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Leptin and glucose homeostasis in the Zebrafish
Get PDFObesity and type 2 diabetes mellitus (T2DM) are among the most common and costly health-related issues worldwide, and both have been declared a global epidemic by the World Health Organization. A key characteristic of these conditions is disturbed leptin signalling. Leptin, named after the Greek word for ‘lean’ (λεπτός), is a hormone that plays a key role in energy homeostasis. Although known best for its adipostatic role in mammals, leptin also regulates glucose homeostasis independent of effects on adipostasis. To date, the mechanism through which leptin exerts its glucoregulatory actions remains largely unknown. This thesis investigates whether leptin regulates glucose homeostasis via the canonical WNT pathway in the zebrafish (Danio rerio). Non-mammalian leptin studies have been conducted for only a little bit over a decade, and still comprise but a tiny fraction (<2%) of the total research in this field. However, these studies are needed not only to answer comparative questions, but uncovering the evolutionary origins of leptin function will clarify our understanding of how this hormone functions in the human body.
Using the CRISPR/Cas9 mutagenesis system, stable mutant zebrafish lines were successfully generated (chapter 2). These fish had a homozygous knockout of either the leptin-a gene (lepa), the leptin-b gene (lepb) or the leptin receptor gene (lepr). Next, protocols were established to investigate glucose homeostasis in adult zebrafish (chapter 3). Existing procedures for intraperitoneal injection and blood sampling were adapted to create a protocol for intraperitoneal glucose tolerance tests in adult zebrafish. In addition to this acute hyperglycaemic challenge, a glucose-immersion protocol was established to allow for the investigation of artificially-induced persistent hyperglycaemia as well.
Combining the mutant fish generated in chapter 2 with the protocols established in chapter 3, the effects of leptin- and leptin receptor deficiency were characterized in adult zebrafish (chapter 4). It was confirmed that under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in the zebrafish. However, in times of nutrient excess leptin was found to regulate body weight and standard length, and glucose homeostasis was impaired.
Next, the canonical WNT pathway was investigated as a potential mediator of the glucoregulatory effects of leptin. Taking advantage of the optical transparency of zebrafish larvae, a transgenic zebrafish line expressing fluorescence upon WNT pathway activation was used to demonstrate that leptin directly activates the canonical WNT pathway in vivo, specifically in the hypothalamus (chapter 5). Finally, various pharmacological manipulations of the canonical WNT pathway were performed in leptin-mutant fish and wild type controls to demonstrate that leptin regulates glucose homeostasis via the canonical WNT pathway (chapter 6).
Together, these findings show a novel essential role of the canonical Wnt pathway in the neuroendocrine control of glucose homeostasis in zebrafish. Furthermore, these data highlight that leptin may primarily have evolved as a glucoregulatory hormone with its role of an adipostat acquired later in evolution. Finally, the glucoregulatory action of leptin is mediated via the Wnt pathway - an essential mechanism that appears preserved throughout the vertebrate phylum
Complication profiling and Evolution of pelvic exenteration surgery performed in high volume tertiary referral centres over thirty years
Get PDFObjective: To examine the changes in exenterative surgery over three decades analysing oncological outcomes and whether changes in surgical approach have led to improved patient outcomes
Background: Advances in surgical technology, perioperative care and pattern of disease recurrence have coincided with an evolutionary change in exenterative surgery.
Methods: A review of prospectively maintained databases of pelvic exenteration surgery from 1988 – 2018 at two high volume specialised institutions. The total cohort was divided into three major time points (1988- 2004, 2005-2010 and 2011 to 2018) to allow comparative analysis. Primary endpoints were overall survival in primary and recurrent disease at each time point. Secondary endpoints included anastomotic leak, blood transfusion, ileus, wound infection rates and evolution of case complexity. Data were analysed using R with a p<0.05 considered significant.
Results: Six hundred and seventy patients underwent exenterative surgery. In 2011–2018 there was an increase in resection of recurrent malignancy with a continuous increase in gastro-intestinal malignancies resected over each time period(p<0.001,<0.01) and a reduction in gynaecological malignancy(p<0.001). A significant increase in sacrectomy, pelvic sidewall resection and ileal conduit reconstruction was observed (p<0.01,<0.001). In 2005–2010 patients had increased rates of ileus and anastomotic leak(p<0.05). Patients undergoing resection for primary disease had improved overall survival at time points 1998-2004 and 2011–2018 compared to those with recurrent disease(p=0.007,<0.001). Overall survival was significantly improved in patients with primary versus recurrent disease(p=0.022).
