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Towards predictable tuning of spin crossover
No full textSpin crossover (SCO) active metal complexes are highly versatile materials thanks to their sensitivity to tiny physical or chemical environmental changes. This property makes them very useful for a wide range of applications: employable for experimental studies as molecular switches or for theoretical studies investigating the M-L bonds. In both cases, these studies aim to develop strategies of predictably tuning them.
Chapter One. An introduction to the SCO phenomenon: from gradual to cooperative SCO; various methods of monitoring the SCO transition. A summary of some literature examples of SCO-active systems is given. An overview of the published achievements in predicting the SCO phenomenon, including an introduction to the computational models deployed across the years. The EDA-NOCV model, employed in this field for the first time in this PhD thesis, is introduced. Finally, the aims of this study are presented.
Chapter Two. The synthesis and characterisation of four new non-symmetrical ligands, 3-(2-(5-Z-pyridyl))-4-tolyl-5-phenyl-1,2,4-triazole (LpytZ, Z = CF3, Br, F, Me), and the corresponding [FeII(LpytZ)2(NCBH3)2] complexes are presented. All four of these new complexes are SCO-active in the solid state and in CDCl3 solution, but T1/2 tuning by the meta-Z substituents was very modest. Three literature families were also tested, successfully extending the generality of using the 15N NMR chemical shift δNA of the coordinated nitrogen atom of the free ligand as measure of the T1/2 in the resulting Fe(II) complex.
Chapter Three. Theoretical study of a family of five iron(II) SCO-active [Fe(Lazine)2(NCBH3)2] (Lazine = 3-(2-azinyl)-4-tolyl-5-phenyl-1,2,4-triazole) and of the related five LS [Fe(Lazine)3(BF4)2]. The EDA-NOCV model was applied to molecular fragments to provide quantitative assessment of the σ- and π-bonding. A new corrected [Mn+ + L6] fragmentation was implemented which promises to enable a general approach suitable for any ML6 system. Finally, the σ- and π-bonding character is strongly correlated with the experimental T1/2 of the SCO-active [Fe(Lazine)2(NCBH3)2] complexes.
Chapter Four. Theoretical study of the M-L bond in a family of sixteen SCO-active differently para-X substituted [Fe(bppX)2]2+ complexes (bppX is 2,6-di(pyrazol-1-yl)-4-X-pyridine). Results of the EDA-NOCV revealed the σ-strength of the bppX ligand is correlated with σp+(X), δNA(bppX), experimental T1/2([Fe(bppX)2]2+) and calculated AILFT ΔO([Fe(bppX)2]2+). Results are explained at the molecular level by investigating the orbital population of the valence orbitals of the coordinating nitrogen involved in the aromatic π-system (pπ) and in the Fe-N bond (sp2(Fe)). From the observed correlations, the unknown σp+ parameter for two substituents (X = SOMe, SO2Me) is predicted.
Chapter Five. First theoretical study on [CoII(dpzca)2] SCO in the solid state, aiming to establish a computational protocol able to predict experimental T1/2 in pressure-activated SCO. The first part of the study validated a DFT protocol at p = 1 bar. The protocol was then extended and trialled up to 4300 bar. Results revealed good reproduction of the experimental results up to 2100 bar; but beyond this pressure, the theoretical and experimental findings diverge. Theoretical data suggest a possible phase change for the crystalline structure of HS [CoII(dpzca)2] at higher pressures than 2100 bar; this would explain why the implemented computational protocol lost validity
The Self Divided: The Problems of contradictory claims to Indigenous peoples’ self-determination in Australia
No full textWhen the United Nations General Assembly endorsed the United Nations Declaration on the Rights of Indigenous Peoples (‘UNDRIP’) in 2007, many extolled it for recognising Indigenous peoples’ right of self-determination. Although there is some consensus that all peoples have self-determination, as a legal claim it has routinely inspired criticism that it will lead to contradictory claims, interpretations, and further political contestation. Ten years after UNDRIP's endorsement, some Indigenous claims of self-determination in Australia are contradictory. While contradictory claims may not be inherently problematic, in the context explored in this article, there are problematic effects. This article examines how contradictory self-determination claims arose in response to Australia's recent Native Title Amendment (Indigenous Land Use Agreements) Act 2017 (Cth) (‘2017 Amendments’), which amended and weakened the Native Title Act 1993 (Cth) (‘NTA’). It also evaluates the consequences of those contradictory claims and argues for renewed critical assessment of who legitimately determines the self who claims self-determination.Peer Reviewe
On the functions of palladium (II) cages
Full text linkThis thesis consists of four chapters.
