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This thesis is an account of the synthesis of novel iron(II) and ruthenium(II) complexes using 2,6-di(1H-pyrazol-3-yl)pyridine based ligands and their efficacy as spin crossover complexes and transfer hydrogenation catalysts respectively. Chapter 1 is an introduction to the spin crossover and transfer hydrogenation detailing significant literature examples, important influencing factors and applications. Chapter 2 details the synthetic pathways towards all novel ligands used in this work, including numerous attempted pathways and problems which were overcome. Chapter 3 describes the properties of a number of iron(II) complexes made using 2,6-di(alkylpyrazol-3-yl)pyridine ligands. It is shown here that subtle steric and electronic factors about the iron(II) coordination sphere hinder spin crossover. Chapter 4 is a comparison of spin crossover properties of iron(II) complexes containing 2,6-di(1H-pyrazol-3-yl)pyridine ligands bearing a number of different substituents on different positions. The spin crossover behaviour of these complexes is rationalised based on crystal structures, powder x-ray diffraction and magnetic susceptibility measurements. Chapter 5 is an explanation of the differing magnetic susceptibilities of a number of solid solutions of the complex [FeL2]X2 (L = 2,6-di(1H-5-methylpyrazol-3-yl)pyridine). Chapter 6 shows the effectiveness of a number of ruthenium(II) pincer type complexes as catalysts for the transfer hydrogenation of acetophenone derivatives. Chapter 7 is an account of all the synthetic procedures used in this work and includes the full analysis of all novel compounds synthesised.

In recent years, geography and the wider social sciences have been animated by a conceptually reinvigorated concern for materialist thought. Within cultural geography, this emerging movement - which is often referred to as a 'new' or 'vital' materialism - has sought to understand the materiality of human life in relation to the lively capacities of a more-than-human world. Contrary to entrenched humanist narratives, these new materialist trajectories refute the anthropocentric assumption that humans are metaphysically exceptional beings, defined by their capacity to transcend nonhuman nature. The following thesis contributes to these debates through an experimental engagement with the notion of a 'transcendental empiricism,' as defined in the philosophy of Gilles Deleuze. My contention is that, whilst current materialist trajectories accept the critique of anthropocentrism at a superficial level, they often do not go far enough in considering the implications of this critique for social science; the most radical of which is that we can no longer approach human life from the Kantian position of a transcendental subject. With Deleuze, I argue that a materialism worthy of the name must begin from the supposition of a transcendental field, that is, a plane of nature that admits of neither subject nor object. My concern, then, is to explore the implications of transcendental empiricism for contemporary materialist thought. Venturing beyond Deleuze, I find resonances of this strange empiricism in the conceptual landscapes of A.N. Whitehead, Gilbert Simondon and Felix Guattari. Taking each in turn, I show how these three renditions of nature's transcendental field generate new kinds of questions regarding the materiality of human life. I achieve this task through a range of empirical lenses, which include singular objects, technical ecologies and aesthetic encounters. I conclude the thesis by affirming the capacity for transcendental empiricism to radicalise materialist thought through its commitment to immanence.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.

With the dual challenge of peak oil and climate change, there is a crucial need to gain public support for different energy options. This thesis explores the role that critical narrative rationality can play in attending to this pressing public policy issue. It argues that ,human communication and rationality is inextricably bound up with narrative forms of discourse, and that the recognition of this narrative dimension of rationality can serve important critical functions. The thesis develops a theoretical approach to narrative that can contribute to the post-positivist analysis of policy processes. Through an engagement with Mary Douglas's cultural theory, this argument is developed further in relation to the divergent 'mythologies' associated with energy and climate issues. The indispensability of narrative reasoning in structuring future uncertainties and shaping policy scenarios is also revealed. Narrative is also addressed as an innovative public engagement methodology for publics to contribute to imagining futures, and a novel Deliberative Storytelling Workshop with an orientation toward lay forms of narrative discourse is described. During the workshop, professional storytelling, visualisation exercises and policy scenarios were all used to elicit and stimulate public imaginations and future stories regarding the human and cultural aspects of life in 2050. The thesis demonstrates how this process provided a rich set of public understandings of the future and concerns over energy and climate change, and helped to reveal participants' situated, relational and storied forms of knowledge. The thesis concludes by arguing that such an approach, with its emphasis on narrative rationality, could provide opportunities for a public sociology that gives a greater democratic voice to lay publics in upstream futures work, and offers potentially fruitful synergies that bridge the division between expert and lay knowledges and understandings.

