An investigation of the role of TRIB2 in steady state and stressed haematopoiesis

TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1-3). Here, we studied murine haematopoiesis after Trib2 ablation under steady state and proliferative stress conditions, including genotoxic and oncogenic stress. At the steady state, we found that TRIB2 loss did not adversely affect peripheral blood cell counts and populations. No detectable significant differences were found in the populations of haematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity due to the increased proliferation of Trib2-/- developing thymocytes which give rise to increased number of mature thymic subsets. During stressed haematopoiesis, Trib2-/- developing thymocytes demonstrate hypersensitivity to 5-fluorouracil-induced cell death. Nevertheless, Trib2-/- mice exhibit accelerated thymopoietic recovery post 5-fluorouracil treatment due to increased cell division kinetics of developing thymocytes. In an experimental murine T-cell acute lymphoblastic leukaemia (T-ALL) model, Trib2-/- mice had reduced latency in vivo which associated with aggressive T-ALL phenotypes and impaired activation of mitogen-activated protein kinase. Gene set enrichment analysis showed that TRIB2 expression is elevated in immature subtype of human T-ALL enriched with mitogen-activated protein kinase signalling. However, TRIB2 expression is suppressed in mature subtype of human T-ALL. Thus, TRIB2 emerges as a novel regulator of thymocyte cellular proliferation, important for the thymopoietic response to genotoxic and oncogenic stress, and possessing tumour suppressor function. In Drosophila, Tribbles promotes degradation of String which is an orthologue of mammalian CDC25 phosphatases in order to arrest cell cycle during embryonic development. Here, we showed that the role of Tribbles-induced degradation of String is evolutionarily conserved in TRIB2. We found that TRIB2 interacts with CDC25B/C but not CDC25A isoform. Overexpression of TRIB2 promotes polyubiquitination and degradation of CDC25C. Hence, future works are warranted to examine TRIB2-CDC25C interaction in the context of developing thymocytes and in T-cell acute lymphoblastic leukaemia, the malignant counterpart.

Life history of a native emydid turtle (Malaclemys terrapin centrata) on the remote oceanic islands of Bermuda

Diamondback terrapins (Malaclemys terrapin) are native to the remote oceanic islands of Bermuda and presently inhabit only four small brackish water ponds on a private golf course. The life history of this species is poorly understood on Bermuda and so the aim of this study was to fill these knowledge gaps, to compare the results with what is known from other areas in the North American range, and to inform the development of a local management plan. The results of a mark-recapture census revealed that ca. 100 individuals ≥81 mm straight carapace length live on Bermuda, of which nearly half (48.5%) were considered sexually mature. The population is dominated by females (sex ratio 2.9:1) and annual recruitment over the three year period was found to be extremely low (approximately two terrapins). Female diamondback terrapins in Bermuda nest almost exclusively within a limited number of sand bunkers on the golf course. Nesting commenced in late March or early April and ended in late August. Peak oviposition was observed in May and June. Clutch size averaged 5.1 eggs (range 0-10; SD 2.4) and the incubation period averaged 61.8 days (range 49-83; SD 10.5). Delayed emergence was documented, with 43.8% of the hatchlings remaining in their natal nests over the winter months. The mean annual hatching success rate was determined to be 19% (range 17.6-21; SD 1.9). Radio-telemetry was used to investigate the movements and survivorship of postemergent hatchling diamondback terrapins. The results indicated that mangrove swamps and grass-dominated marshes adjacent to the ponds are important developmental habitats for hatchlings. Yellow-crowned night herons (Nyctanassa violacea) were found to be significant predators of small terrapins during spring emergence. Small aquatic gastropods comprised 66.7% of the faecal samples analysed from the Bermudian population. Scavenged fish and vertebrate animal remains, terrestrial arthropods, polychaete worms and bivalves were consumed in lesser amounts. Sediment from the pond environment was found in 74% of the faecal samples analysed and is believed to have been incidentally ingested while foraging for the small benthic gastropods. Eco-toxicological analyses of the pond sediment, prey and terrapin eggs showed that the Bermudian diamondback terrapins live and feed in wetland habitats characterised by chronic, multifactorial contamination; principally total petroleum hydrocarbons, polycyclic aromatic hydrocarbons and a variety of heavy metals. This study found that some of those contaminants are accumulating in the gastropod prey as well as being transferred to terrapin eggs. This may be reducing the incidence of successful embryonic development for this species in Bermuda and may likely contribute to the observed low hatching rates. These collective findings indicate that the Bermudian population is very vulnerable to local extirpation.