Chapter 1 provides an overview of metallosupramolecular chemistry, detailing the three main approaches to self-assembled metallosupramolecular architectures: the ligand direct approach, the symmetry interaction approach, and the weak link approach. The various applications of these systems are discussed with regards to their molecular recognition properties. A series of [Pd2(L)4]4+ cages designed and produced by the Crowley group is introduced and their achievements and failings of binding the inorganic anticancer drug cisplatin are outlined. The necessity to address the failings of these systems is the foundation for one of the aims of this project. The other goals of this project are centred around structurally similar, but functionally different [Pd2(L)4]4+ cages. These are investigated as molecular reaction vessels and their potential to enhance the rate of [4+2] Diels Alder cycloaddition reactions are studied.
Chapter 2 describes the global struggle against cancer, and how inorganic drug molecules such as cisplatin are used to combat the fatal disease. Advances in the use of metallosupramolecular architectures as drug delivery vectors are discussed, in particular [Pd2(L)4]4+ cage systems. Work building on the cages first developed by the Crowley group is detailed. Terminal coordinating pyridine groups have been substituted for quinoline and isoquinoline units in the new cages. The quinoline system no longer has the ability to bind cisplatin due to a twisting caused by steric clashes of the quinoline units. As these units are situated over the external faces of the palladium(II) metal ions, they provide protection from biological nucleophiles and therefore increase the kinetic robustness of the cage. This is reflected in the increased, sub-micromolar anticancer activity, the highest activity of any [Pd2(L)4]4+ system to date. However, like cisplatin, the system displayed very little discrimination between cancerous cells and healthy tissue.
Chapter 3 introduces a new [Pd2(L)4]4+ cage system whereby the central pyridine ring is replaced with a rotationally flexible, redox active ferrocene moiety. X-ray crystallography confirmed the structure of both the BF4- salt and the PF6- salt. The coordination chemistry of an alteration of the ferrocene-based ligand, whereby a 3-pyridyl donor is replaced by a 4-pyridyl donor, is also studied. Again, a single architecture was generated (and confirmed through X-ray crystallography), in this case a [Pd3(L)6]6+ prism. The exploration of the host-guest properties of both systems revealed them to interact strongly with the toluenesulfonate anion. While the cages retained the redox properties of ferrocene, the ferrocene units in each ligand did not interact with each other, and the electrochemical signals were unperturbed upon the introduction of the guest.
Chapter 4 details the history and current state of artificial enzymes, with particular emphasis on metallosupramolecular structures and more specifically, how [Pd2(L)4]4+ cages accelerate the rate of bimolecular [4+2] Diels Alder cycloaddition reactions. The work presented in this chapter is a continuation of a project started by the Lusby group in which the original [Pd2(L)4]4+ cage developed by Crowley was shown to not only accelerate the cycloaddition, but also alter the chemo- and regio-selectivity of the products. Electronic alterations to the cage have been explored in this section with regard to the catalytic activity. The new cages maintained their hosting abilities however altering the electronics only seemed to diminish the catalytic behaviour of the system when compared to the parent cage
Genetic manipulation of Plasmodium cynomolgi as a model for investigating Plasmodium vivax drug resistance
No full textPlasmodium vivax is the most widely distributed cause of human malaria, and thought to be the most difficult to diagnose and treat. Until recently this species has largely been neglected compared to the most significant cause of fatal human malaria, Plasmodium falciparum. One of the reasons behind this comparative neglect is that, unlike P. falciparum, P. vivax is not amenable to continuous in vitro culture. The absence of a tractable continuous culture method has hampered mechanistic biological studies of P. vivax, and in particular has frustrated the development of methods for the genetic manipulation of this species.