This dissertation explores, through three case studies, the relationship between the lexical semantics of clausal embedding (CE) predicates, and the syntactic and pragmatic restrictions on their complements, such as the (in)ability of certain predicates to embed declarative clauses or nominal expressions. I propose that these restrictions arise from interactions between fine-grained aspects of predicate meaning and the linguistic environment in which those predicates occur, rather than being stipulated into predicates' lexical entries via restrictions on permissible semantic types or semantic categories of their arguments (e.g. Grimshaw 1979, Pesetsky 1982, 1991) or resulting from polysemy or ambiguity of CE predicates (e.g. George 2011). First, I examine the Estonian verb mõtlema, which has a believe-like interpretation with an embedded declarative, and a wonder-like interpretation with embedded declaratives. I show that this behavior can be derived straightforwardly only if declaratives and interrogatives are typewise identical, i.e. sets of sets of worlds (Hamblin 1973, Ciardelli et al. 2013), and \textit{mõtlema} denotes an ontologically primitive attitude of 'contemplation'. Second, I analyze the behavior of English believe, which cannot embed interrogatives except under a combination of modal and nonveridical operators, such as can't. I propose that the apparent 'lifting' of selectional restrictions of believe in some contexts, as well as other unexpected properties of the can't believe construction, can be understood compositionally: believe can in fact compose with interrogative clauses in principle, but doing so normally results in systematically trivial meanings (Theiler et al. 2019). By placing believe under the right combination of operators, this triviality can be obviated (see Mayr 2019). Finally, I develop a semantic account of verbs like believe and trust, which can embed variety of nominal expressions alongside clauses, posing a compositional puzzle (Djärv 2019). I propose that these predicates are a kind of weak response-stance predicates (Cattell 1978), in that they presuppose an evidential source of the relevant attitude, which can be spelled out as a direct object. I take this to suggest a tight link between argument structure of belief predicates and their external syntactic distribution.

Healthcare sector emits over 4% of global carbon emissions. In the UK NHS have recently committed to achieving net zero carbon emissions by 2040. Facilitating sustainable consumption of energy, waste and everyday consumables is an important part of reaching that goal. Using examples from the NHS, this practitioner doctorate project aims to discover the drivers, barriers, and levers for facilitating sustainable consumption practices among healthcare employees. The thesis adopts a practice theory perspective and a complementary framework to achieve three objectives: explore healthcare employees' current attitudes and consumption patterns; identify opportunities for improving sustainability of everyday consumption; propose recommendations on how to encourage it. Firstly, a novel methodology for applying practice theory to analyse sustainable workplace practices was proposed and implemented. It has demonstrated the benefits of conducting an in-depth analysis of context as part of designing a pro-environmental organisational change project. It was also useful for analysing routine and inconspicuous types of workplace consumption, such as energy, waste and everyday consumables. Secondly, analysis of NHS staff survey has revealed clusters of employees depending on their pro-environmental actions. Understanding the differences between those clusters can be useful to create tailored sustainability strategies. Finally, an ethnographic study conducted in the sponsoring acute hospital trust have confirmed that healthcare employees are concerned with climate change and have positive attitudes towards personal and organisational action. However, lack of allocated time, financial resources or space to implement pro-environmental initiatives remain barriers to change. More needs to be done to improve waste and energy infrastructure and reduce procurement of paper, plastic and single use items. Organisations must help employees develop a wider set of pro-environmental skills that are relevant to their role. And a systematic approach to developing, implementing and maintaining sustainable consumption practices must be adopted.