L’ingénierie protéique servant autrefois à comprendre les relations structures-fonctions des protéines connait un tournant majeur depuis plusieurs années. L’ingénierie protéique évolue pour créer des nouvelles fonctions protéiques : c’est la naissance de l’ingénierie protéique moderne. L’objectif de ma thèse a consisté à mettre en place et caractériser deux approches indépendantes d’ingénierie protéique dans le domaine du vaccin et du diagnostic. Le premier projet consistait à générer des ligands protéiques à partir d‘échafaudages moléculaires (des alternatifs aux anticorps) en couplant le ribosome display au NGS et en développant des outils d’analyses bio-informatiques. Des sélections contre des cibles protéiques d’origine bactérienne et virale ont conduit à l’identification de ligands Affibodies affins (µM au nM). Leur caractérisation a validé leur potentiel comme outil de recherche et de réactif diagnostique. Ces études ont permis de valider la plateforme de génération des ligands mise en place, en augmentant l’exploration de l’espace de diversité des interactions des ligands. Le second projet portait sur le développement d’une plateforme de présentation et de vectorisation à partir de particules d’encapsuline. Elles ont été génétiquement modifiées pour présenter de manière répétée à leur surface l’ectodomaine de la protéine de matrice M2 (M2e) du virus Influenza A H1N1 tout en encapsulant une protéine hétérologue : l’eGFP. Les nanoparticules modifiées sont correctement formées et encapsulent l’eGFP. Des souris immunisées par ces particules induisent une réponse anticorps spécifique contre l’épitope M2e et l’eGFP. L’utilisation de ces nanoparticules comme plateforme vaccinale de présentation et de vectorisation est prometteuse et ouvre la voie pour d’autres applications en biotechnologie / In the past, protein engineering used to understand function and structure relationship. But since few years, protein engineering was used to create new protein functions: modern protein engineering was born. The aim of my thesis was to set up and characterize two approaches of protein engineering in diagnostic and vaccine field. The first project was to generate artificial binder using protein scaffolds as an alternative to antibodies by coupling ribosome display (RD) to NGS and developing bio-informatics tools. Screening and selection against bacterial and viral targets have led to affibody binder’s identification with an affinity range from µM to nM. Their characterization has validated their potential as research tools and protein reagents for diagnostic assay. Coupling ribosome display to high throughput sequencing as means to directly identify selected binder coding sequences, enormously enhance binder discovery depth. The second project was to generate an innovative nanocarrier based on encapsulin nanoparticle, for customized peptide display and cargo protein vectorization. Encapsulin particles from T.maritima were genetically modified for simultaneous display of the matrix protein 2 ectodomain of the influenza H1N1 A virus and heterologous protein eGFP packaging. Genetically engineered encapsulin nanoparticles were well-formed and abled to efficiently load eGFP. Immunogenicity studies revealed antibody responses against both the surface epitope and the loaded cargo protein. Taken together, this display system is a versatile tool for rational vaccine design and paves the way for new applications in the research fields of vaccine, antimicrobial research and other biotechnological applications

Ett sedan länge betraktat problem inom politisk ekonomi är om, och i så fall i vilken utsträckning, politiska partier påverkar ekonomiska policyutfall. Syftet med studien är att undersöka om det rådande politiska majoritetsförhållandet i kommunfullmäktige har en effekt på kostnaderna för och kvaliteten inom en verksamhet som kommit att hamna allt högre på den politiska dagordningen under coronapandemin – äldreomsorgen. Studien tar avstamp i teoretiska utgångspunkter i form av medianväljarteoremet och citizen candidate-modellen. I syfte att estimera effekten av den politiska majoriteten på äldreomsorgen tillämpas en skarp regression discontinuity (RD) design för två kostnadsmått och två kvalitetsmått; antalet fallskador bland personer 80 år och äldre per 1 000 invånare samt brukarbedömning avseende äldreomsorg i särskilt boende. Resultatet visar att en vänsterblocksmajoritet är associerad med drygt 23 procent högre kostnader för äldreomsorg i kronor per invånare samt drygt sex procentenheters lägre nivå i fråga om brukarbedömning än jämfört med andra partikonstellationer. Resultaten är dock inte stabila över olika ekonometriska specifikationer och ytterligare studier skulle därmed behövas för att säkrare kunna belägga ett eventuellt samband.