As reverse genetics is the gold standard for the investigation of putative drug resistance markers, our current inability to easily manipulate the genome of P. vivax has impeded our understanding of drug resistance in vivax malaria. Efforts to verify putative markers of drug resistance in vivax malaria are of particular importance given the recent emergence and spread of multiple drug resistance throughout Southeast Asia. To date, the identification of molecular markers of P. vivax drug resistance has wholly relied on ex vivo and clinical drug susceptibility studies. While a range of molecular markers have been proposed, including mutations in mdr1, crt, and dhfr-ts, these phenotypic sensitivity studies are subject to a range of confounding factors (i.e., patient immunity, geographic gene fixation). Until we can verify these putative markers using reverse genetics, our understanding of vivax drug resistance and the mechanistic action of antimalarials against P. vivax will remain obscured.
We have been fortunate to be at the forefront of a revolution in the in vitro continuous culture of P. cynomolgi, a sister species to P. vivax which shares many of its distinctive features (including dormant hypnozoite stages), and a high degree of genetic similarity. Considering the lack of a method for the continuous culture of P. vivax, the use of the P. cynomolgi Berok model provides for an exciting opportunity investigate P. vivax drug resistance markers through reverse genetics. While P. cynomolgi has been transfected with episomal plasmids using ex vivo and in vivo methods, the future of such work, which relies heavily on the use of nonhuman primates, faces increasing ethical and practical obstacles. Although the tractable continuous culture method for P. cynomolgi has resulted in a range of important phenotypic studies, conditions for the genetic manipulation of this species have not been optimised. Indeed, no in vitro episomal or integrative genetic manipulation has been reported in this species.
As no framework existed for the integrative genetic manipulation of P. cynomolgi, we undertook training in a well-established method for Plasmodium genetic manipulation in the Fidock Lab (Columbia University, NY) using CRISPR/Cas9. We used this system to investigate the role of putative drug markers for artemisinin sensitivity, and trained in the construction, selection, and phenotypic assessment of recombinant P. falciparum parasites. This experience allowed us to design a CRISPR/Cas9 system for the genetic manipulation of P. cynomolgi.
Our experience working with the well-established P. falciparum system highlighted the requisite attributes for the successful genetic manipulation of Plasmodium species. These include culture scalability, long term, contamination free culture, and the ability to enrich mature asexual forms for transfection. Therefore, the next focus of this study was to optimise the P. cynomolgi Berok continuous culture for reverse genetics. As many of the conditions used for P. falciparum were not compatible with P. cynomolgi culture, we identified viable alternatives for our model. These included a cost-effective substitution for nonhuman primate serum in culture media (horse serum and Albumax), a combination of antibiotics amenable for contamination prevention in P. cynomolgi cultures (penicillin and cefquinome), and a practical method for schizont enrichment (Nycodenz gradient centrifugation).
These basic culture condition improvements facilitated the first in vitro selection of an episomal expression plasmid in P. cynomolgi. Although initial transfections of episomal plasmids were successfully undertaken using the Bio-Rad Gene Pulser, we found that larger plasmids were more efficiently delivered using the Amaxa Nucleofector 4D. The later system was used to deliver CRISPR Cas9 plasmids for the insertion of P. vivax putative resistance markers in mdr1, dhfr-ts, and k13. Of these, we were only able to successfully insert the mdr1 Y976F mutation, which has been associated with chloroquine resistance in ex vivo and epidemiological drug resistance studies of vivax malaria. This marks the first recorded integrative genetic manipulation of P. cynomolgi. Preliminary phenotypic assays indicate that the Y976F mutation does not alter the sensitivity of P. cynomolgi to chloroquine, mefloquine, or lumefantrine. A small decrease in amodiaquine sensitivity was observed, which requires further investigation.