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles released by cells that facilitate the intercellular transfer of proteins and nucleic acids. This intrinsic delivery property of EVs, together with their low immunogenicity and potential for repeat administration, makes them promising delivery vehicles for novel therapeutics. The loading of cargo molecules into EVs constitutes one of the main challenges limiting clinical translation of EV-based therapies. Post-translational modification (PTM) of proteins with ubiquitin-like protein 3 (UBL3) has been previously implicated in the sorting of specific proteins into EVs. Initially, we assessed the utility of UBL3 as an EV-scaffold to enhance EV-loading of Cas9/sgRNA complexes by fusing Cas9 to UBL3 (to mimic the UBL3 PTM). This approach was shown to be successful in terms of the loading of Cas9 and sgRNA into the EVs, although no functional activity of the complexes was observed in the recipient cells after EV transfer. PTMs such as myristylation, ubiquitination, and prenylation have also been shown to play a role in EV-protein sorting. We were motivated to identify additional PTMs (and other sequence features) associated with EV loading that may be exploited for therapeutic purposes. To this end, we developed a novel bioinformatics analysis: integrated proteomics and feature annotation analysis (IPFA) that enabled the identification of EV-enriched annotated PTMs, protein motifs, and domains by combining protein feature annotations from UniProt with experimentally-observed EV-enriched proteins observed in proteomics data. This analysis identified several promising features, of which N-glycosylation was selected as the most promising PTM associated with EV protein enrichment. Specifically, the majority of N-glycosylation-annotated transmembrane proteins were EV-enriched, and nearly half of all EV-enriched transmembrane proteins were annotated as being N-glycosylated. Candidate protein features that were identified with the IPFA analysis were then experimentally assessed for their capacity to mediate functional cargo protein delivery. PTTG1P (Pituitary tumor-transforming gene 1 protein-interacting protein), a single-pass transmembrane protein with two annotated N-glycosyation sites, was selected as a putative EV-sorting scaffold. PTTG1IP was found to be highly enriched in EVs in an N-glycosylation dependent manner. Highly efficient functional delivery of Cre recombinase to reporter cells and mouse xenograft tumors was accomplished using PTTG1IP as an EV scaffold. Indeed, cargo molecules fused to PTTG1IP scaffolds were successfully delivered to recipient cell cultures with improved efficiency relative to the commonly used CD63-based scaffold. The PTTG1IP platform was further utilized for successful Cas9 and Cas9/sgRNA complex delivery to reporter cells. Several PTTG1IP variants with improved properties for therapeutic delivery were also developed (i.e., reduced size, enhanced loading potential, and enhanced intrinsic cleavage for cargo release), demonstrating the potential for further engineering PTTG1IP beyond its native human amino acid sequence. To achieve functional delivery, PTTG1IP-cargo fusion constructs were designed to include self-cleaving sequences (i.e., an intein from Mycobacterium tuberculosis or an intrinsic cleavage sequence derived from PTTG1IP itself) to facilitate cargo release from the EV scaffold. Furthermore, co-expression of the fusogenic protein VSVG (vesicular stomatitis virus glycoprotein) was utilized to enhance cellular uptake and promote endosomal escape. In summary, PTTG1IP was identified as a suitable EV-loading scaffold using IPFA, and its ability to efficiently package and deliver protein and protein/RNA complexes was demonstrated in both cell cultures and in vivo. Notably, PTTG1IP EV-sorting was shown to be dependent on its N-glycosylation PTMs, providing a validation of the robustness of the IPFA approach. The PTTG1IP-based platform offers significant advantages over other EV-loading strategies, (such as favorable membrane topology, the potential for further engineering, and functional delivery capability), which will enable the development of improved EV-based therapeutics.