Rett Syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, are the primary cause of the disorder. Despite the dominant neurological phenotypes that characterise RTT, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as growth retardation (reduced height and weight), skeletal deformities (scoliosis/kyphosis), reduced bone mass and low energy fractures are also common yet under-reported clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any such defects, I have conducted series of histological, imaging and biomechanical tests of bone using an accurate genetic (functional knockout) mouse model of RTT. Initial experiments using a GFP reporter mouse line demonstrated the presence of MeCP2 in bone cells and the effective silencing on the gene in functional knockout mice. Different aspects of the study were conducted in different types of bone tissues that were especially suited for individual assays. For instance, biomechanical three point bending tests were conducted in long bone (femur) whilst trabecular geometry measures were measured in spinal vertebrae. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation (XCI), revealed, lighter and smaller long bones and significant reductions in cortical bone mechanical properties (~ 39.5% reduction in stiffness, 31% reduction in ultimate load and 37% reduction in Young’s modulus respectively in Mecp2stop/y male mice; %) and material properties (microhardess reduced 12.3% in Mecp2stop/y male mice and 14% inMecp2stop/+ female mice) as compared to age wild type control mice. Micro structural analysis conducted using µCT also revealed a significant reduction in cortical (54% reduction in cortical thickness, 30% in bone volume, 20% in total area, and 38% in marrow area) and trabecular (~30% in trabecular thickness) bone parameters as compared to age matched wild-type controls MeCP2-deficent mice. Histological analysis using Sirius red staining as a marker of collagen revealed a ~25% reduction in collagen content in MeCP2 deficient mice as compared to age matched wild type controls. In experiments designed to establish the potential for reversal of MeCP2-related deficits, unsilencing of Mecp2 in adult mice by tamoxifen-induced and cre-mediated excision of a stop cassette located at the endogenous Mecp2 locus (male; Mecp2stop/y, CreER and female; Mecp2+/stop, CreER), resulted in a restoration of biomechanical properties towards the wild-type levels. Specifically, Male Mecp2stop/y, CreER mice displayed improvement in mechanical properties (stiffness 40%, ultimate load 10%, young’s modulus 61% and micro hardness 12%) and structural bone parameter (trabecular thickness 80%) as compared to Mecp2stop/y male mice. Female Mecp2+/stop, CreER, displayed a significant improvement (19%) in microhardess measures as compared to Mecp2 deficient mice. Overall, the results of my studies show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies for Rett Syndrome.

With the current crisis of organ shortage and an increasing number of dialysis patients,studies directed at ameliorating such a substantial organ discrepancy are of considerable importance to the transplant community. The use of extended criteria donation has helped to compensate the disparity of organs however, we are still a long way from achieving satisfactory targets. Still there are many organs from older donors that are discarded primarily on the basis of chronological age. It is here that biological age may display a crucial role in allowing the transplant team to characterize donor organs with greater accuracy. Indeed both biological and chronological age are very closely related and ECD criteria are based very much on the latter, albeit with other clinical variables. However the biomarker of ageing CDKN2A which is suitably represented by Baker and Sprott’s criteria, displays closer variabilities with post-operative transplant function, at least up to one year. Telomere length known as the “Gold standard” biomarker of ageing does not display as robust a role in predicting organ function as CDKN2A. The classification of organs represented in this text from category I-IV serves merely as a guide to future studies and is yet to be validated in larger clinical trials. It is however a simple and rapid assessment tool (Gingell-Littlejohn et al PLOS One 2013). Relatively advanced cellular senescence was displayed in the mutant AS/AGU rat kidney when compared to the parent AS strain. This was exploited in a unique animal model to study the effects of ischaemia reperfusion injury on the mutant kidney, hence mimicking to a certain degree the transplant related injuries in ECD kidneys. Although ischaemic times in the model were moderate in nature, there was nonetheless a difference in the tolerance to IR injury between parent and mutant strain as evidenced by increased p16 and p21 staining in AS/AGU rats. Such a model therefore is exclusive in that interventions to improve ECD renal function post-transplantation can accurately and conveniently be represented and studied at a pre-clinical level. Anti-ischaemic compounds have been the subject of much debate over the years, however a single “Holy Grail” compound able to completely abolish the injurious effects of IR injury has never been elicited. mTOR inhibitors however, display several cellular effects and act as potent immunosuppressants. They (AZ-6) have also been shown to partially mediate the detrimental effects of IR injury on the native kidneys of AS rats in a specifically designed animal model as shown. Further studies encompassing transplanted kidneys from mutant AS/AGU rats exposed to such a promising agent would be of undoubted importance to the clinical field of transplantation, potentially leading to immeasurable economic and patient benefits.