The role of mitogen activated protein kinase phosphatase-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons: relevance to Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of midbrain dopaminergic neurons from the substantia nigra pars compacta(SNpc), which results in motor, cognitive and psychiatric symptoms. Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Inflammation may also be associated with the neuropathology of PD due to evidence of increased levels of proinflammatory cytokines such as interleukin-1β (IL-1β) within the SNpc. Because of the specific loss of dopaminergic neurons in a discreet region of the brain, PD is considered a suitable candidate for cell replacement therapy but challenges remain to optimise dopaminergic cell survival and morphological development. The present thesis examined the role of MKP-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons. We show that MKP-1 is expressed in dopaminergic neurons cultured from embryonic day (E) 14 rat ventral mesencephalon (VM). Inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6- hydroxydopamine (6-OHDA) is mediated by p38, and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. Dopaminergic neurons transfected to overexpress MKP-1 displayed a more complex morphology and contributed to neuroprotection against the effects of 6-OHDA. Therefore, MKP-1 expression can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Neural precursor cells (NPCs) have emerged as promising alternative candidates to fetal VM for cell replacement strategies in PD. Here we show that phosphorylated (and thus activated) p38 and MKP-1 are expressed at basal levels in untreated E14 rat VM NPCs (nestin, DCX, GFAP and DAT-positive cells) following proliferation as well as in their differentiated progeny (DCX, DAT, GFAP and βIII-tubulin) in vitro. Challenge with 6-OHDA or IL-1β changed the expression of endogenous phospho-p38 and MKP-1 in these cells in a time-dependent manner, and so the dynamic balance in expression may mediate the detrimental effects of neurotoxicity and inflammation in proliferating and differentiating NPCs. We demonstrate that there was an up-regulation in MKP-1 mRNA expression in adult rat midbrain tissue 4 days post lesion in two rat models of PD; the 6-OHDA medial forebrain bundle (MFB) model and the four-site 6-OHDA striatal lesion model. This was concomitant with a decrease in tyrosine hydroxylase (TH) mRNA expression at 4 and 10 days post-lesion in the MFB model and 10 and 28 days post-lesion in the striatal lesion model. There was no change in mRNA expression of the pro-apoptotic gene, bax and the anti-apoptotic gene, bcl-2 in the midbrain and striatum. These data suggest that the early and transient upregulation of MKP-1 mRNA in the midbrain at 4 days post-6-OHDA administration may be indicative of an attempt by dopaminergic neurons in the midbrain to protect against the neurotoxic effects of 6-OHDA at later time points. Collectively, these findings show that MKP-1 is expressed by developing and adult dopaminergic neurons in the midbrain, and can promote their morphological development. MKP-1 also exerts neuroprotective effects against dopaminergic neurotoxins in vitro, and its expression in dopaminergic neurons can be modulated by inflammatory and neurotoxic insults both in vitro and in vivo. Thus, these data contribute to the information needed to develop therapeutic strategies for protecting midbrain dopaminergic neurons in the context of PD.

Team-teaching for inclusive learning: Purposes, practices and perceptions of a team-teaching initiative in Irish post-primary schools