Responsive Predicates (ResPs)–those clausal-embedding predicates which permit both declarative or interrogative complements, like know and say–pose a puzzle for compositional semantics. If declarative clauses denote propositions and interrogative clauses denote questions, it is difficult to explain how ResPs may select both types of clauses. Prior accounts diverge on how to best rectify this inconsistency, from reducing interrogatives to propositions (Karttunen 1977, Ginsburg 1995, Spector & Egré 2015, a.o.), to reducing declaratives to questions (Uegaki 2016) or dispensing altogether with the assumption that the denotations of declaratives and interrogatives are of different types, as in Inquisitive Semantics (e.g. Theiler et al 2016).In this thesis I bring novel data from Estonian to bear on this theoretical debate. In Estonian, a class of ResPs whose denotations concern contemplation, such as mõtlema ’think, consider’, convey what radically different meanings depending on the type ofcomplement it takes: When paired with a declarative complement, mõtlema canonically indicates representational belief like English think, but with an interrogative complement, it indicates ignorance toward the true answer to the embedded question, like English wonder.I demonstrate that a proposition-embedding denotation for mõtlema is not compatible with its full range of meanings. Instead, I propose a question-embedding semantics for motlema, which relates an individual to the set of issues they are contemplating. I show how this denotation, combined with standard Gricean reasoning, can naturally derive the superficially distinct interpretations of mõtlema across contexts. Building upon the analysis by Uegaki (2016), I argue that a proposition-embedding semantics for ResPs is not sufficient to capture the Estonian facts, whereas a question-embedding semantics is. Furthermore, I suggest that this approach is also compatible with the uniform treatment of declaratives and interrogatives of Inquisitive Semantics.

Neuromuscular diseases are a broad group of debilitating disorders that impair muscle functioning. Duchenne muscular dystrophy (DMD) is the most common hereditary form of neuromuscular disease, for which there is currently no cure. DMD is caused by null mutations in the dystrophin gene (DMD), which encodes an essential cytoskeletal protein required for myofibre integrity and function. The leading DMD therapeutic strategy is antisense oligonucleotide-mediated 'exon skipping', however, this requires lifelong repeat administration. Single intervention strategies for the permanent correction of disease-causing mutations in the genomic DNA (such as CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) are important avenues for DMD therapeutic development. An AAV9-delivered CRISPR/Cas9 system was employed to excise a disease-causing Dmd exon in the severely affected double knockout (dKO) mouse (lacking dystrophin and its developmental paralogue, utrophin). Dystrophin protein expression was partially restored in the muscle of dKO mice, although no extension in lifespan was observed. Within-fibre patchiness of dystrophin expression was observed in longitudinal skeletal muscle sections, an as of yet unreported consequence of CRISPR-mediated Dmd gene editing in myofibres. This provides an insight into the myonuclear domain structure of dystrophic fibres, and raises concerns about the possible functionality of gene edited myofibres that contain interspersed dystrophin-positive and -negative regions. The development of non-viral therapeutic strategies focused on improving muscle architecture are also of particular interest for neuromuscular diseases. Extracellular vesicles (EVs) are membrane-enclosed nanoparticles involved in natural cell-cell communication, believed to transfer pro-myogenic and anti-fibrotic factors in the muscle niche. Both myotube- and myoblast-derived EVs enhanced myogenic differentiation at lower, rather than higher, doses in recipient C2C12 muscle cell culture. Highly pro-myogenic effects observed in non-vesicular transfer experiments were attributed to the co-purification of insulin within the isolation medium, highlighting important methodological considerations for the EV field. Strategies that enable the specific activation of gene expression by translational modulation are currently lacking. Upstream open reading frames (uORFs) present within the 5ʹ untranslated regions of mRNA transcripts function in cis to repress translation of the downstream primary ORF. The in situ disruption of uORFs would be therapeutically beneficial for upregulating expression of a disease-relevant protein or protective factor. Validation of a panel of bioinformatically predicted uORFs revealed that not all are functional, including two uORFs with relevance to neuromuscular disease (i.e. SMN2 and UTRN for SMA and DMD respectively). A complex interplay of uORF features was found to influence uORF activity, where initiation of uORF translation was essential for functionality. The biophysical properties of poly-amino acid residues in synthetic uORF micropeptides were found to be major determinants of uORF activity. A synthetic poly-acid uORF sequence was highly repressive, and truncations of this sequence resulted in predictable levels of transgene attenuation. Such uORFs could be useful in biotechnology for the development of tunable, inducible, and tissue-specific transgene expression systems. Finally, CRISPR/Cas13b-mediated RNA editing was applied for the upregulation of a reporter transgene by uORF start codon disruption.