Feline Osteoarthritis (OA) is a pathological change of a diarthrodial articulation which primarily occurs in older cats. The aims of this study were:- 1) to define the radiographic features of OA in the cat for each individual appendicular joint; 2) to relate the radiographic features to the gross pathologic and histopathologic features; 3) to explore underlying causes of OA in cats, 4) to identify the presence of Protease Activated Receptor-2 (PAR-2) and matriptase in feline articular cartilage and synovial membrane and to determine their role in OA pathogenesis. The present study has defined five radiographic features of OA for each appendicular joint:- presence of osteophytes, enthesiophytes, areas of abnormal mineralisation,synovial effusion and joint remodelling. The study furthermore suggested that increases in radio-opacity beneath the semilunar notch, along the femoral trochlea, beneath the tibial plateau and on the femoral head/neck are also important radiographic features. The radiographic prevalence was highest in the elbow (23.9%, 93/389) and stifle (23.9%,93/389) joints, followed by the hip (21.1%, 82/389), tarsal (17.7%, 69/389), shoulder(6.7%, 27/389) and carpal (6.4%, 25/389) joints. The results from this study demonstrate that the presence of a radiographically apparent supinator sesamoid bone(SSB), meniscal mineralisation (MM) and two fabellae are related to cartilage pathology and can be indicators of OA. Prevalence rates for gross pathology changes were highest in the elbow (20.2%,102/506) joint, followed by the stifle (19.6%, 99/506), hip (18.4%, 93/506), shoulder (17.8%, 90/506), tarsal (15.0%, 76/506), and carpal (9.1%, 46/506) joints. Eight key gross pathologic features were identified- cartilage discolouration, cartilage fibrillation,cartilage ulceration, cartilage erosion, osteophytes, thickening of joint capsule, synovium discolouration and joint remodelling. The radiographic and gross pathologic total scores were positively correlated in each appendicular joint and the joint most likely to have cartilage damage without radiographic evidence of OA is the shoulder (71.1%, 64/90) followed by the elbow (39.1%, 9/23), hip (32.4%, 11/34), stifle (26.1%,6/23), carpal (23.1%, 21/91) and tarsal (14.9%, 7/47) joints. Four possible underlying conditions that lead to secondary OA were identified:- radioulnar incongruity, hip dysplasia (HD), cranial cruciate ligament (CCL) disease and primary meniscal mineralisation. The identification of PAR-2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding supporting the hypothesis that serine proteases are involved in the articular cartilage degradation seen in feline OA.

The selection for specific physical characteristics by dog breeders has resulted in the expression of undesirable phenotypes, either directly or indirectly related to the physical characteristic selected for. One conformation that was considered desirable is extreme brachycephalia, which is associated with secondary physical changes adversely affecting the airways, eyes and central nervous system. Using a large population of pet dogs having diagnostic magnetic resonance imaging (MRI) studies, I demonstrated that the most commonly used historical head phenotype indices (Stockard and Evans indices) can be determined on MR images. I furthermore conformed that olfactory bulb angulation can be used as an alternate for classification of dog into brachycephalic, mesaticephalic and dolichocephalic head shapes, with similar results to the historical indices. The advantages of olfactory bulb angulation are that it only requires a single midline MR image and inclusion of the entire nose is not required. Using the historical indices and olfactory bulb angulation I then examined the effect of increasing brachycephalia on the appearance and dimensions of the nasal and cranial cavity. I established that progressive ventral rotation of the olfactory bulb (increasing brachycephalia) resulted in an alteration in the shape and a reduction in cross-sectional area of the nasopharynx. Similarly, increasing brachycephalia resulted in a reduction in the dorsal area of the ethmoturbinates and a corresponding reduction in the midline area of the olfactory bulb, providing a potential explanation for reduced olfactory acuity in brachycephalic dogs. Finally, I examined the effect of head phenotype on the structures of the middle fossa, the 3rd ventricle, quadrigeminal cistern and interthalamic adhesion. Head phenotype had a lesser effect on these structures, while brain disease (in particular ventriculomegaly) has a substantial effect, the recognition of which I described. These results confirm the potential of olfactory bulb angulation and orientation for objectively determining head phenotype using in vivo MRI, in particular determining the degree of brachycephalia. The study also quantified the effect of brachycephalia on the nasal cavity and rostral and middle cranial fossae dimensions. The objective quantification of head phenotype provides a useful tool for selection of breeding animals to normalise extreme brachycephalia. This might reduce the incidence of the adverse effects associated with extreme brachycephalia.