Conclusion: There has been a significant improvement in survival in patients undergoing pelvic exenteration surgery from primary disease. Case complexity has increased without significant morbidity.
BACKGROUND: The oncological role of pelvic exenteration for locally advanced and recurrent pelvic malignancies arising from the anorectum, gynaecological or urological systems is now well established. Despite this, the surgical community has been slow to accept pelvic exenteration, undoubtedly owing to concerns about high morbidity and mortality rates based on historical data. Therefore, the aims of this study were to assess the general major complications and predictors of morbidity following exenterative surgery for locally advanced and recurrent pelvic malignancies.
METHODS: Data were collected from prospective databases at two high-volume institutions specialising in beyond TME surgery for locally advanced and recurrent pelvic malignancies between 1990 and 2015. The primary outcome measures were major complications (Clavien-Dindo 3 or above) and predictors for morbidity.
RESULTS: A total of 646 consecutive patients requiring exenterative surgery for local advanced pelvic malignancies were identified. The median age was 63 years (range 19-89 years), and the majority were female patients (371; 57.4%). Five hundred and forty patients did not suffer a major complication (83.6%) following pelvic exenterative surgery. One or more major complications were observed in the remaining 106 patients (16.4%). The most common major complications were intra-abdominal collection (43.7%; n=59/135) and wound infection (14.1%; n=19/135). The overall inpatient mortality rate was 0.46% (n=3/646). Independent predictors for major morbidity following exenterative surgery for locally advanced or recurrent pelvic malignancies were squamous cell carcinoma of anus, sacrectomy, past history of peripheral vascular disease and requirement for blood transfusion.
CONCLUSION: Our series adds to the increasing evidence that good outcomes can be achieved for pelvic exenterative surgery in locally advanced and recurrent pelvic malignancies. A coordinated approach in specialist centres for beyond TME surgery demonstrates this is a safe and feasible procedure, offering low major complication rates
The impact of thumb carpometacarpal osteoarthritis and the effectiveness of splinting
Get PDFBackground: Thumb carpometacarpal (CMC) osteoarthritis (OA) is a common, disabling condition, affecting substantial numbers of working and older-age people. It is the single commonest site affected by OA yet its unique characteristics, distinct from general hand OA, have received little individual attention – far less than hip and knee OA. In particular, little is known from patients’ perspectives about the impact of thumb CMC OA. Furthermore, international guidelines recommend splinting as a non-surgical, non- pharmacological treatment option for CMC OA; however, evidence supporting splinting in people with thumb CMC OA is sparse and inconsistent.
The aims of this research were to: 1) explore the impact of thumb CMC OA from the perspective of people living with the condition; 2) investigate the effectiveness of splinting interventions for thumb CMC OA.
Methods: Three main studies were conducted: 1) a pragmatic qualitative study exploring the impact of thumb CMC OA in 30 individual interviews of people with the condition; 2) a systematic review with meta-analyses of previous studies reporting on the effectiveness of splinting for thumb CMC OA; 3) a feasibility study for a future fully-powered randomised controlled trial (RCT) investigating the effectiveness of a soft splint intervention combined with standardised best practice usual care vs best practice usual care alone comparator intervention. Design of the feasibility study was based on the findings from the qualitative study and the systematic review.
Results: The qualitative study identified five main themes representing five inter-related levels of health impact: negative experience of symptoms, functional limitations, restricted social activities and roles, negative thoughts and feelings, and altered sense of self. Pain, including pain at night, was the major concern. CMC OA impact was influenced by: dominant hand involvement; cold climate; people’s financial, social, and societal support; and attitudes to the condition. Many areas of impact are unidentified and missing in currently recommended patient-reported outcomes. The study found a strong desire for access to high-quality information about self-management and effective non-surgical, non-pharmacological treatment options.
