Image format (Laws, Adlington, Gale, Moreno-Martínez, & Sartori, 2007), ceiling effects in controls (Fung et al., 2001; Laws et al., 2005; Moreno-Martínez, & Laws, 2007; 2008), and nuisance variables (Funnell & De Mornay Davis, 1996; Funnell & Sheridan, 1992; Stewart, Parkin & Hunkin, 1992) all influence the emergence of category specific deficits in Alzheimer‟s dementia (AD). Thus, the predominant use of line drawings of familiar, everyday items in category specific research is problematic. Moreover, this does not allow researchers to explore the extent to which format may influence object recognition. As such, the initial concern of this thesis was the development of a new corpus of 147 colour images of graded naming difficulty, the Hatfield Image Test (HIT; Adlington, Laws, & Gale, 2009), and the collection of relevant normative data including ratings of: age of acquisition, colour diagnosticity, familiarity, name agreement, visual complexity, and word frequency. Furthermore, greyscale and line-drawn versions of the HIT corpus were developed (and again, the associated normative data obtained), to permit research into the influence of image format on the emergence of category specific effects in patients with AD, and in healthy controls. Using the HIT, several studies were conducted including: (i) a normative investigation of the effects of category and image format on naming accuracy and latencies in healthy controls; (ii) an exploration of the effects of image format (using the HIT images presented in colour, greyscale, and line-drawn formats) and category on the naming performance of AD patients, and age-matched controls performing below ceiling; (iii) a longitudinal investigation comparing AD patient performance to that of age-matched controls, on a range of semantic tasks (naming, sorting, word-picture matching), using colour, greyscale, and line-drawn versions of the HIT; (iv) a comparison of naming in AD patients and age-matched controls on the HIT and the (colour, greyscale and line-drawn) images from the Snodgrass and Vanderwart (1980) corpus; and (v) a meta-analysis to explore category specific naming in AD using the Snodgrass and Vanderwart (1980) versus other corpora. Taken together, the results of these investigations showed first, that image format interacts with category. For both AD patients and controls, colour is more important for the recognition of living things, with a significant nonliving advantage emerging for the line-drawn images, but not the colour images. Controls benefitted more from additional surface information than AD patients, which chapter 6 shows results from low-level visual cortical impairment in AD. For controls, format was also more important for the recognition of low familiarity, low frequency items. In addition, the findings show that adequate control data affects the emergence of category specific deficits in AD. Specifically, based on within-group comparison chapters 6, 7, and 8 revealed a significant living deficit in AD patients. However, when compared to controls performing below ceiling, as demonstrated in chapters 7 and 8, this deficit was only significant for the line drawings, showing that the performance observed in AD patients is simply an exaggeration of the norm.

Murals have been the formal visual interpretation of the cultural, social and political life of all ages. Throughout they have been consistently combined with their architectural setting, for example, in ancient Egyptian tombs, in Renaissance churches and on the external walls of buildings in Mexico in the twentieth century. This is a central feature of mural painting. However many contemporary murals do not integrate with their architectural settings, in other words, do not fulfil the site-specificity of the architectural spaces for which they were made. This means that the most important aspect that distinguishes murals from other types of painting is absent. I studied and analysed a number of murals produced in the Italian Renaissance, Baroque and Rococo as this particular period is considered to be not only one of the most significant in the history of art but also a period in which painting and architecture were very closely allied as practices. In particular the radical developments in painting of pictorial space took place along side the developments in architecture. I argue that Renaissance murals could be described, using the terminology of contemporary art, as site-specific art. By identifying the relationship between pictorial space, architectural space and compositional structure I was able to test, through my own practice, the importance of these relationships in understanding the site-specificity of the compositional structure of murals. To address the issue of sitespecificity in murals, I investigated and developed a set of compositional structures through my mural practice that could be applied in the design, execution, and teaching of contemporary mural design. I have developed the notion of a deconstructive method of mural design in which the illusory space of the mural derives its compositional structure from the architectural space in which it sited. I have applied it, tested it and refined it through the execution of a number of hypothetical and live mural commissions. I believe that the approach to the study and practice of mural design I have developed from the perspective of a practice lead researcher contributes to the furtherance of mural design as both a profession and field of study. In particular the identification of compositional structures in mural design and the proposal of a deconstructive method contributes to our understanding of what a mural is as well as current notions of site-specificity in contemporary art.