With the advent of facial transplantation some academic authors have suggested that faces are significant for humans and that identities are located corporeally within faces and therefore transplantable. However, there has been little evidence to support these claims, particularly from a qualitative, theoretically informed social science background. Responding to this hiatus, in this thesis I set two interconnected research objectives: • to examine socio-cultural values associated with human faces in predominantly Western societies using secondary sources; • to explore the relationship between acquired facial ‘disfigurement’ and embodied identity shift using a narrative methodology. The first objective was addressed in full through an analytical review of largely Western secondary sources. It has become clear that faces, as part of bodies, are imbued with a variety of socio-cultural meanings on multiple levels. Individually people experience the world through their body and their face, making it a significant site for perception and sense making. On a societal level, faces and facial appearance have been associated with social reproduction (Giddens, 1991). For example, inaccurate and harmful historical associations between facial appearance and moral character still pervade British society. And, utilising the concept of faciality (Deleuze and Guattari, 1987, p.168) Twine (2002), Dudley (2002) and Benson (2008) have illustrated that the faces of people in sub-sections of society, generally those with very little power, can be conceptualised negatively and used to serve the interests of powerful elites. In terms of the second objective, most facial ‘disfigurement’ research has been completed using quantitative methods, resulting in partial knowledge and the disconnection of persons. Through the use of a phenomenological epistemology, embodiment position and a narrative methodology I have put the experiences of the 13 participants at the heart of the research. The analysis chapters focus on the participants’ embodied identities before, during and after an acquired facial ‘disfigurement’. In terms of conclusions, I have found that faces are important however, identities are not located within them but created and reshaped through embodied life experiences. I have also found that the relationship between embodied identity shift and acquired facial ‘disfigurement’ is one of contested negotiation between wider socio-cultural facial values, transitional/liminal identity states during and after the event(s) and the aim of previous identity restoration.

Regulatory T cells (Treg) are enriched in tumour tissue relative to other compartments. Anti-tumour immunity is promoted through their depletion. It is hypothesised that Treg are recruited to human colorectal cancer (CRC) via a specific combination of chemokine receptors and integrins, blockade of which reduces tumour Treg recruitment, ameliorating the anti-tumour immune response. A systematic examination was conducted of receptors expressed by CRC-isolated Treg and the cognate ligands expressed by CRC. The effects of receptor inhibition were tested in murine models of colorectal cancer. Human CRC-infiltrating Treg exhibit a specific chemokine receptor signature, expressing significantly higher levels of CCR5 than conventional T cells. CRC expresses the ligands for CCR5 at significantly higher levels than distal tissue. Isolated Treg migrated towards CCR5 ligands in vitro and suppressed allogeneic T cell proliferation. CCR5 inhibition in murine models of CRC led to delayed tumour growth but had no effect on tumour Treg infiltration compared with vehicle control. CCR5 inhibition is unlikely to provide any significant reduction in the infiltration of Treg into human CRC. Given the effects CCR5 inhibition had on tumour growth, CCR5 antagonists command further investigation into their potential role as novel therapeutic agents in the treatment armoury against human CRC.

Modern cobalt-chromium (CoCr) alloy compositions, for hip implants, were developed to resist the issues of wear and corrosion; however they still succumb to degradation. While the literature is vast, there is still a lack of understanding of the variability in implant-metal derivatives generated, and the effect such products can have on biological components other than just cells. In this thesis the effect of Co ions on type I collagen (main component of the extracellular matrix) was investigated. The conformation of the triple-helix was maintained, however the time taken for fibril formation to complete increased with Co concentration. In addition, with increasing Co, the collagen matrix became more heterogeneous and cellular attachment and proliferation was reduced. It is likely that Co ions are interacting with a C-O (hydroxyl) group. An overlooked population of degradation products was also investigated. They were found to be highly dependent upon the local environment. Media composition resulted in changes to the morphology, while pH directed the initiation of precipitation. A pH < 5 resulted in no observed pellet. In addition, the presence of Co ions in the media resulted in a change of Cr speciation. Finally, an approach is presented for sub-micron (600nm) x-ray absorption near edge spectroscopy (XANES) mapping of ex vivo tissue. Sub-micron XANES maps contained at least 4 spectra, determined through principal component analysis and clustering. A 5x5 pixel region was averaged for comparison to the 3μm beam approach. Both spectra contained similar features representative of chromium phosphate suggesting that XANES with a micron-sized beam (standard approach) cannot represent the full chemical variability present within the tissue.

Total disc arthroplasty (TDA) can be used to replace a degenerated intervertebral disc in the spine. There are different designs of TDAs, but one of the most common is a ball-and-socket combination. Contact between the bearing surfaces of such designs can result in high frictional torque, which can then result in wear and implant loosening. This study was designed to determine the effects of change in design factors, such as dimensions and material combinations, on friction and wear of ball-and-socket TDAs. Friction tests were carried out on generic models with ball radii 10, 12, 14 and 16 mm. Three material combinations were investigated; metal-on-metal, metal-on-polymer and for the first time polymer-on-metal. Wear tests were performed on metal-on-polymer Charité® TDAs and generic metal-on-metal models to compare the wear rate under the same conditions. Friction test results showed that polymer-on-metal TDAs create less friction than metal-on-polymer and metal-on-metal TDAs. Wear test results showed that under the same conditions, metal-on-metal TDAs create 23 times less wear debris than metal-on-polymer. The results were in agreement with studies on total hip arthroplasty (THA). The results of this work suggest possible alternatives for future TDA designs.