This thesis examines how the commedia dell'arte manifested in the English imagination in the period 1571-1611. This form of professional, semi-improvised performance practice emerged from Italy in the mid-sixteenth century to capture the hearts of audiences across Europe and introduce regional dramaturgies to the literary and theatrical innovations of the Cinquecento. There are frequent references to the masked stock characters and extemporising routines of the arte in extant English print, and scholars have often found parallels between the dramas and performance practices of English playwrights and Italian practitioners. However, these studies are complicated by the lack of direct exposure to the arte in performance. The Italians made only a handful of visits to England in the 1570s, after which they would not set foot on English soil again for over twenty years. Utilising methods from contemporary transcultural theory, this thesis approaches the commedia dell'arte as a culturally porous theatrical apparatus of constituent parts that became disentangled from the whole during their migratory journey. These constituent parts were transferred at different rates, at different times and through different means, and as such were adapted and repurposed to the demands of specific cultural and political moments along the way. In other words, the commedia dell'arte did not pass into the English imagination all at once, a direct exchange from one culture to another through sustained exposure to Italian performance, but rather percolated through a series of piecemeal translations and appropriations. As such, this thesis contends that the commedia dell'arte found in extant print and manuscript records in the period has little resemblance to or bearing on the activities of the Italian practitioners on the continent. Rather, it was highly situated and syncretic, constructed by writers and dramatists to interrogate and reflect anxieties over difference, belonging, and what it meant to be English.

The purpose of this study was to determine the perceived value of certification to research administration professionals and demographic characteristics. This study sought to determine whether those who have attained the Certificate in Research Administration (CRA) perceive a benefit to their careers and why most research administrators do not attempt certification. The primary research question studied is concerned with the relationship between perceived value of the CRA to research administration professionals and demographic characteristics. The survey instrument, Research Administrator Survey, was electronically distributed to 277 research administrators based in the Southeast region of the National Council of University Research Administrators (NCURA). A total of 230 surveys were completed and returned for a usable return rate of 83%. The research revealed that CRAs consistently perceived the value of certification to be greater than non-certified research administrators; however, the majority of both CRAs and non-certified research administrators perception was that becoming certified would, or did, enhance their knowledge as research administrators. Overall, the majority of non-certified research administrators reported that their reason for not attempting certification was that they did not believe there was any benefit to becoming certified, but when all the other reasons for not attempting certification are closely examined, the responses taken as a whole indicate that as many non-certified research administrators may perceive a benefit to becoming certified as those who do not perceive a benefit. In summary, the data indicated that non-certified research administrators do perceive some benefit to certification. It is recommended that the Research Administrators Certification Council (RACC) attempt to be more closely aligned with the with National Council of University Research Administrators (NCURA) and the Society of Research Administrators International (SRA) since research administrators report having extremely positive professional development experiences with these organizations. It is further recommended that research be conducted to determine if curriculum at the university level should be developed in research administration management. Finally, it is recommended that NCURA and SRA engage in research to determine how many people are involved in the profession of research administration to help make decisions in regard to continuing adult education.