All evidence for splinting was of low quality. Splints cause a moderate-to-large reduction in pain (SMD -0.7 [95% CI -1.04, -0.35], < 0.0001) and small-to-moderate improvement in function (SMD -0.42 [-0.77, -0.08], p = 0.02) in the medium-term (3-12 months). No effect exists in the short-term. The review identified: variability in self-reported outcomes, case definitions, and rationale for splinting; low and variable splint dosage; lack of standardised usual care; unassessed QoL; and inappropriate study designs.
In the feasibility study, all primary outcomes surpassed the a priori thresholds for feasibility. Of thirty enrolled participants, 29 (97%) were retained at the 4-week and 6-month follow-ups. Interventions were acceptable and safe. Preliminary clinical findings suggested greater improvements in pain in the splint group vs comparator intervention in the short-term.
Conclusions: Thumb CMC OA has a profound impact on a person’s health and well-being. Splinting is an acceptable and promising intervention although good quality evidence to support its use is lacking. A full RCT of splinting in addition to standardised best practice usual care for thumb CMC OA pain is feasible but should be preceded by exploration of dose effect and optimisation of outcome measures
An Inter-Species Investigation into the Role of Cytochrome c in Apoptosis
No full textCytochrome c is a protein that has important roles in electron transport and cell death (apoptosis). In its role during the intrinsic apoptosis pathway, cytochrome c is released from the mitochondria for it to interact with the cytosolic protein Apaf-1 at its WD40 domain. The binding event between cytochrome c and Apaf-1 triggers the formation of the heptameric apoptosome which activates caspase enzymes for cell death. The first naturally occurring mutation in cytochrome c, characterised as a substitution of glycine to serine at position 41 (G41S), was identified in a New Zealand family with inherited thrombocytopenia. This mutation was found to cause an increase in the ability of human cytochrome c to activate caspases. The same G41S mutation when introduced into mouse cytochrome c exhibited the opposite effect with a decrease in caspase activation compared to mouse wildtype. The molecular mechanisms that underly these species-specific effects of the G41S mutation are unknown, though the dynamics of the 40-57 Ω-loop has been suggested to play a role. This thesis reports analysis of molecular dynamics simulations of mouse WT and G41S cytochrome c compared against human WT and G41S cytochrome c simulation data. Characterisation of the global stabilities of mouse and human cytochrome c variants and trialled expression of a Apaf-1 WD40 domain construct are also reported.
Expression trials of the Apaf-1 WD40 domain construct ∆N594 in insect cell lines showed high expression but little solubility upon purification. Future work involving the redesign of a WD40 domain construct will be required to achieve a useable construct for investigating the interaction of Apaf-1 with cytochrome c. Molecular dynamics simulations of mouse WT and G41S cytochrome c showed no clear difference in mobility to human WT cytochrome c with these three variants not exhibiting any large conformational shifts. Human G41S shows a large increase in mobility, especially in the 40-57 Ω-loop region, over that of human WT cytochrome c. This indicates species-specific effects of the G41S mutation on mobility. Analysis of the global stabilities of WT and G41S cytochrome c of each species showed there is no difference in Tm. Human WT, mouse WT and mouse G41S exhibited the same thermodynamic parameters. Human G41S however showed a decrease in enthalpy and increase in entropy, much lower than the other three variants, indicating hG41S has a more disordered native structure. This aligns with the observations from the molecular dynamics simulations. It is likely that the increased mobility of human G41S may explain the increase in caspase activation seen with this variant. However, the dynamics do not explain the decrease in caspase activation seen with mouse G41S. Species variation in the WD40 domain of Apaf-1 may play a role in determining this effect, where mouse and human Apaf-1 may allow different ranges of mobility for interacting residues on cytochrome c to achieve tight binding
Constitutive surveillance and social media
Get PDFStarting from the premise that surveillance is the ‘dominant organising practice’ of our time (Lyon et al 2012: 1), this thesis establishes a framework of ‘constitutive surveillance’ in relation to social media, taking Facebook as its key example. Constitutive surveillance is made up of four forms: economic, political, lateral, and oppositional surveillance. These four surveillance forms – and the actors who undertake them – intersect, compound, and confront one another in the co-production of social media spaces. The framework of constitutive surveillance is structured around a Foucauldian understanding of power, and the thesis shows how each surveillance form articulates strategies of power for organising, administering, and subjectifying populations. After outlining the four surveillance forms, each chapter unpacks the relationship of one form to social media, building throughout the thesis an extensive critical framework of constitutive surveillance
Part(s) (w)hole: Manifestations of shame in lesbian poetry from Ireland and New Zealand since 1982
Get PDFThis creative/critical PhD examines the work of lesbian poets from Ireland and Aotearoa/New Zealand in order to demonstrate the creative energy made possible by their shared experience of and response to shame.
