Introdução: A avaliação de fragilidade requer medidas mensuráveis de alguns critérios. Em nosso meio, sabe-se que a utilização destas medidas, em larga escala, não será facilmente operacionalizada por dificuldades logísticas. Diante disso, estuda-se a possibilidade de identificação da síndrome de fragilidade por meio de questões subjetivas. Objetivo: Validar componentes subjetivos para avaliação de fragilidade. Método: Este estudo é parte do Estudo SABE - Saúde, Bem-estar e Envelhecimento, realizado no município de São Paulo, Brasil. Trata-se de um corte transversal, com 433 idosos (idade 75 anos), em 2009. Foi adotado o Fenótipo de fragilidade proposto por Fried e colaboradores como padrão-ouro (avaliando objetivamente 5 critérios: perda de peso não intencional, fadiga relatada, redução da força de preensão, redução da velocidade de caminhada e baixa atividade física). Neste modelo, o idoso com um ou dois componentes foi considerado frágil, e com três ou mais era frágil. A avaliação subjetiva foi realizada por meio de questões dicotômicas referentes a cada componente. Calculou-se confiabilidade, sensibilidade, especificidade e valores preditivos positivo e negativo, para análise psicométrica da avaliação subjetiva. Resultados: A avaliação subjetiva é confiável e válida. Para os idosos classificados como pré-frágeis a sensibilidade foi de 89,7 por cento e especificidade de 24,3 por cento ; enquanto para os frágeis, a sensibilidade foi de 63,2 por cento e especificidade de 71,6 por cento . Ao analisar o processo de fragilização (pré-frágil+frágil) quase 90 por cento dos idosos frágeis foram detectados na avaliação subjetiva, 85,2 por cento foram preditos positivamente e 32,7 por cento foram preditos negativamente. Conclusão: A avaliação subjetiva de fragilidade é uma boa ferramenta para identificar processo de fragilidade em idosos / Introduction: The evaluation of frailty measures requires some measurable criteria. In our environment, it is known that the use of these measures on a large scale is not easily operationalized, due to logistical difficulties. Thus, we study the possibility of identifying the syndrome of frailty through subjective questions. Objective: To validate the subjective components for evaluation of frailty. Method: This study is part of the SABE Study - Health, Well-being and Ageing, held in São Paulo, Brazil. This is a cross sectional study of 433 elderly (age 75 years) in 2009. We adopted the phenotype of frailty proposed by Fried and colleagues as a gold standard (measuring objectively 5 criteria: unintentional weight loss, fatigue reported, reduced grip strength, reduced walking speed and low physical activity). In this model, elderly with one or two components were considered frail, and those with three or more were considered frail. Subjective evaluation was performed using dichotomous questions for each component. We calculated the reliability, sensitivity, specificity and positive and negative predictive values for psychometric analysis of subjective evaluation. Results: The subjective evaluation is reliable and valid. For the pre-frail elderly the sensitivity was 89.7 per cent and specificity of 24.3 per cent , while for the frail, the sensitivity was 63.2 per cent and specificity of 71.6 per cent . When analyzing frailty process (pre-frail+frail) almost 90 per cent of the frail elderly were detected in the subjective assessment, 85.2 per cent were predicted positively and 32.7 per cent were predicted negatively. Conclusion: The subjective evaluation of frailty is a good tool to identify frailty process in elderly

