEMA; reduction, readiness, response and recovery. Considering the fundamental role that land-use planning has for hazard management, this research has analysed the lessons learnt from the Christchurch and Kaikōura earthquakes and assess the effectiveness and appropriateness of the current hazard and emergency framework. The research found significant issues within this framework that extend from tension and disconnection in the guiding legislation (RMA and CDEMA) leading to non-optimal development and management decisions, deeply ingrained resourcing issues that affect both central and local government with debilitating effects for the development and implementation of best practice and national and regional differences that call for increased centralisation to ensure greater levels of consistency of planning processes and outcomes. In order to overcome these issues in the framework, this research has suggested a number of pathways to help achieve best practice in the future. Aligning with the eventual reforms or possible repeal of the RMA, there must be more connection and communication between the RMA and CDEMA, prior to and after disasters. The significant resourcing issues that characterise the sector must be addressed for practices such as targeted hazard and risk assessment and proactive planning to take place. This research has also suggested that more centralisation must be achieved in the form of an NPS for natural hazards, a national recovery office or both, however, it is imperative that this does not result in the loss of autonomy for local governments, especially in small regions
In vivo Characterisation of Plant Cysteine Dioxygenase
Get PDFPlants not only produce oxygen, but also require molecular oxygen to live. Mitochondrial respiration in plants heavily relies on oxygen, and when there is a lack, the plant undergoes an energy crisis and other stresses, and a balancing act begins to ensure survival. When the plant undergoes hypoxia, plant-specific group VII Ethylene Response Factors (ERF-VIIs) regulate hypoxic response to aid plant survival. These transcription factors promote expression of hypoxia-response genes. These transcription factors are regulated through the N-end pathway. This pathway determines the fate of these proteins based on the amino acid on the N-terminus.Under normoxia, a group of enzymes called Plant Cysteine Dioxygenases (PCO) catalyse the oxidation of the N-terminal cysteine found in the ERF-VII RAP2.12 from Cys to Cys-sulfinic acid with molecular oxygen as the co-substrate. This triggers a degradation pathway for RAP2.12. Arginyl transferase (ATE1) arginylates the acidic N-terminus, and is sent to the proteasome to get degraded. Under hypoxic conditions, there is no molecular oxygen available, and PCO is unable to catalyse the oxidation of RAP2.12.PCO1 was expressed in Escherichia coli cells and purified using StrepTrap™ HP. This purified protein was used to define which PCO1 cysteines are potentially in a disulfide bond. Disulfide bonds are important in redox signalling in the cytoplasm, and some cysteines were found to be “blocked” and could potentially be bound in disulfide bonds. It was concluded that the cysteines found in PCO are interacting with either another cysteine to form disulfides, and it was hypothesised that this was important for redox signalling. Purified PCO was tested using ferrozine and Ellman’s assays, and was unclear if it was kinetically active. PCO1 constructs were fused with green fluorescent protein (GFP) using Multi-site Gateway® Cloning to visualise which part of the protein localises PCO1 into the nucleus. GFP-PCO constructs were transiently expressed in tobacco leaves and were visualised under confocal microscopy. Full length PCO was found to localise exclusively in the nucleus, while the N-terminus (NTD) and C-terminus (CTD) localised in both the nucleus and the cytoplasm. The nuclear localisation signal (NLS) could not be defined exactly, and both the NTD and the CTD are needed to localise the protein in the nucleus. Using a similar approach, RAP2.12 constructs were fused with tandemTOMATO to develop a RAP-reporter system in tobacco leaves, visualised under confocal microscopy and quantified using fluorescence detection. PCOs play a significant role in plant hypoxia response, and characterising these enzymes could contribute to understanding how plants survive submergence tolerance. This is important for agriculture, especially with increasing flooding events, and combining this knowledge with biotechnology, submergent tolerant plants can be developed, to combat the consequences of climate change and population growth
Modelling the structures of metallic clusters
Get PDFMetallic nanoparticles (clusters) are collections of metallic atoms that are bound together to form structures between 1 - 500 nm in diameter. Metallic clusters have potential in many practical applications due to their size dependent chemical and physical properties. In order to fully utilise their potential, it is vital that the various structural features that clusters can exhibit are understood. This thesis explores the various ways that the structures of clusters are studied computationally. This includes:
- Elucidating the structures of Au and Pt clusters between 55 and 309 atoms in size and comparing these to experimental results,
- Creating an auxiliary method for improving the explorational ability of the potential energy surface by global optimisation algorithms towards the global minimum,
- Investigating the structures of Au55, Au85, and Au101 clusters over time using dynamical simulation methods, and
- Understanding the structures of Cu clusters between 55 and 147 atoms in size and their electrocatalytic properties towards the reduction of CO2 towards biofuels
Comparative anatomy and functional morphology of the forelimb in cetaceans from New Zealand
No full textThe transition of early semi-aquatic archaeocetes to fully aquatic modern whales and dolphins is documented by a wealth of fossil evidence, and one of the most profound and well-studied macroevolutionary events in mammalian history. However, despite a fairly comprehensive understanding of the cetacean evolutionary pathway, only very few modern accounts have amalgamated arrangements of the soft tissue in the shoulder and forelimb with evolutionary aspects.
In this study, a total of 62 specimens comprising 15 species have been investigated and morpho-functional characteristics of the shoulder and forelimb integrated into behavioural, physiological and phylogenetic aspects. A thorough literature research complemented these findings.
In Chapter 2 Caperea new osteological characteristics, and for the first time myological arrangements, are described for the pygmy right whale (Caperea marginata), the sole member of the family Neobalaenidae. Some unique traits, only seen in fossil Cetotheriidae, give credence to a possible close affiliation with this extinct family. Other morphological attributes of the forelimb are the unique ability amongst whales to change the contour of the flipper and possibly also correlated functional attributes.
Chapter 3 Berardius investigates for the first time soft tissue arrangements of the shoulder and forelimb in Arnoux’s beaked whale (Berardius arnuxii, Ziphiidae). Despite highly derived morphological, physiological and behavioural adaptations to extreme deep-diving, Arnoux’s beaked whales reveal astonishingly unspecialised flipper attributes.
Chapter 4 Functional anatomy presents detailed descriptions of soft tissue arrangements in two dolphins (Grampus griseus, Cephalorhynchus hectori) and one porpoise (Phocoena dioptrica). These and other findings of this study are then analysed and combined with previously published accounts of the cetacean and mammalian forelimb, inclusive fossil specimens. The resulting conclusions are then presented within a phylogenetic context detailing possible locomotory adaptations of early archaeocetes and arrangements of flipper morphology in fully aquatic later archaeocetes and modern cetaceans