<b>Introduction</b>: Kinesiology-style manual muscle testing (kMMT) is a non-invasive assessment method used by various types of practitioners to detect a wide range of target conditions. It is distinctly different from the muscle testing performed in orthopaedic/neurological settings and from Applied kinesiology. Despite being estimated to be used by over 1 million people worldwide, the usefulness of kMMT has not yet been established. The aim of this thesis was to assess the validity of kMMT by examining its accuracy and precision. <b>Methods</b>: A series of 5 diagnostic test accuracy studies were undertaken. In the first study, the index test was kMMT, and the target condition was deceit in verbal statements spoken by Test Patients (TPs). The comparator reference standard was a true gold standard: the actual verity of the spoken statement. The outcomes of the muscle tests were interpreted consistently: a weak result indicated a Lie and a strong result indicated a Truth. A secondary index test was included as a comparator: Intuition, where Practitioners used intuition (without using kMMT) to ascertain if a Lie or Truth was spoken. Forty-eight Practitioners were recruited and paired with 48 unique kMMT-naïve TPs. Each Pair performed 60 kMMTs broken up into 6 blocks of 10, which alternated with blocks of 10 Intuitions. For each Pair, an overall percent correct was calculated for both kMMT and Intuition, and their means were compared. Also calculated for both tests were sensitivity, specificity, positive predictive value and negative predictive value. The second study was a replication of the first, using a sample size of 20 Pairs and a less complex procedure. In the third study, grip strength dynamometry replaced kMMT as the primary index test. In the fourth study, the reproducibility and repeatability of kMMT were examined. In the final study, TPs were presented with emotionally-arousing stimuli in addition to the affect-neutral stimuli used in previous studies, to assess if stimuli valence impacted kMMT accuracy. <b>Results</b>: Throughout this series of studies, mean kMMT accuracies (95% Confidence Intervals; CIs) ranged from 0.594 (0.541 – 0.647) to 0.659 (0.623 - 0.695) and mean Intuition accuracies, from 0.481 (0.456 - 0.506) to 0.526 (0.488 - 0.564). In all studies, mean kMMT accuracies were found to be significantly different from mean Intuition accuracies (p ≤ 0.01), and from Chance (p < 0.01). On the other hand, no difference was found between grip strength following False statements compared to grip strength following True statements (p = 0.61). In addition, the Practitioner-TP complex accounted for 57% of the variation in kMMT accuracy, with 43% unaccounted for. Also, there was no difference in the mean kMMT accuracy when using emotionally-arousing stimuli compared to when using affect-neutral stimuli (p = 0.35). Mean sensitivities (95% CI) ranged from 0.503 (0.421 - 0.584) to 0.659 (0.612 - 0.706) while mean specificities (95% CI) ranged from 0.638 (0.430 - 0.486) to 0.685 (0.616 - 0.754). Finally, while a number of participant characteristic seemed to influence kMMT accuracy during one study or another, no one specific characteristic was found to influence kMMT accuracy consistently (i.e. across the series of studies). <b>Discussion</b>: This series of studies has shown that kMMT can be investigated using rigorous evidence-based health care methods. Furthermore, for distinguishing lies from truths, kMMT has repeatedly been found to be significantly more accurate than both Intuition and Chance. Practitioners appear to be an integral part of the kMMT dynamic because when replaced by a mechanical device (i.e. a grip strength dynamometer), distinguishing Lies from Truth was not possible. In addition, since specificities seemed to be greater than sensitivities, Truths may have been easier to detect than Lies. A limitation of this series of studies is that I have a potential conflict of interest, in that I am a practitioner of kMMT who gets paid to perform kMMT. Another limitation is these results are not generalisable to other applications of kMMT, such as its use in other paradigms or using muscles other than the deltoid. Also, these results suggest that kMMT may be about 60&percnt; accurate, which is statistically different from Intuition and Chance; however it has not been established if 60&percnt; correct is "good enough" in a clinical context. As such, further research is needed to assess its clinical utility, such as randomised controlled trials investigating the effectiveness of whole kMMT technique systems. Also, future investigators may want to explore what factors, such as specific Practitioner and TP characteristics, influence kMMT accuracy, and to investigate the validity of using kMMT to detect other target conditions, using other reference standards and muscles other than the deltoid. <b>Summary</b>: This series of diagnostic test accuracy studies has found that kMMT can be investigated using rigorous methods, and that kMMT used to distinguish Lies from Truths is significantly more accurate that both Intuition and Chance. Further research is needed to assess kMMT’s clinical utility.