While this study has demonstrated that P. cynomolgi is amenable to genetic manipulation in vitro, there are some important challenges and concerns relating to this model which still need to be addressed. Nonetheless, the undeniable similarity between the genome and phenotype of P. cynomolgi and P. vivax warrants further investment into the development of a more tractable system for the genetic manipulation of this species
A Probe for Conformational Change of Hsc70 in Mammalian Cells under Stress Conditions
Full text linkHsc70 is a constitutively expressed member of the heat shock protein family. It has an important role in proteostasis and can stabilise nonnative proteins before being further matured in refolding, disaggregation, and degradation processes. Hsc70 switches between ATP and ADP bound states, and the hydrolysis of ATP drives the reaction for folding of the unfolded peptide. Förster resonance energy transfer (FRET) was used to report this conformational shift.
Creation of the FRET probe used variant 3T of Hsc70, which has three cysteines. One is buried in the conformation, and two are surface exposed on different domains. Maleimide Alexa Fluor (AF) dyes were applied to make covalent additions to the reduced cysteines, and conformational change was tracked by FRET. A luciferase refolding assay showed that although these mutations do not interfere with conformational change, they hinder refolding capabilities with substrate peptides.
Double labelled Hsc70 3T was transduced into HEK293 cells with varying conditions. It was found that unfiltered samples, imaged with Opti-MEM in the wells, and at the suggested PULSin concentration by the manufacturer was enough to detect fluorescence of AF488 and AF594. Heat shock can be used to determine how these conformations differ under stress and has been observed in live cell microscopy. This makes FRET a suitable technique to study conformational change of Hsc70 3T under cellular stress. All steps to establish a method to study FRET in live cells were optimised and could be done if a confocal microscope were available
Review of Online Food Delivery Platforms and their Impacts on Sustainability
Full text linkDuring the global 2020 COVID-19 outbreak, the advantages of online food delivery (FD) were obvious, as it facilitated consumer access to prepared meals and enabled food providers to keep operating. However, online FD is not without its critics, with reports of consumer and restaurant boycotts. It is, therefore, time to take stock and consider the broader impacts of online FD, and what they mean for the stakeholders involved. Using the three pillars of sustainability as a lens through which to consider the impacts, this review presents the most up-to-date research in this field, revealing a raft of positive and negative impacts. From an economic standpoint, while online FD provides job and sale opportunities, it has been criticized for the high commission it charges restaurants and questionable working conditions for delivery people. From a social perspective, online FD affects the relationship between consumers and their food, as well as influencing public health outcomes and traffic systems. Environmental impacts include the significant generation of waste and its high carbon footprints. Moving forward, stakeholders must consider how best to mitigate the negative and promote the positive impacts of online FD to ensure that it is sustainable in every sense
Role of langerin+ cells in skin pathologies
No full textLangerhans cells (LCs) are epidermal immune cells that express C-type lectin receptor langerin along with a subset of dermal dendritic cells. Langerin+ cells play a pivotal role in skin immunity. Previous research has suggested that LC may be involved in skin repair, but little is known about the importance of LC/ langerin+ cells or their specific role in wound healing processes. In this study the impact of ablation of langerin+ cells on healing of a full-thickness excision wound were investigated using the langerin-DTR LC-depletable mouse. Strikingly, depletion of langerin+ cells resulted in more rapid reduction in wound area. Accelerated wound healing in the langerin+ cell depleted group was characterized by enhanced neo-epidermis and granulation tissue formation, and increased cellular proliferation and angiogenesis within these newly formed tissues. The accelerated healing in the absence of langerin+ cells were associated with increased levels of GM-CSF and F4/80+ cells within the granulation tissue.