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder characterised by severe muscle weakness. Progressive muscle loss ultimately culminates in fatality due to failure of the heart and diaphragm muscles, typically in the 2nd-4th decade of life. The genetic cause of DMD is disruption of the DMD gene encoding for the dystrophin protein. Dystrophin is important for cytoskeletal structure and normal muscle function, and it plays a vital role in maintaining membrane stability and signalling functions. However, the downstream molecular events that follow dystrophin loss are incompletely understood. Moreover, pharmacodynamic biomarkers that can inform on disease progression are severely lacking. This thesis aimed to identify the effects of the dystrophin protein absence on the cardiac transcriptome, skeletal muscle proteome and extracellular microRNA (miRNA) secretome with pathological progression. To improve the understanding of the dystrophic cardiomyopathic advancement in the various anatomical regions of the heart, transcriptomic arrays were performed on RNA taken from the distinct chambers to identify novel pathways involved in pathology. Analysis identified early molecular changes in the right atrium and ventricle, mostly related to cell migration and cardiac remodelling. Furthermore, Sfrp2 (secreted Frizzled-related protein 2), a major regulator of the Wnt signalling pathway, was found to be highly differentially expressed between control and dystrophic hearts. It was observed that expression of the non-canonical c-Jun N-terminal kinase (JNK)-arm of the Wnt cascade was reduced. Interestingly, cellular fractionation demonstrated decreased levels in the cytoplasmic/extracellular region and increased expression surrounding the nucleus, which suggests a potential mislocalisation of the sFRP2 protein in dystrophic hearts. Protein expression in skeletal muscle was characterised by high-resolution mass spectrometry-based proteomics in two dystrophic mouse models over time. Analysis of the two models at different time points allowed for protein identification irrespective of mutation or age. The results highlighted the involvement of many proteins in DMD, such as Rho GTPases, SLC25A24, RICTOR, CAV3 and MVP. Expression of major vault protein (MVP) increased during differentiation of myoblasts in vitro and interestingly, Mvp knockdown was observed to potentially influence Dmd expression levels, suggesting a novel role for MVP in DMD pathology. The miRNA secretome was examined to investigate the relationship between skeletal muscle dystrophin expression and extracellular miRNA abundance. Interestingly, while muscle-related miRNAs (myomiRs, indicative of muscle turnover) were not restored to wild-type levels in mdx-XistΔhs mice, which express low levels of dystrophin from birth (~8-44%), other miRNAs such as miR-193b-3p, miR-370-3p and miR-483-3p did exhibit a decrease in extracellular abundance. In contrast, exon skipping treatment achieving similar dystrophin re-expression levels was able to completely restore serum miRNA levels. The disparity between the mdx-XistΔhs and mdx mice treated with exon skipping was attributed to uneven dystrophin distribution at the sarcolemma in the former. The observed patchy, within-fibre mosaicism was unable to restore extracellular myomiR abundance, and by extension muscle turnover. These results are directly relevant to the development of therapeutic strategies for dystrophin re-expression. In conclusion, this work has identified novel molecular pathways involved in cardiac and skeletal muscle dystrophic pathology. Furthermore, the results have demonstrated the importance of uniform dystrophin distribution at the sarcolemma for pathological correction. Together, these findings provide new insights into the molecular pathology of DMD with implications for therapy.

Woodcock are widely distributed over Tennessee, but are most common during fall and spring in the Great Valley, Central Basin, and throughout western Tennessee. Harvest records indicate that peak fall migration occurs in mid-November. In 1977 and 1978, spring migration began during mid-February, and ended during mid-March. Peak migration occurred in late February or early March, but this chronology may not be representative of normal years. Testes lengths averaged 9.2 mm, indicating that male woodcock are in breeding condition by mid-February. Follicle development suggested that 44 percent of the females collected in February 1977 and 52 percent of the females collected in February 1978 were rapidly approaching nesting. The breeding season began during mid-February and lasted until late May. Peak nesting occurs from early to mid-March. Temperatures near 0°C curtailed breeding activity. Courtship activity began an average of 12.6 minutes after sunset; mean light intensity was 3.2 foot candles. Stands of second growth hardwoods found in poorly drained areas were favored diurnal coverts. Singing grounds were frequently early succession old fields, although pastures and other open areas were also used. Most nests were in young hardwood stands and were less than 50 m from a singing ground. Four of 5 broods were in honeysuckle thickets and were less than 50 m from streams.