<p>Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.</p> / Dissertation

The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets.               The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design.             The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry.

The twisted-wing parasites (Strepsiptera) are entomophagous insect order with cosmopolitan distribution. There are about 600 known species up to date. In spite of this, they have very broad host spectrum. Strepsiptera parasites in seven insect groups (Thysanura, Blattodea, Mantodea, Orthoptera, Hemiptera, Diptera, Hymenoptera). The mutual relationship between genera or even between species are not known, except for the species list and the host specification. Moreover, there is an anambiguous use of their species concept. Some authors consider Strepsiptera as the specialists and they match almost every host species with one separate strepsipteran parasite. The opposite concept is to consider strepsiptera as the generalists. The presence of the crypctic species also affect our understanding of the diversity of Strepsiptera. Therefore, the knowledge of Strepsiptera phylogeny provide us the important information about species diversity of studied group as well as about their coevolution with their hosts. On the basis of molecular analyses of three genes constructed the phylogeny genus Stylops. This genus has the wider spetrum of the host species from other strepsipterans of Stylopidae, It is obvious, that strepsipterans of genus Stylops are mainly specialised on their host subgenus. There was detected two...

Les glycosaminoglycannes (GAGs) sont des molécules polysaccharidiques polysulfatées intervenant dans des processus aussi variés que la prolifération, différenciation ou migration cellulaire, la coagulation sanguine ou l‟infection virale. Il est généralement admis qu‟une séquence particulière de GAG doit être associée à une fonction biologique spécifique. Les structures chimiques globales des GAGs sont connues. Cependant, contrairement au séquençage des gènes ou des protéines, la détermination de la séquence saccharidique exacte impliquée dans une fonction biologique particulière n‟est encore pas possible. Le séquençage « glycomique » constitue donc un enjeu majeur. L‟une des technologies les plus novatrices pour aborder ce problème de séquençage des GAGs semble être l‟impression moléculaire. En effet, elle permet d‟obtenir des polymères (MIPs pour Molecular Imprinted Polymer) spécifiquement imprimés par la forme structurale d‟une molécule cible.En nous appuyant sur des travaux antérieurs réalisés avec des modèles saccharidiques sulfatés simples, nous avons appliqué cette technologie à la reconnaissance de glycannes sulfatés complexes d‟intérêt biologique tels qu‟une héparine de bas poids moléculaire ou un mimétique ayant une activité anticoagulante. Il a été démontré une reconnaissance spécifique et sélective selon la molécule étudiée à l‟aide de MIPs spécialement conçus pour chaque GAG. De plus, nous avons obtenu des MIPs qui, en immobilisant temporairement un sucre, permettraient leur substitution de façon stéréospécifique. La détermination des conditions optimales de synthèse des MIPs s‟est avéré une étape nécessaire à l‟obtention d‟une bonne reconnaissance. Ces travaux ouvrent des perspectives d‟application de la technique d‟impression moléculaire à l‟analyse des séquences de GAGs d‟intérêt biologique / Glycosaminoglycans (GAGs) are polysulfated polysaccharide molecules involved in many biological processes such as cellular proliferation, differentiation or migration, blood clotting or viral infection. It is generally admitted that a particular GAG sequence is connected to a specific biological function. Depending on their composition in disaccharides, GAGs are classified into subfamilies whose overall chemical structures are known. Unlike gene or protein sequencing, determination of the exact saccharidic sequence involved in a particular biological function is not yet