RNA-seq analysis revealed that there was an increase in genes associated with muscle contraction and myofibril assembly pathways but a loss in genes associated with keratinization pathways was observed in the DT treated mice at day 0 post – wounding which could be contributing to the accelerated healing. Deconvolution of RNAseq data revealed that a potential increase in the percentage of eosinophils at D0 which was sustained throughout the healing process in the depleted group. An earlier infiltration of dendritic cells and M2 macrophages in the depleted group at earlier time points could be setting up the wound microenvironment more conducive for healing. Down regulation and delayed expression of genes in the complement cascade, neutrophil degranulation pathways support an inhibitory role for langerin+ cells during inflammatory phase of wound healing. Therapies that suppress langerin+ cells or their function may therefore have utility in progressing the healing of wounds in humans.
Based on the inhibitory role of langerin+ cells on proliferation and angiogenesis in the wound, we predicted that in a highly proliferated and vascular condition such as a tumour, langerin+ cell numbers would decrease. However, in human squamous cell carcinoma tissues, we did not observe significant correlation between proliferation and angiogenesis in the tumour with langerin+ cells, tumour size of depth, indicating that the SCC microenvironment is not similar to the wounds. In contrast to our hypothesis, the distribution of langerin+ cells increased as the tumour size increased, suggesting that increased langerin+ cells might aid in tumorigenesis or the tumour microenvironment could be inhibiting the migration of langerin+ cells. Similar to the wounding experiments, GM-CSF and IL-17 exhibited opposing levels of expression in the centre and periphery of the tumour. Unlike in the wound healing tissues, langerin+ cells did not significantly correlate with other factors such as proliferation, angiogenesis, GM-CSF or IL-17, suggesting that langerin+ cells could be playing different roles according to the microenvironment. Further single cell sequencing in both mouse and human wounds and tumours would be highly beneficial to reduce the granularity of their roles in the varying skin microenvironment. Use of mouse models that ablate only the LCs or the langerin+ DCs can help elucidate their specific roles in the skin
Functional Beverages in Selected Countries of Asia Pacific Region: A Review
Full text linkFunctional beverages have gained increasing market share over the last decade. As the Asia Pacific region is one of the largest and most important markets for functional foods, it is critical when developing and promoting new products that food manufacturers/marketers have a good understanding of the Asia Pacific market, including the legislative requirements and consumers’ perceptions of functional beverages. A literature review was undertaken to elucidate legislation criteria and consumers’ perceptions of functional beverages in Asia Pacific countries. Topics reviewed included the origin and definitions of functional foods and beverages; the legislative criteria for functional foods and beverages in four representative countries—Australia, New Zealand, China, and Japan; and consumers’ perceptions of functional beverages. There was no concrete definition of “functional food” or “functional beverage” region-wide and correspondingly, the legislative terms and regulatory frameworks for functional foods and beverages varied from country to country and showed divergence due to cultural differences. The systematic review of consumer perceptions of functional beverages showed that product acceptance and purchase intention for different functional beverages was heterogeneous among consumers in the Asian Pacific Region, with many factors playing a role including product attributes (e.g., functional attributes, sensory attributes, and product form) and consumer perceptions (e.g., health motivation, trust in food industry, and food neophobia). The findings from this review will help guide product development and inform marketing strategies for functional beverages targeting the Asia Pacific region by providing information on legislation and consumers’ perceptions
Te Tautarinui o Matariki: a whānau health compass
Full text linkIn 2019 a historic claim was lodged with the Waitangi Tribunal under the Health and Service Outcomes Kaupapa Enquiry. Two main findings emerged; firstly, Māori have the poorest outcomes of any ethnic group in New Zealand, and secondly, Māori have suffered persistent inequities within the New Zealand health system (Waitangi Tribunal 2019). This research project aimed to address these findings by developing an iwi (tribal) whānau (family) model of health and well-being.