The creative part of the PhD comprises a poetry collection, Small Town Quare, and a solo piece of poet’s theatre, Catlicks. In these creative works, I reflect on and respond to my own experiences of shame as an Irish-New Zealand lesbian poet.
The critical part of the thesis pinpoints how four other lesbian poets, two each from Ireland and Aotearoa/New Zealand, explore and respond to shame in their work. I argue against universalist readings of their poetry and, instead, show the importance of attending to their particular lesbian experiences of and responses to shame. While Mary Dorcey’s work has been read in universalizing ways, I show how she writes against normative notions of the lyric tradition and strives to find a place for her antinormative desires in poetry. Her poetry is therefore most fruitfully read for its engagement with the particularities of lesbian experience, including those of shame. Similarly, I read Heather McPherson’s poetry for the experience of shame that hides under the cover of her rage, which is politicised by her lesbian feminist reorientation and bolstered by a community of like-minded women. In reading Cherry Smyth’s poetry, I show how experiences of shame in her work extend beyond her sexuality and intertwine with questions of home and identity that emerge from her Northern Irish upbringing and diasporic life in England. Rhian Gallagher’s lesbian love poems cascade over the boundaries of private and public spheres to show how environment structures intimacy through the revealing and concealing poetics of shame. Finally, the exegesis explores how my own creative practice builds on and responds to the work of these four poets. In the exegesis, I break down the processes of my one-woman show Catlicks and full-length poetry collection Small Town Quare to show how shame intercepts and invigorates my creative process.
The creative/critical structure of this PhD is intended to interconnect the personal and creative elements of shame to show how it manifests in the lives and poetry of lesbian women
Sexual Dignity in Rape Law
Get PDFForthcoming, to be published in the Yale Journal of Law & Feminism.Dignity is a famously contested concept, suggesting its deployment as a legal principle should be closely scrutinized. This Article sets out a functional and contextual analysis of dignity as an organizing principle underpinning rape law, which I term “sexual dignity”. Based on sexual violence theory, I trace the “democratization” of sexual dignity over time, as dignity and attendant rights of autonomy and equality have gradually extended from man to the (qualified) woman to women as a group, and identify an emerging contemporary feminist consensus on the meaning of sexual dignity. This framework is then applied to a critical review of how judges across common law jurisdictions understand and use dignity in decisions on rape. The caselaw of sexual dignity illustrates that dignity is a usefully capacious concept for exploring and condemning the multiplicity of rape’s harms and wrongs. However, uncritical engagement with sexual dignity can be harmful, with implications both for rape law and for the regulation of sexual behaviour generally. As such, I argue that robust and reflective engagement with sexual dignity is both necessary and productive.Peer Reviewe
Copper & friends: Luminescence & triazole based switching
No full textThis thesis describes attempts towards producing a new motif for Cu(I/II) switching that incorporates CuAAC synthesised 1,2,3-triazoles. A variety of techniques is used to explore the properties of new and model ligands, their potential for Cu(I/II) switching and photoswitching. The thesis consists of five chapters, where the work presented in Chapter 2 and Chapters 3/4 was carried out concurrently.
Chapter 1 introduces switching by first discussing several important biological switches. Switching in terms of this thesis is defined and synthetic switches are broadly categorised by the method with which switching is achieved, i.e. chemical, electrochemical or photophysical/photochemical. The photophysics of transition metal complexes is briefly described before an overview of Cu(I/II) switching is provided. Emphasis is placed on the pioneering work of Sauvage and co-workers, and on the HETPHEN strategy reported by Schmittel and co-workers. The previous work from these groups provided the inspiration for the research described in chapters 2-4, where the goal was to move away from polypyridyl systems to the synthetically more facile “click” triazoles while also exploring the potential for a new Cu(I/II) photoswitch.