Background. Schools unequivocally privilege solo-teaching. This research seeks to enhance our understanding of team-teaching by examining how two teachers, working in the same classroom at the same time, might or might not contribute to the promotion of inclusive learning. There are well-established policy statements that encourage change and moves towards the use of team-teaching to promote greater inclusion of students with special educational needs in mainstream schools and mainstream classrooms. What is not so well established is the practice of team-teaching in post-primary settings, with little research conducted to date on how it can be initiated and sustained, and a dearth of knowledge on how it impacts upon the students and teachers involved. Research questions and aims. In light of the paucity and inconclusive nature of the research on team-teaching to date (Hattie, 2009), the orientating question in this study asks ‘To what extent, can the introduction of a formal team-teaching initiative enhance the quality of inclusive student learning and teachers’ learning at post-primary level?’ The framing of this question emerges from ongoing political, legal and educational efforts to promote inclusive education. The study has three main aims. The first aim of this study is to gather and represent the voices and experiences of those most closely involved in the introduction of team-teaching; students, teachers, principals and administrators. The second aim is to generate a theory-informed understanding of such collaborative practices and how they may best be implemented in the future. The third aim is to advance our understandings regarding the day-to-day, and moment-to-moment interactions, between teachers and students which enable or inhibit inclusive learning. Sample. In total, 20 team-teaching dyads were formed across seven project schools. The study participants were from two of the seven project schools, Ash and Oak. It involved eight teachers and 53 students, whose age ranged from 12-16 years old, with 4 teachers forming two dyads per school. In Oak there was a class of first years (n=11) with one dyad and a class of transition year students (n=24) with the other dyad. In Ash one class group (n=18) had two dyads. The subjects in which the dyads engaged were English and Mathematics. Method. This research adopted an interpretive paradigm. The duration of the fieldwork was from April 2007 to June 2008. Research methodologies included semi-structured interviews (n=44), classroom observation (n=20), attendance at monthly teacher meetings (n=6), questionnaires and other data gathering practices which included school documentation, assessment findings and joint examination of student work samples (n=4). Results. Team-teaching involves changing normative practices, and involves placing both demands and opportunities before those who occupy classrooms (teachers and students) and before those who determine who should occupy these classrooms (principals and district administrators). This research shows how team-teaching has the potential to promote inclusive learning, and when implemented appropriately, can impact positively upon the learning experiences of both teachers and students. The results are outlined in two chapters. In chapter four, Social Capital Theory is used in framing the data, the change process of bonding, bridging and linking, and in capturing what the collaborative action of team-teaching means, asks and offers teachers; within classes, between classes, between schools and within the wider educational community. In chapter five, Positioning Theory deductively assists in revealing the moment-to-moment, dynamic and inclusive learning opportunities, that are made available to students through team-teaching. In this chapter a number of vignettes are chosen to illustrate such learning opportunities. These two theories help to reveal the counter-narrative that team-teaching offers, regarding how both teachers and students teach and learn. This counter-narrative can extend beyond the field of special education and include alternatives to the manner in which professional development is understood, implemented, and sustained in schools and classrooms. Team-teaching repositions teachers and students to engage with one another in an atmosphere that capitalises upon and builds relational trust and shared cognition. However, as this research study has found, it is wise that the purposes, processes and perceptions of team-teaching are clear to all so that team-teaching can be undertaken by those who are increasingly consciously competent and not merely accidentally adequate. Conclusions. The findings are discussed in the context of the promotion of effective inclusive practices in mainstream settings. I believe that such promotion requires more nuanced understandings of what is being asked of, and offered to, teachers and students. Team-teaching has, and I argue will increasingly have, its place in the repertoire of responses that support effective inclusive learning. To capture and extend such practice requires theoretical frameworks that facilitate iterative journeys between research, policy and practice. Research to date on team-teaching has been too focused on outcomes over short timeframes and not focused enough on the process that is team-teaching. As a consequence team-teaching has been under-used, under-valued, under-theorised and generally not very well understood. Moving from classroom to staff room and district board room, theoretical frameworks used in this research help to travel with, and understand, the initiation, engagement and early consequences of team-teaching within and across the educational landscape. Therefore, conclusions from this study have implications for the triad of research, practice and policy development where efforts to change normative practices can be matched by understandings associated with what it means to try something new/anew, and what it means to say it made a positive difference.

Expression and function of the neurotrophic factors GDF5 and GDNF in the nigrostriatal system during development and in rat models of Parkinson's disease

Growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD), and may represent promising new therapies for PD. The aim of the present study was to investigate the endogenous expression and function of GDF5 and GDNF in the nigrostriatal dopaminergic system during development and in rat models of PD. Examination of the temporal expression patterns of endogenous GDF5, GDNF, and their respective receptors, in the developing and adult nigrostriatal dopaminergic system suggest that these factors play important roles in promoting the survival and maturation of midbrain dopaminergic neurons during the period of postnatal programmed cell death. The relative levels of GDF5 and GDNF mRNAs in the midbrain and striatum, and their individual temporal expression patterns during development, suggest that their modes of actions are quite distinct in vivo. Furthermore, the sustained expression of GDF5, GDNF, and their receptors into adulthood suggest roles for these factors in the continued support and maintenance of mature nigrostriatal dopaminergic neurons. The present study found that endogenous GDF5, GDNF, and their receptors are differentially expressed in two 6-hydroxydopamine-induced lesion adult rat models of PD. In both terminal and axonal lesion models of PD, GDF5 mRNA levels in the striatum increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased at 10 and 28 days post-lesion. Thus, despite the fact that exogenous GDF5 and GDNF have similar effects on midbrain dopaminergic neurons in vitro and in vivo, their endogenous responses to a neurotoxic injury are quite distinct. These results highlight the importance of studying the temporal dynamic changes in neurotrophic factor expression during development and in animal models of PD.