The research question “can the underpinning tikanga and mātauranga of way-finding navigation transfer into whānau health context?” was informed by kaupapa Māori theory and methodology, research tools that assume tikanga Māori (customs, values), te reo Māori (language) and mātauranga Māori (knowledge, skills) are legitimate ways for Māori to conduct research with and for their own. Participants (individual and collectives) were deemed experts in their respective fields and included three master way-finding navigators, two waka and tikanga experts, one whānau collective who were experts in maramataka (Māori lunar calendar) and 12 whānau collectives.
The aka matua (vine upon which everything hangs) of the research was based on the five key components that align to connectedness: whakapapa (origins and layers), pūrākau (narratives and stories), way-finding (voyaging), the maramataka, and tikanga. These layers were each built into Te Tautarinui o Matariki modelled on the way-finding star compass that was developed alongside and used in whānau domains.
Te Tautarinui o Matariki reflects the origins of Māori health where subsequent layers of whakapapa – namely knowledge and meanings including the researcher’s own – were added. The model supports whānau whakamana (empowerment) and tino rangatiratanga (self-determination) enabling whānau to both individually and collectively self-identify and monitor their health needs. Moreover, the model provides a potential mechanism for whānau to foster their own solutions by providing culturally familial resources, knowledge and tools.
The main findings of this research highlighted that whānau consistently drew upon whakanoa (the state of transition) as a strategy for their health and well-being. Whakanoa (transition) examples ranged from large to small and included karakia (incantations), meals and coffee, water (ocean, river, pool or bath), exercise, waiata (music) and breathing. Strategies were used for different transitions, such as to start and finish their day, or to transition between work and home.
The maramataka also had a significant impact on whānau health and well-being, identifying patterns of highs and lows in the likes of mood, energy a food consumption. Whānau reported that observation of various cycles helped them to identify circumstances, behaviour and products that would either enhance or decrease their well-being.
Outcomes from the research confirmed that Māori continue to be disproportionally affected by the burden of disease as a result of systemic inequities. Access to tools that are whānau-inspired, culturally informed and easy obtainable can help greatly in examining and improving Māori whānau health outcomes.
Te Tautarinui o Matariki was mainly used in this piece of research to identify particular health and well-being patterns for whānau. Further research will help refine its use so whānau not only identify but also generate suitable and appropriate strategies that ultimately improve Māori whānau health and well-being outcomes.
Using the Systems-Practice Framework to Understand Food Allergen Management Practices at College Catering Operations: A Qualitative Study
Full text linkThe number of individuals with food allergies or intolerances attending catered university residential colleges is increasing, and safe dining options are required to minimize the risk of allergic reactions and food-induced death. This qualitative research study sought to advance professional knowledge of the factors affecting allergen management practices, particularly pertaining to college foodservices. Three catered residential colleges affiliated with a major university in New Zealand were selected as research sites. The study used an ethnographic approach and systems-practice theory as a framework for data collection and organizing results. Data collection techniques included document analyses (3 hours per site), observations (6 to 8 hours per site), focus groups with foodservice workers (30 to 45 minutes per site, n¼16), and interviews with foodservice managers (45 to 90 minutes per interview, n¼5). Notes and transcripts were coded through the process of thematic analysis using NVivo for Mac software, version 11.1.1, to identify factors affecting allergen management practices. The main factors affecting allergen management practices at college foodservices included information provided by residents about dietary requirements; communication between residents and foodservice staff; systems for allergen management; attitude of foodservice staff; and college size. Detailed dietary information, effective communication with residents, sufficient resources, clarification of responsibilities, and thorough systems are required for staff to perform safe allergen management practices. Ultimately, successful implementation was predominantly determined by staff attitude. Foodservice managers are advised to identify motivators and address barriers of staff attitudes toward allergen management practices to promote successful implementation