possible with the available technological tools. "Glycomics" is a real challenge nowadays. One of the most innovative technologies to achieve this goal seems to be the molecular imprinting. Indeed, it provides polymers (MIPs for Molecular Imprinted Polymer) imprinted by the structural form of a target molecule.Based on previous studies performed with simple sulfated saccharides, this technology has been applied to the recognition of complex sulfated glycans. MIPs were achieved demonstrating specific and selective recognition for a Low Molecular Weight Heparin or a synthetic anticoagulant mimetic. Other MIPs were able to temporally immobilize sugars which make them available for stereo-specific modifications. Screening of optimal synthesis conditions of MIPs appeared a necessary step to obtain a specific and selective recognition. These studies open further possibilities to analyze GAG sequences carrying biological functions by the molecular imprinting technology

Doctor of Philosophy / Department of Biology / Alexander Beeser / Large-scale genomic approaches have demonstrated many atypical dual specificity phosphatases (DUSPs) are differentially expressed or mutated in cancer. DUSPs are proteins predicted to have the ability to dephosphorylate Ser/Thr and Tyr residues, and the atypical DUSP subgroup contains at least 16 members with diverse substrates that include proteins, nucleic acids, and sugars, and some of the atypical DUSPs function in the cell not as phosphatases but as scaffolds in signal transduction pathways. Of the atypical DUSPs, DUSP12 is one of the most evolutionarily conserved with homologs found in organisms ranging from yeast to humans. DUSP12 is of particular interest as it has been identified to be one of only two candidate genes for the target of a genetic amplification found in liposarcomas. Furthermore, DUSP12 may be an oncogene in that over-expression of dusp12 in cell culture promotes apoptosis resistance, cell motility, and the up-regulation of two established oncogenes, the hepatocyte growth factor receptor (c-met) and integrin alpha 1 (itga1). Additionally, DUSP12 may protect from apoptosis by functioning as a regulator of stress-induced translation repression and stress granule formation that may be due to its interaction with the DEAD Box RNA Helicase, DDX3.

A raiva é uma encefalomielite aguda causada por um vírus da ordem Mononegavirales, família Rhabdoviridae, gênero Lyssavirus e espécie Rabies virus (RABV). É um vírus RNA de fita simples, senso negativo que acomete todas as espécies de mamíferos. Seu diagnóstico é obtido de modo definitivo por técnicas laboratoriais. A imunofluorescência direta (IFD) é uma prova de triagem rápida, considerada padrão ouro pela OMS e OIE, pois possui alta sensibilidade e especificidade diagnóstica. Esta técnica utiliza como teste confirmatório o isolamento viral em camundongos (IVC) ou em cultura de células (IVCC). Uma vez que o RABV não infecta todas as estruturas do sistema nervoso central (SNC) de modo uniforme, a detecção deste agente pode ter resultados variáveis. Algumas situações exigem o desenvolvimento e implantação de metodologias alternativas, tais como transcrição reversa seguida de reação em cadeia da polimerase (RT-PCR), RT-PCR em tempo real (RT-qPCR) e imunohistoquímica (IHQ), que possuem vantagens e apresentaram resultados bastante promissores. Para tanto, foram utilizadas amostras de SNC[tálamo, córtex, hipocampo, cerebelo, tronco encefálico (bulbo, ponte e mesencéfalo) e medula cervical]de 127 bovinos provenientes da Seção de Diagnóstico da Raiva do Instituto Pasteur, durante o período de março de 2015 a abril de 2016. As diferentes regiões do SNC dos animais foram identificadas e alíquotas foram separadas para a realização das técnicas de identificação viral, totalizando 689 fragmentos. A presença viral foi observada pela IFD e confirmada tanto pelo IVC quanto pelo IVCC. A distribuição do vírus pelas diferentes porções do SNC foi observada pelas técnicas de IFD, IHQ e RT-PCR para detecção