Chapter 2 outlines previous work by Crowley and co-workers on Cu(I/II) switchable 1,1'-disubstituted ferrocene complexes. CuAAC “click” chemistry is then described, its mechanism presented and its potential to be a promising alternative to the traditionally used polypyridyl systems in Cu(I/II) switching is discussed. A description of the switching systems targeted in this thesis is provided, highlighting the different components of the switch that should provide selectivity for Cu(I) or Cu(II) ions. Following this the synthesis and coordination properties of several model systems, that reflect the different components of the switches, are explored as a means of determining the efficacy of the desired switching systems. Upon confirming the model systems to behave in the expected manner, the synthesis of the switch ligands through “click” chemistry and Pd cross couplings is presented. The coordination properties of the switch ligands with Cu(I) and Cu(II) ions is explored. Followed by a series of competition experiments being performed due to the tridentate pockets within the switch ligands being hypodentate, resulting in the binding of Cu(I) ions at this site. Finally, an interesting result in which an oxo-bridged diCu(II) complex was obtained, reminiscent of hemocyanin, is discussed as well as its potential for future study.
Chapter 3 explores the electrochemical and photophysical properties of a small family of 2-pyridyl-1,2,3-triazole Cu(I) complexes. The use of 2-pyridyl-1,2,3-triazole (pytri) ligands for the development of photophysically active transition metal complexes is broadly examined, before emphasis is placed on previous studies of 2-pyridyl-1,2,3-triazole Cu(I) complexes. The overarching goal of the work presented in the chapter was to attempt to develop a photoswitchable Cu(I/II) system. To achieve this a study of the properties of Cu(I) pytri complexes was undertaken. Given the parent [Cu(pytri)(diMesbpy)](PF6) complex proved to be non-emissive the effects of adding a known chromophoric substituent (TPA) and a change of ancillary ligand were examined.
Chapter 4 moves away from 1,2,3-triazole ligands to look at a 6,6'-diTPA-2,2'-bipyridine (diTPAbpy) ligand as an alternative ancillary ligand in the HETPHEN strategy. Again, the overall intention was towards producing a photoswitchable Cu(I/II) system. Previous literature on analogous ligands are discussed, though surprisingly this 6,6' disubstituted ligand had not been reported despite studies on both its 4,4' and 5,5' analogues. As such the synthesis of diTPAbpy is described and its coordination chemistry with 4- and 6-coordinate transition metals explored. The compounds electronic properties are also examined.
Chapter 5 gives a summary of the work presented in the thesis, while also providing several avenues for further study. These include adjustments to the switch ligands to improve selectivity, alternative chromophoric substituents for photoswitching and the possible use of ligands within the thesis to produce supramolecular architectures
A strategic settler colonial research agenda: turning the microscope to move beyond indigenous resistance.
Get PDFThis research report is a tryptic of essays that: deconstruct how indigenous narratives are
understood and defined by the academy; analyses whether indigenous narratives have
moved toward self-determination or are stuck in modes of survival and resistance; and,
advocates and proposes a structure for the development of a strategic settler colonial
research agenda aimed at dismantling oppressive structures of settler colonialism. The
ultimate goal of the research is to support the movement of indigenous peoples out of
narratives defined by survival and resistance. Each essay can stand alone but create a fuller
picture when read together. The first two essays are foundational and serve as broad reviews
of the literature and the third synthesises and responds to the ideas that emerge in them. If
they were paintings hanging on a wall, the first two essays would sit either side, with the third
placed in the middle
Exploring Neuropathology in delta122p53 Mouse Models
No full textOverview: A role for p53 isoforms in largely promoting cancer has been established. One isoform, ∆133p53, promotes numerous aspects of cancer namely inflammation, proliferation and invasion. Δ133p53 functions are also involved in reducing cell death while promoting survival, leading to brain tumours and other diseases in the brain, such as neurodegeneration. A mouse model that mimics ∆133p53 function, ∆122p53, has aided the understanding of ∆133p53 function. This study explores a role for ∆133p53 in neuropathology which was investigated using two ∆122p53 models: the original ∆122p53 model where ∆122p53 was present in every cell type and a new model, MOGLI, where ∆122p53 expression was restricted to glial cells.
Hypothesis: If Δ133p53 is involved in neurodegeneration, histological assessment of the original Δ122p53 mice and MOGLI mice will show neuropathological evidence.