Poon Kwok Man. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 118-123). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / DECLARATION --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1. --- Antibody structure and diversity --- p.1 / Chapter 1.2. --- Antibody genes --- p.5 / Chapter 1.3. --- The antibody response to phosphorylcholine --- p.10 / Chapter 1.3.1. --- Group I antibodies --- p.11 / Chapter 1.3.2. --- Group II antibodies --- p.14 / Chapter 1.3.3. --- Fine specificity of group I antibodies --- p.14 / Chapter 1.4. --- Anti-phosphorylcholine antibody structure --- p.15 / Chapter 1.5. --- Recombinant antibody --- p.22 / Chapter 1.5.1. --- Phage biology --- p.24 / Chapter 1.5.2. --- Phage-displayed antibodies --- p.29 / Chapter 1.5.3. --- Helper phage --- p.32 / Chapter 1.6. --- Objectives and scope of study --- p.34 / Chapter CHAPTER 2 --- METHODOLGY / Chapter 2.1. --- Antibody --- p.41 / Chapter 2.1.1. --- Hybridoma culture --- p.41 / Chapter 2.1.2. --- Production of antibody by induction of ascitic fluid --- p.41 / Chapter 2.1.3. --- Antibody purification --- p.41 / Chapter 2.1.3.1. --- Ammonium sulfate precipitation --- p.42 / Chapter 2.1.3.2. --- Affinity purification by Protein A-sepharose --- p.42 / Chapter 2.1.4. --- Production of Fab fragment by papain digestion --- p.43 / Chapter 2.2. --- Antigens --- p.43 / Chapter 2.2.1. --- Preparation of TsAg form infected ICR mouse --- p.44 / Chapter 2.2.2. --- Purification of Trichinella spairalis PC antigen --- p.44 / Chapter 2.2.2.1. --- Preparation of Mab2 affinity column --- p.44 / Chapter 2.2.2.2. --- Purification of TsAg --- p.45 / Chapter 2.2.3. --- Preparation of PC-HSA --- p.45 / Chapter 2.2.3.1. --- Preparation of p-diazonium phenylphosphorylcholine (DPPC) --- p.45 / Chapter 2.2.3.2. --- Conjugation of PC to HSA --- p.45 / Chapter 2.2.4. --- Commercial available antigens --- p.46 / Chapter 2.2.4.1. --- Pneumovax® 23 --- p.46 / Chapter 2.2.4.2. --- Lipopolysaccharide --- p.46 / Chapter 2.2.5. --- Standardization of PC-antigens --- p.46 / Chapter 2.3. --- Cloning of Mab2-scFv into phage display form --- p.47 / Chapter 2.3.1. --- Total RNA extraction --- p.50 / Chapter 2.3.2. --- cDNA synthesis --- p.50 / Chapter 2.3.3. --- Heavy chain variable region gene amplification --- p.51 / Chapter 2.3.4. --- Light chain variable region gene amplification --- p.51 / Chapter 2.3.5. --- Joining of heavy and light chain gene with linker --- p.52 / Chapter 2.3.6. --- Ligation of scFv gene with pCANTAB-5E vector --- p.52 / Chapter 2.3.7. --- Transformation --- p.53 / Chapter 2.3.7.1. --- E.coli strains --- p.53 / Chapter 2.3.7.2. --- E.coli cell preparation for electroporation --- p.54 / Chapter 2.3.7.3. --- Electroporation --- p.54 / Chapter 2.3.7.4. --- Competent E.coli preparation by CaCl2 --- p.55 / Chapter 2.3.7.5. --- Heat shock --- p.55 / Chapter 2.4. --- Expression of phage display scFv --- p.55 / Chapter 2.5. --- Enrichment and screening of Mab2-scFv phage --- p.56 / Chapter 2.5.1. --- Biopanning --- p.56 / Chapter 2.5.2. --- Restricition fragment analysis --- p.58 / Chapter 2.5.3. --- PCR screening --- p.58 / Chapter 2.5.4. --- DNA sequencing --- p.58 / Chapter 2.5.4.1. --- Manual sequencing --- p.58 / Chapter 2.5.4.2. --- Auto sequencing --- p.59 / Chapter 2.6. --- Mutagenesis --- p.59 / Chapter 2.6.1. --- Preparation of Uracil containing ssDNA --- p.60 / Chapter 2.6.2. --- Phosphorylation of mutagenic oligonucleotide --- p.60 / Chapter 2.6.3. --- Hybridization and secondary strand synthesis...…… --- p.60 / Chapter 2.6.4. --- Transfection and screening of mutants --- p.61 / Chapter 2.7. --- Expression of soluble scFv-E-tag --- p.61 / Chapter 2.7.1. --- SDS-PAGE analysis --- p.62 / Chapter 2.7.2. --- Anti-E-tag ELISA --- p.62 / Chapter 2.8. --- ELISA binding assay --- p.63 / Chapter 2.8.1. --- Specificity of Mab2 antibody Fab --- p.63 / Chapter 2.8.1.1. --- Carrier specifcity assay --- p.63 / Chapter 2.8.1.2. --- Free hapten inhibition assay --- p.64 / Chapter 2.8.2. --- Specificity of the scFv --- p.64 / Chapter 2.8.2.1. --- Antigen binding assay --- p.65 / Chapter 2.8.2.2. --- Free hapten inhibition assay --- p.65 / Chapter 2.8.2.3. --- Inhibition on Ts2 and Mab2 antibody assay --- p.65 / Chapter 2.9. --- Affinity assay --- p.66 / Chapter 2.10. --- Mutants analysis --- p.66 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1. --- Cloning VH and VL gene of Mab2 into scFv --- p.67 / Chapter 3.1.1. --- Amplification of variable region of H and L chain --- p.67 / Chapter 3.1.2. --- Biopanning --- p.70 / Chapter 3.1.3. --- Genetic composition of isolated clones --- p.70 / Chapter 3.2. --- Mutagenesis --- p.84 / Chapter 3.3. --- Expression and characterisation of wild-type scFv --- p.88 / Chapter 3.3.1. --- ScFv soluble protein --- p.88 / Chapter 3.3.2. --- Phage displayed scFv --- p.91 / Chapter 3.3.3. --- Standardization of PC antigens --- p.91 / Chapter 3.3.4. --- Binding acticity of scFv --- p.94 / Chapter 3.3.4.1. --- Influence of the avidity on carrier specificity binding --- p.96 / Chapter 3.4. --- Antigen specificity --- p.99 / Chapter 3.4.1. --- Free hapten inhibiton --- p.99 / Chapter 3.4.2. --- Inhibition on the binding of Ts2 --- p.102 / Chapter 3.4.3. --- Binding affinity --- p.104 / Chapter 3.5. --- Binding activities of mutants --- p.106 / Chapter CHAPTER 4 --- GENERAL DISCUSSION --- p.109 / REFERENCE --- p.118