do gene N em 40 animais que foram considerados positivos. Os 40 bovinos positivos na IFD confirmaram sua positividade na IHQ e IVC, apresentando concordância de 100% dos resultados. Entretanto houve diferença em relação aos resultados dos isolamentos virais, uma vez que, dos 38 animais positivos que foram submetidosaambas as técnicas, três foram negativos para a presença do RABV no IVCC, indicando uma concordância de 92,1%, (3538) quando comparado ao IVC. Em relação às técnicas moleculares houve diferenças, a RT-PCR apresentou positividade de 100% (4040), enquanto que a RT-qPCR observou-se que 97,5% (39/40) dos bovinos foram positivos. Na IFD, de modo geral, não houve disparidade entre os fragmentos em relação à positividade, porém ocorreu diferença quando se analisou a intensidade de fluorescência entre os fragmentos. Quando comparadas, a IHQ e a IFD apresentaram resultados semelhantes, embora a IHQ tenha apresentado positividade em 100% fragmentos (209209) e a IFD em 99,51% dos fragmentos (205206). Conclui-se, pelos resultados obtidos, diferenças na intensidade da distribuição viral e na concentração de RNA viral nas diferentes porções do SNC (padrões de neuroinvasividade) em bovinos; fato este que poderia interferir nos resultados obtidos no diagnóstico de rotina da raiva. / Rabies is an acute encephalomyelitis caused by a virus belonging to the Mononegavirales order, Rhabdoviridae family, Lyssavirus genus and Rabies virus (RABV) species. It is a single-stranded RNA virus, negative sense that affects all species of mammals. The definitively diagnosis is obtained by laboratory techniques. Direct fluorescent antibody test (dFAT) is a rapid screening, considered the gold standard by WHO and OIE, as it has high diagnostic sensitivity and specificity. However, it´s recommended the mouse inoculation test (MIT) or rabies tissue culture infection test (RTCIT) as a confirmatory test. Once RABV does not infect all central nervous system (CNS) structures uniformly, the detection of this agent may have varying results Some situations require the development and implementation of alternative methods such as RT-PCR, RT-qPCR and immunohistochemistry (IHC), which have advantages and showed very promising results. Therefore, CNS structures [thalamus, cortex, hippocampus, cerebellum, brainstem (medulla, pons and midbrain) and cervical cord] from 127 cattle were selected from the Rabies Diagnostics Section of the Instituto Pasteur during the period March 2015 to April 2016. The different CNS structures were identified and aliquots were separated for carrying out the viral identification techniques, totalizing 689 structures. The viral presence was observed by dFAT and confirmed by both the MIT and the RTCIT. The distribution of the virus by different portions of the CNS was observed by dFAT, IHC and RT-PCR for detection of the N gene in 40 animals considered positive. The 40 positive cattle in the dFAT confirmed their positivity on IHC and MIT, with agreement of 100% of the results. However there was a difference from the viral isolation since the 38 positive animals tested in both techniques, three were negative for the presence of RABV in RTCIT indicating an agreement of 92.1% (35/38) when compared to MIT. The dFAT, in general, showed no disparity among the fragments relative to their positivity, however, a difference was observed when analyzing the fluorescence intensity between the fragments. When compared IHC and dFAT presented similar results, although the IHC has presented a 100% positivity in the fragments (209/209) and 99.51% in dFAT (205/206).Regarding the molecular techniques, RT-PCR showed positivity of 100% (40/40), meanwhile RT-qPCR showed 97,5% (39/40) among the positive animals studied. In conclusion, the results suggest that there is variability in the intensity of virus distribution and in the virus concentration in different parts of the CNS (neuroinvasiveness patterns) in cattle. This fact could affect the results obtained in the diagnosis of rabies routine.