Methods: Mice cohorts were aged and euthanized on showing symptoms such as seizures or major loss in body weight, or after a decided time had passed. Brains were harvested and processed into formalin fixed paraffin embedded (FFPE) blocks. Brains of the original ∆122p53 mice, where ∆122p53 is expressed in every cell type, were investigated for neuropathology using routine histology staining (Haematoxylin and Eosin (H&E)), special staining (iron deposition using Perls Prussian blue), and immunohistochemistry (glial cell glial fibrillary acidic protein, GFAP), T lymphocyte (CD3) and M2 macrophage/activated microglia (CD163) markers. Mice heterozygote for full-length p53 and a p53 null allele (p53+/-) were used as controls. The survival of MOGLI model, where ∆122p53 was restricted to glial cells, was investigated. Brain tissue from the MOGLI mice (GCE ∆122p53+/+ and GCE ∆122/p53+) and its controls (GCE m∆pro+/+, GCE m∆pro+/-, m∆pro+/- and GCE wild type+/+ mice) were also investigated for neuropathology as the original model with additional special staining for myelin loss (Luxol fast blue staining), and in situ hybridisation analyses for proinflammatory cytokine expression (IL-6 and IFN) using RNAscope®. Lastly, this study also aimed at analysing protein cell surface expression data from Δ133p53 expressing monoclonal cell lines to check if any proteins implicated in neurodegenerative pathology are increased or decreased on the Δ133p53 cell surface compared to parent cell line (H1299: A human non-small cell lung carcinoma cell line), which acted as the control. Statistical analysis on all the relevant data obtained throughout the study was performed using Prism 9 software.
Results: The original ∆122p53 mouse model, which had not shown any physical neurological symptoms, had neuropathology on analysis of H&E staining with the presence of a greater number of microcysts compared to the control mice (p = 0.0014). Immunohistochemistry and iron staining did not reveal the underlying cause of the microcysts nor display any evidence for another type of neuropathology. The MOGLI test mice developed an array of neurological symptoms, suggesting glioblastoma development. Of the 43 MOGLI test mice, 42 mice displayed at least 1 neurological symptom or adverse effect that required euthanasia, while none of the 11 control mice displayed any signs of neurological distress. MOGLI mice had reduced survival compared to the control mice (p < 0.0001). A sex difference in overall survival was found for MOGLI test mice, in which male mice heterozygote and homozygote for
∆122p53 had a reduced median survival time compared to female homozygote (p = 0.0386) and heterozygote (p = 0.00178) MOGLI mice. Haematoxylin and Eosin staining showed that glioblastomas or other tumours were not present in the MOGLI mice. Instead MOGLI mice had other features of neuropathology i.e., foamy macrophage cells (8/43, 18%), mononuclear aggregates (5/43, 12%) and hypercellularity (13/43, 30%). These features were absent in control animals. An unknown brown pigment was also seen in some MOGLI test (19/43, 44%) as well as control mice (6/11, 55%). Immunohistochemistry, iron staining, myelin staining and RNAscope® were not able to clarify the underlying cause or characterise further the above neuropathologies.
Cell surface protein expression analysis of Δ133p53 expressing cell lines revealed 34 upregulated proteins and 32 downregulated proteins in Δ133p53 expressing cells compared to the control. Of the altered proteins, 10 upregulated and 9 downregulated proteins were myelin sheath proteins. Among the upregulated proteins, clathrin (CLTC) showed highest expression and among the downregulated proteins CCT2 had no expression at all.
Conclusion: This study identified neuropathologies in the original Δ122p53 and the MOGLI mouse model, but did not find the underlying cause of the pathologies. Nonetheless, it revealed some new avenues to explore. It showed the presence of microcysts in the ∆122p53 mouse model which have not been reported previously. Foamy macrophages, a characteristic of lipid storage diseases, were seen in some MOGLI mice. Inflammatory hypercellularity and mononuclear aggregates were seen in some MOGLI mice, but were never observed occurring together. An unidentified brown pigment, of unknown significance, was seen in some of the MOGLI test as well as control mice, with varying quantity distribution across the genotypes and sexes. Difference in survival times was also observed between the MOGLI test and control mice, with significant variations between sexes for the MOGLI test mice. Lastly, protein expression analysis of Δ133p53 expressing cell lines revealed upregulation of proteins involved in vesicle transport and molecular trafficking