Extracellular microRNAs (ex-miRNAs) and serum proteins are promising biomarkers for a variety of pathological conditions, including muscular dystrophies and, Duchenne Muscular Dystrophy (DMD) in particular. miR-1, miR-133 and miR-206 are muscle-specific miRNAs (myomiRs) that regulate myoblast proliferation and differentiation. These myomiRs are highly abundant in serum of DMD patients and dystrophic mdx mice and have therefore been investigated as biomarkers of dystrophic disease. The biological significance of miRNAs present in the extracellular space is currently not well understood. The work presented here demonstrates that ex-myomiRs are selectively released during periods of myogenic differentiation in cell culture and in vivo. Consequently, their release can be independent of dystrophic pathology, thus indicating that the presence of myomiRs in the serum may serve a physiological function in some contexts. In summary, serum myomiR abundance appears to be a function of the regenerative/degenerative status of the muscle, overall muscle mass, tissue expression levels and myomiR stability in serum. These findings have implications both for miRNA biology in normal muscle physiology as well as for the use of ex-myomiRs as biomarkers for DMD. Secondly, size-exclusion chromatography was identified as a useful methodology to fractionate biofluids and thereby separate extracellular vesicles (EV) from protein complexes and lipoproteins. Using this tool, it was demonstrated that ex-myomiR carriers in cell culture supernatant and murine serum are predominantly non-vesicular, and furthermore, that their release is independent of ceramide-mediated vesicle secretion. In addition, it was found that EVs isolated from dystrophic mice are smaller and more numerous compared to EVs derived from wild-type mice, thereby indicating a role for dystrophic pathology in the regulation of vesicle biogenesis and/or secretion. There is currently an urgent need for minimally invasive, therapy-monitoring DMD biomarkers for use in clinical trials. Serum protein biomarkers are desirable since they can typically be quantified by clinical biochemistry assays, which are technically facile and allow for rapid screening of large numbers of patients. Previously, identification of such biomarkers by mass spectrometry has been limited due to analytical challenges resulting from the massive complexity of the serum proteome. Here, an aptamer-based proteomics approach was utilised to profile 1,129 proteins in the serum of wild-type, mdx mice and, mdx mice treated with exon skipping therapy to restore dystrophin expression. A number of novel, therapy-responsive biomarkers were identified, and the leading candidate (ADAMTS5) was also found to be significantly elevated in DMD patient serum. In conclusion, this work demonstrates that myomiR release accompanies myogenic differentiation and shows that serum myomiR levels are influenced by a number of physiological and pathological factors. Furthermore, multiple novel, therapy-responsive protein biomarkers were identified in the serum of the mdx mouse with potential utility in clinical trials for DMD.

The phosphatases PTEN and INPP4B are frequently deregulated in human cancer and have been proposed to act as tumor suppressor genes by coordinately antagonizing PI3K/AKT signaling. While the function of PTEN has been extensively studied, little is known about the underlying molecular mechanisms by which INPP4B exerts its tumor suppressive function. Additionally, its role in tumorigenesis in vivo has not been studied. Here, we show that a partial or complete loss of the phosphatase function of Inpp4b morphs benign thyroid adenoma lesions observed in Pten heterozygous mice into lethal and metastatic follicular-like thyroid cancer (FTC) (Chapter 2). Importantly, analysis of human thyroid cancer cell lines and specimens reveals that INPP4B expression is downregulated in FTC. Mechanistically, we have found that INPP4B, but not PTEN, is enriched in the early endosomes of thyroid cancer cells, where it blocks PI3K-C2 mediated AKT2 activation and in turn tumor proliferation and anchorage-independent cell growth. Taken together, these data identify INPP4B as a novel tumor suppressor in FTC oncogenesis and metastasis through localized regulation of PI3K/AKT pathway at the endosomes. Further, we present evidence that INPP4B downregulation cooperates with PTEN loss in prostate cancer progression and metastasis (Chapter 3). In vivo, a partial loss of Inpp4b cooperates with Pten haploinsufficiency to promote prostate tumorigenesis. In vitro, we have found that knockdown of INPP4B in cell lines increased their migratory and invasive properties. Overall, our studies have greatly increased our understanding of the molecular mechanisms of the tumor suppressive functions of INPP4B and provided in vivo evidence for the cooperation of Inpp4b with Pten haploinsufficiency in both thyroid and prostate tumorigenesis, Medical Sciences