This thesis argues that caffeine use offered a survival advantage to our ancestors and that moderate use continues to offer modern humans benefits. Caffeine ingestion, through the blocking of adenosine receptors, elicits broad elements of the mammalian threat response, specifically from the ‘flight or fight’ and ‘tend and befriend’ repertoires of behaviour: in effect, caffeine hijacks elements of the stress response. If the effects of caffeine had been discovered recently, rather than being available to Homo sapiens since Neolithic hunter gatherer times, it is likely that caffeine would be considered a ‘smart’ drug. More caffeine is being ingested today than ever previously recorded. Caffeine use is found across all age groups, all socio-economic strata, most ethnic groups, and is being used increasingly by the medical and pharmaceutical industries and by the armed forces. Yet despite this wide usage and a substantial body of research literature, there is at present no clear pattern or plausible model for the way caffeine achieves its effects. There is much contradiction in the literature and ambiguity as to why caffeine use should improve performance on some tasks, impair it on others and have no effect on other tasks, for some but not all of the time. The present work argues, through an examination of the specificity of caffeine’s operation, that these effects are not arbitrary but elicited by the nature of the tasks, in particular that caffeine ingestion affects those processes and behaviours which improve the probability of survival under perceived threat or stress. This is argued through the perspective of evolutionary psychology and relies theoretically on Polyvagal Theory. The argument generates testable hypotheses and empirical support for the thesis is garnered from nine experiments on card-sorting, verbal and numerical processing, local and global categorization, field dependence-independence, the Stroop task, tests of visuo-spatial ability, and from a correlational study of caffeine use and personality traits. It is concluded that moderate caffeine use in healthy adults promotes behaviours likely to be adaptive under perceived threat or stress. Limitations of both theory and empirical work and are discussed, together with potential practical applications and suggestions for further work.

DNA-binding proteins bind to specific sequences to direct their activity to defined loci in the genome. Regulation of gene expression, for example, is dependent on the recognition of specific DNA sequences by transcription factors (TFs). These TFs receive input from cellular signals to control panels of genes to meet the needs of the cells. Critical to this function is the recognition and binding of TFs to the correct DNA sequence. The main focus of this thesis is to quantitatively determine how proteins, including TFs, distinguish DNA sequences, and to understand how DNA sequence affect their function. Primarily using the Glucocorticoid receptor (GR) as the model TF, I developed novel methods to measure the DNA binding specificity over long binding sites. These methods: 1) Distinguished the sequence specificity of GR and closely related androgen receptor (AR), which helped to both account for differential genomic localization between the two factors, and explained how GR can functionally substitute for AR in castration-resistant prostate cancer (Chapter II); 2) Explored the effect of DNA sequence on GR-regulated transcription through the specification of monomeric versus dimeric binding. Sequence motifs that bias GR binding toward the monomeric state were discovered (Chapter III); 3) Demonstrated a conserved role of intrinsic specificity in directing the degree of GR genomic occupancy in vivo in a fixed chromatin context (Chapter V); 4) Quantitatively modeled and decoupled the DNA binding and cleavage specificities of CRISPR-Cas9 system, providing a rapid pipeline to characterize the genome-editing reagents (Chapter IV). In summary, we showed here that DNA binding specificity is only the initial step in directing the activity of the bound protein. Beyond the affinity-based recruitment, DNA sequences can regulate the protein activity through alternative mechanisms, such as modulating the binding cooperativity, or directly serving as an allosteric ligand for protein function that is independent of DNA binding affinity.