Student voice in Irish post-primary schools: a drama of voices

This research is an exploration of the expression of student voice in Irish post-primary schools and how its affordance could impact on students’ and teachers’ experiences in the classroom, and at whole-school level through a student council. Student voice refers to the inclusion of students in decisions that shape their experiences in classrooms and schools, and is fundamental to a rights-based perspective that facilitates students to have a voice and a say in their education. Student voice is essential to the development of democratic principles, active citizenship, and learning and pedagogy. This qualitative research, based in three post-primary case-study schools, concerns teachers in eighteen classrooms engaging in dialogic consultation with their students over one school year. Teachers considered the students’ commentary and then adjusted their practice. The operation of student councils was also examined through the voices of council members, liaison teachers and school principals. Theorised within socio-cultural (social constructivist), social constructionist and poststructural frames, the complexity of student voice emerges from its conceptualisation and enactment. Affording students a voice in their classroom presented positive findings in the context of relationships, pedagogical change and students’ engagement, participation and achievement. The power and authority of the teacher and discordant student voices, particularly relating to examinations, presented challenges affecting teachers’ practice and students’ expectations. The functional redundancy of the student council as a construct for student voice at whole-school level, and its partial redundancy as a construct to reflect prefigurative democracy and active citizenship also emerge from the research. Current policy initiatives in Irish education situate student voice in pedagogy and as dialogic consultation at classroom and whole-school level. This work endorses the necessity for and benefit of such a positioning with the author further arguing that it should not become the instrumental student voice of data source, accountability and performativity.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Studies on factors relating to the development of defects in commercial cheese

Defects in commercial cheese result in a downgrading of the final cheese and a consequential economic loss to the cheese producer. Developments of defects in cheese are often not fully understood and therefore not controllable by the producer. This research investigated the underlying factors in the development of split and secondary fermentation defect and of pinking defects in commercial Irish cheeses. Split defect in Swiss-type cheese is a common defect associated with eye formation and manifests as slits and cracks visible in the cut cheese loaf (Reinbold, 1972; Daly et al., 2010). No consensus exists as to the definitive causes of the defect and possible factors which may contribute to the defect were reviewed. Models were derived to describe the relationship between moisture, pH, and salt levels and the distance from sample location to the closest external block surface during cheese ripening. Significant gradients within the cheese blocks were observed for all measured parameters in cheeses at 7 day post/after manufacture. No significant pH gradient was found within the blocks on exit from hot-room ripening and at three months post exit from the hot-room. Moisture content reached equilibrium within the blocks between exit from hot-room and 3 months after exit from hot-room while salt and salt-to-moisture levels had not reached equilibrium within the cheese blocks even at three months after exit from hot-room ripening. A characterisation of Swiss-type cheeses produced from a seasonal milk supply was undertaken. Cheeses were sampled on two days per month of the production year, at three different times during the manufacturing day, at internal and external regions of the cheese block and at four ripening time points (7 days post manufacture, post hot-room, 14 days post hot-room and 3 months in a cold room after exit from hot-room). Compositional, biochemical and microbial indices were determined, and the results were analysed as a splitplot with a factorial arrangement of treatments (season, time of day, area) on the main plot and ripening time on the sub-plot. Season (and interactions) had a significant effect on pH and salt-in-moisture levels (SM), mean viable counts of L. helveticus, propionic acid and non-starter lactic acid bacteria, levels of primary and secondary proteolysis and cheese firmness. Levels of proteolysis increased significantly during hot-room ripening but also during cold room storage, signifying continued development of cheese ripening during cold storage (> 8°C). Rheological parameters (e.g. springiness and cohesiveness) were significantly affected by interactions between ripening and location within cheese blocks. Time of day of manufacture significantly affected mean cheese calcium levels at 7 days post manufacture and mean levels of arginine and mean viable counts of NSLAB. Cheeses produced during the middle of the production day had the best grading scores and were more consistent compared to cheeses produced early or late during day of manufacture. Cheeses with low levels of S/M and low values of resilience were associated with poor grades at 7 days post manufacture. Chesses which had high elastic index values and low values of springiness in the external areas after exit from hot-room ripening also obtained good commercial grades. Development of a pink colour defect is an intermittent defect in ripened cheese which may or may not contain an added colourant, e.g., annatto. Factors associated with the defect were reviewed. Attempts at extraction and identification of the pink discolouration were unsuccessful. The pink colour partitioned with the water insoluble protein fraction. No significant difference was observed between ripened control and defect cheese for oxygen levels and redox potential or for the results of elemental analysis. A possible relationship between starter activity and defect development was established in cheeses with added coulourant, as lower levels of residual galactose and lactose were observed in defective cheese compared to control cheese free of the defect. Swiss-type cheese without added colourant had significantly higher levels of arginine and significantly lower lactate levels. Flow cell cytometry indicated that levels of bacterial cell viability and metabolic state differed between control and defect cheeses (without added colourant). Pyrosequencing analysis of cheese samples with and without the defect detected the previously unreported bacteria in cheese, Deinococcus thermus (a potential carotenoid producer). Defective Swiss-type cheeses had elevated levels of Deinococcus thermus compared to control cheeses, however the direct cause of pink was not linked to this bacterium alone. Overall, research was undertaken on underlying factors associated with the development of specific defects in commercial cheese, but also characterised the dynamic changes in key microbial and physicochemical parameters during cheese ripening and storage. This will enable the development of processing technologies to enable seasonal manipulation of manufacture protocols to minimise compositional and biochemical variability and to reduce and inhibit the occurrence of specific quality defects.