T-cell acute lymphoblastic leukemia (T-ALL) is a lethal decrease that is usually caused by hyper-activation of PI3K signaling, generally featured with loss of Pten. In this thesis, using genetically engineered murine model of T-ALL initiated by Pten-loss, we studied the roles of different PI3K catalytic subunit isoforms in T cell specific PTEN null induced T-ALL. We show that neither \(p110\alpha\) nor \(p110\beta\) isoform is critical for the maintenance of the T cell specific PTEN loss induced T-ALL, suggesting potential critical role of the PI3K \(p110\delta\) and \(p110\gamma\) in PTEN-null induced T-ALL. Highly elevated c-MYC oncoprotein abundance is found in most cases of T-cell acute lymphoblastic leukemia (T-ALL) and plays a key pathogenic role in this disease. Notably, the co-occurrence of c-MYC overexpression and PTEN-deficiency is common in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanistic link between the two events resulting in this correlation has remained unclear and unexploited therapeutically. Using the genetically engineered murine model of T-ALL initiated by Pten-loss and featuring spontaneous overexpression of c-MYC as well as a panel of PTEN-deficient c-MYC-high human T-ALL cell lines, we found that the abundance of c-MYC in these cells is critically dependent on hyper-activation of PI3K-mTOR-S6K1 signaling. While multiple mechanisms have been proposed to allow PI3K signaling to regulate c-MYC expression, we found that the details of c-MYC regulation in T-ALL are unique, not occurring via the regulation of c-MYC stability by phosphorylation or the common translational initiation machinery via eIF4E. We demonstrate that the PI3K signaling pathway robustly regulates translation of MYC via eIF4A, a eukaryotic translation initiation factor with intrinsic RNA helicase activity. Notably, eIF4A inhibition, either by RNA interference or via the pharmacological inhibitor hippuristanol, efficiently suppresses MYC translation and tumor cell proliferation via a mechanism specifically dependent on the 5'UTR of c-MYC. Our study establishes a novel eIF4A-dependent mechanism of MYC translation essential in T-ALL, and identifies eIF4A as a promising therapeutic target in c-MYC-dependent hematological malignancies.

In response to signals mediated by receptor tyrosine kinases (RTKs), G protein-coupled receptors or oncogenes, the two ubiquitously expressed isoforms of class IA phosphatidylinositol-3-kinases (PI3Ks), \(p110\alpha\) and \(p110\beta\), generate lipid second messengers at plasma membrane, which elicit multiple signal transduction cascades that regulate a broad range of cellular processes such as cell survival, proliferation, adhesion, motility, and transformation. Despite their similarity in sequence, expression pattern and regulatory subunits, growing evidence suggests that \(p110\alpha\) and \(p110\beta\) have distinct and redundant functions in normal physiological and disease conditions. For instance, activating mutations in \(p110\alpha\) have been frequently found in human tumors, while mutations in \(p110\beta\) have not been reported. \(p110\alpha\) is required for tumor formation induced by oncogenic RTKs, RAS, or polyoma middle T antigen (MT), whereas \(p110\beta\) seems to be essential for PTEN-deficient tumors. The objective of my dissertation has been to investigate the mechanisms underlying isoform selectivity and functional redundancy of \(p110\alpha\) and \(p110\beta\). First, we performed tandem affinity purification and mass spectrometry to look for \(p110\alpha\)- and \(p110\beta\)-interacting proteins. We found that CRKL preferentially binds to \(p110\beta\) at least under overexpression conditions. We further demonstrated that CRKL is an important regulator of PI3K activity in \(p110\beta\)-dependent PTEN-deficient tumor cells. Depletion of CRKL attenuates PI3K signaling and cell growth in PTEN-deficient prostate and breast cancer cell lines, but does not impair AKT activation in HER2 amplified and PIK3CA mutant cancer cells. Moreover, downregulation of CRKL does not inhibit AKT activation stimulated by insulin, suggesting that CRKL might regulate \(p110\beta\) activity in PTEN-null tumors independent of RTK stimulation. In the second part of this thesis, we used knockout fibroblasts and pharmacological inhibitors to dissect functional redundancy of \(p110\alpha\) and \(p110\beta\) in PDGF signaling. Our results suggest that both isoforms function downstream of PDGFR (abundant receptors): \(p110\alpha\) is the major isoform to mediate weak signals while both play a role when the signal is strong. When one isoform is ablated, the other isoform can play a compensatory role in the signal transduction. Such functional redundancy of class IA PI3K isoforms may have implications for the choice of the selectivity of PI3K inhibitors in cancer.