Post-translational modification (PTM) of proteins by enzymes such as methyltransferases, kinases and deacetylases play a crucial role in the regulation of many metabolic pathways. Determining the substrate scope of these enzymes is essential when studying their biological role. However, the combinatorial nature of possible protein substrate sequences makes experimental screening assays intractable. To predict new substrates for proteins, various computational approaches have been developed. Our method relies on crystallographic data and a novel multistate computational protein design algorithm. We previously used our method to successfully predict four new substrates for SMYD2 (Lanouette S & Davey J.A., 2015), doubling the number of known targets for this PTM enzyme that has been difficult to characterize using other methods. This was possible by first extracting a specificity profile of Smyd2 using our algorithm and subsequently screening a peptide library for matching sequences. However, our method did not yield successful results when attempting to reproduce specificity profiles of other proteins (64% accuracy on average). Different protein environments have demonstrated limitations in the methodology and lead us to further develop the algorithm on a more thorough dataset. Using our new optimized method, specificity profile predictions increase by roughly 20% (84% accuracy on average), independent of the structural template used. The algorithm was then used to blindly predict a specificity profile for the methyltransferase Smyd3, an enzyme for which limited data is currently available. A library of 2550 peptides was screened with the predicted profile, yielding 123 matching sequences. We randomly chose 64 for experimental validation (SPOT peptide array) of methylation by Smyd3 and found 45 methylated and 19 non-methylated peptides (70% success rate). Finally, we released to the community a web version of the algorithm, which can be accessed as http://viper.science.uottawa.ca.

In many applications, it is common to have multiple diagnostic tests on each subject. When there are multiple tests available, combining tests to incorporate information from various aspects in subjects may be necessary in order to obtain a better diagnostic. For continuous tests, in the presence of a gold standard, we could combine the tests linearly (Su and Liu, 1993) or sequentially (Thompson, 2003), or using the risk score as studied by McIntosh and Pepe (2002). The gold standard, however, is not always available in practice. This dissertation concentrates on deriving classification methods based on multiple tests in the absence of a gold standard. Motivated by a lab data set consisting of two tests testing for an antibody in 100 blood samples, we first develop a mixture model of four bivariate normal distributions with the mixture probabilities depending on a two-stage latent structure. The proposed two-stage latent structure is based on the biological mechanism of the tests. A Bayesian classification method incorporating the available prior information is derived utilizing Bayesian decision theory. The proposed method is illustrated by the motivating example, and the properties of the estimation and the classification are described via simulation studies. Sensitivity to the choice of the prior distribution is also studied. We also investigate a general problem of combining multiple continuous tests without any gold standard or a reference test. We thoroughly study the existing methods for combining multiple tests and develop optimal classification rules corresponding to the methods accommodating the situation without a gold standard. We justify the proposed methods both theoretically and numerically through exten- sive simulation studies and illustrate the methods with the motivating example. In the end, we conclude the thesis with remarks and some interesting open questions extended from the dissertation.

With aging there is a decrease in a person’s ability to perform activities of daily living (ADL) which may be most effectively addressed using training patterns that are biomechanically similar to ADL. Since aquatic exercise offers the opportunity to provide resistance with a high level of safety, the pool may afford the ideal environment for ADL-specific training in an aging population. Purpose: The purpose of this investigation was to compare a traditional aquatic exercise program (TRAD) to an aquatic program tailored to target ADL (ADLspec). Methods: Eighteen independently living individuals (68.7 + 7.5 years) were randomly assigned to a TRAD or ADLspec aquatic exercise group. The exercise groups attended 1 hr exercise sessions, 2 times per week for 8 weeks. ADL ability was assessed using the short version of the Continuous-Scale Physical Functional Performance Test (PFP-10); while strength and power were assessed using the 30s arm curl and 30 sec. chair stand tests. Results: Mixed design ANOVAs revealed a significant group x time interaction for floor sweep time with the ADLspec group outperforming the TRAD and control (CON) groups (p = .043). Additionally, the ADLspec group improved the pan weight and scarf time components of the PFP-10 (p < .020), while the TRAD group improved pan time and laundry time (p < .046). Both training groups showed similar improvements for jacket time, grocery weight, and 6-min walk, (p < .046). The ADLspec and TRAD groups also made similar improvements in upper and lower body strength, as well as lower body power across time, (p < .043). A student’s t-test revealed the TRAD group spent more time exercising during the hour session than the ADLspec group (p < .05). Conclusion: The results indicate that performing an ADLspec aquatic exercise program can increase performance of ADL that require more complex sequential movements; however, ADL more dependent on fitness may be better addressed using a TRAD intervention. These results can be helpful when designing a periodized aquatic training program to increase independence in older persons.

Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now. This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates. In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

Root symbioses with dark septate endophytic fungi (DSE) and arbuscular mycorrhizal fungi (AMF) provide plant tolerance to environmental stresses. This research answers several fundamental questions about the occurrence of these fungi in roots of prairie grasses. Traditional methods and current molecular techniques were combined in order to: 1) define the role and specificity of DSE in plant tolerance to drought; 2) assess the level of host specificity in DSE; 3) document AMF biodiversity and pattern of root colonization at different soil depths; 4) define the influence of soil depth and plant species on the distribution of DSE and AMF in roots and; 5) reveal how DSE and AMF interact in plant roots.<p> Under controlled conditions, DSE isolates showed host preference in colonizing roots and promoting plant growth. They colonized with more intensity the plant species from which they were isolated [Agropyron cristatum L. or Psathyrostachys juncea (Fisch) Nevski subsp. Juncea (Syn: Elymus junceus Fisch)]. Inoculation with five DSE isolates resulted in growth stimulation of the C3 grasses A. cristatum and P. juncea, and growth depression of the C4 grass Bouteloua gracillis (Willd. ex Kunth) Lag. ex Griffiths, under water stress. Plant C concentration suggested that DSE inoculation may have resulted in net C drain from B. gracillis.<p. In the field, soil depth influenced root colonization in A. cristatum, Panicum virgatum L., Nassella viridula Trin and Pascopyrum smithii (Rydb.) A. Löve., while AMF diversity was influenced by the interaction between soil depth and host plant species. Molecular analysis of roots serially sampled during one growing season from the A and B soil horizons, in stands of these grasses, revealed spatial and temporal changes in DSE and AMF community composition, and a significant correlation in DSE and AMF community structure.<p> These results suggest that DSE and AMF are adapted to specific environmental conditions and that root occupation by these fungi is a dynamic phenomenon. It is proposed that temporal variation in root occupation by DSE and AMF impacts plant and ecosystem processes at different times during the growing season.

In the current third wave of internationalization companies from mature markets are investing in emerging markets and increase their foreign activities. For this internationalization process, companies need to enter the market with an appropriate entry mode strategy. Prior research focused mainly on MNC entry modes and also on factors as ownership, location or internalization advantages and not on SMEs and transaction cost theory. This thesis deals with the topic of institutions, transaction costs and entry mode decisions of Swedish SMEs in India. The purpose of this thesis is to understand the managerial perceptions about the influence of institutions on the degree of linkage specificity with which the transaction costs will be explored. This will lead to further knowledge about certain entry mode decisions of Swedish SMEs for the Indian market. This was studied with a qualitative research strategy using a multiple case study method. The empirical data was conducted via secondary data and primary data was collected via interviews with the sales responsible of the four case companies Norden Machinery AB, Slipnaxos AB, Håkansson Sågblad AB and Hedin Lagan AB. Main findings of the research were that institutions are perceived differently by the managers and therefore the institutions dissimilarly influenced the perceptions of the transaction costs. Furthermore, the study revealed that some managers decided their entry mode on the base of the perception of the transaction costs and some managers did not consider transaction costs when entering new markets. All in all it can be stated that transaction costs influence the entry mode of companies. The research is limited by the fact that transaction cost theory in general neglects factors as production costs. Managerial implications are that transactions costs should not be neglected as they help to choose a more successful entry mode and that the assets specificity and the behavioral uncertainties need to be taken into account when deciding upon an entry mode. Theory profits from this thesis as it proved that transaction costs influence entry mode decisions and that the linkage specificity is an important factor to include when combining transaction cost theory with entry mode decisions.