Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained. The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

Based on the view of an agent as an information processing system, and the premise that for such a system it is evolutionary advantageous to be parsimonious with respect to informational burden, an information-theoretical framework is set up to study behaviour under information minimisation pressures. This framework is based on the existing method of relevant information, which is adopted and adapted to the study of a range of cognitive aspects. Firstly, the model of a simple reactive actor is extended to include layered decision making and a minimal memory, in which it is shown that these aspects can decrease some form of bandwidth requirements in an agent, but at the cost of an increase at a different stage or moment in time, or for the system as a whole. However, when combined, they do make it possible to operate with smaller bandwidths at each part of the cognitive system, without increasing the bandwidth of the whole or lowering performance. These results motivate the development of the concept of look-ahead information, which extends the relevant information method to include time, and future informational effects of immediate actions in a more principled way. It is shown that this concept can give rise to intrinsic drives to avoid uncertainty, simplify the environment, and develop a predictive memory. Next, the framework is extended to incorporate a set of goals, rather than deal with just a single task. This introduces the task description as a new source of relevant information, and with that the concept of relevant goal information. Studying this quantity results in several observations: minimising goal information bandwidth results in ritualised behaviour; relevant goal and state information may to some point be exchanged for one another without affecting the agent’s performance; the dynamics of goal information give rise to a natural notion of sub-goals; bottlenecks on goal memory, and a measure of efficiency on the use of these bottlenecks, provide natural abstractions of the environment, and a global reference frame that supersedes local features of the environment. Finally, it is shown how an agent or species could actually arrive at having a large repertoire of goals and accompanying optimal sensors and behaviour, while under a strong information-minimisation pressure. This is done by introducing an informational model of sensory evolution, which indicates that a fundamental information-theoretical law may underpin an important evolutionary catalyst; namely, even a fully minimal sensor can carry additional information, dubbed here concomitant information, that is required to unlock the actual relevant information, which enables a minimal agent to still explore, enter and acquire different niches, accelerating a possible evolution to higher acuity and behavioural abilities.

It’s becoming more and more difficult to make smaller, denser, and faster computer chips. There’s an increasing demand to design new materials to be applied in current lithographic process to get higher patterning performance. In this work, the aqueous developable single molecule resists were introduced, synthesized and patterned. A new group of epoxide other than glycidyl ether, cyclohexene oxide was introduced to functionalize a molecular core and 15 nm resolution was obtained. The directed self-assembly (DSA) of block copolymers as an alternative lithographic technique has received growing interest in the last several years for performing higher levels of pitch subdivision. A 3-step simplified process for DSA by using a photodefinable substrate was introduced by using a functionalized polyphenol with an energy switchable group and a crosslinkable group. Two high χ block copolymers PS-b-PAA and PS-b-PHEMA were successfully designed and synthesized via ATRP with controlled Mw and PDI. The size of the same PS-b-PAA polymer was tunable by varying the thermal annealing time. PS-b-PHEMA shows to be a suitable block polymer for the industry-friendly thermal annealing process. A self-complementary hydrogen-bonding urea group as a center group was used to facilitate the self-assembly of polymers. “Click” chemistry is promising for synthesis of PS-Urea-Urea-PMMA.

A molecular dynamics coarse-grained block copolymer (BCP) model was developed and used to studied directed self-assembly (DSA), especially in regards to applications for semiconductor manufacturing. Most of the thesis is spent investigating the effect that guiding layer properties and block copolymer properties have on line roughness and defect density in a BCP-DSA process. These two effects are perhaps the most critical in making BCP-DSA a cost efficient industrial process. It is found that guiding patterns have little effect on line roughness and in fact that the BCP heals the majority of roughness in the underlying pattern. BCP properties have a larger effect on line roughness. Segregation strength (as measured by χN, where χ is the Flory- Huggins interaction parameter and N is the degree of polymerization) resulted in a larger than expected increase in line roughness when χN was low. Polydispersity resulted in a moderate increase in line roughness. In regards to equilibrium defect density, free energy calculations showed that χ was the primary determining factor, not χN as many expected. Equilibrium defect density was found to decrease exponentially with increasing χ. Defect density is also found to scale exponentially with polydispersity. Concerning defect heal rate, which can increase the real defect rate of a process if said rate is too low, it is found that increasing χN linearly increased the barrier to defect healing, which means that the defect heal rate decreases exponentially. However, for thin films this is only true for χN > ~ 50. Below χN ~ 50, the barrier is approximately constant. These results give excellent guidance to the type of materials and processes necessary to optimize a BCP-DSA process. A simulation technique designed to more efficiently sample over energy barriers called protracted noise dynamics for polymer systems was developed and studied. It was found that a decrease in simulation time of up to 4 orders of magnitude was achieved. The effect of box size on allowable pitches for a lamellar forming BCP was derived and demonstrated. It was found that more elongated boxes yielded more possible pitches and more accurate results. A short study on the effect of multiblock copolymers on the location of the order-disorder transition was also carried out and it was found that multiblock copolymers had small effect on the ODT. The distribution of chain conformations was also calculated.

Relationship of Computer Self-Efficacy and Self-Directed Learning Readiness to Civilian Employees’ Completion of Online Courses. Joan M Lenahan-Bernard, 2014, Applied Dissertation, Nova Southeastern University, Abraham S. Fischler School of Education. ERIC Descriptors: Computer Self-Efficacy, Self-Directed Learning, E-Learning. This study investigated the relationship of computer self-efficacy (CSE) and self-directed learning readiness (SDLR) to civilian employees’ completion of online courses. A mixed methods design was used to answer five research questions: What is the relationship between CSE and federal civilian employees’ completion of online courses? What is the relationship between SDLR and federal civilian employees’ completion of online courses? What do civilian employees identify as the relationship between CSE and SDLR and their decision to complete or not complete online courses? What do civilian employees identify as the relationship between the workplace environment and their decision to complete or not complete online courses? What results emerge from comparing quantitative data and qualitative data regarding the relationship between CSE and SDLR and civilian employees’ decisions to complete online courses? Ninety-eight civilian employees completed the Self-Directed Learning Readiness Scale (SDLRS) and the Computer Users Self-Efficacy (CUSE) Scale. Archival data were used to measure course completion. Descriptive and inferential statistics were used to determine the relationship of CSE and SDLR to the completion of online courses Interviews were conducted to obtain insight into the relationship between CSE and SDLR and civilian employees’ decision to complete online courses. Qualitative data were organized according to the principles of SDLR theory and themes. A merged data display shows responses aligned with questionnaire scores and completion status. Findings showed no statistically significant relationship between the percentage of online courses completed and CSE (r=.04, p=.72, N=98) or between the percentage of online courses completed and SLDR (r=-.15, p=.15, N=98). The relationship between SDLR and CSE was statistically significant (r=.21, p=.035, N=98) which is supported in the literature. Qualitative analysis showed that civilian employees were frustrated by poor design, outdated nature, and irrelevance of most online courses and resources they were expected to use.

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

The purpose of this study was to investigate the effects of explicit instruction combined with video prompting to teach text comprehension skills to students with autism spectrum disorder. Participants included 4 elementary school students with autism. A multiple probe across participants design was used to evaluate the intervention’s effectiveness. Results indicated that the intervention was successful for all participants. All participants mastered the comprehension skills; however, data were highly variable during the acquisition phase. Implications for researchers and practitioners are discussed.

In the first part of this thesis we transfer a result of Guillopé et al. concerning the number of zeros of the Selberg zeta function for convex cocompact Schottky groups to the setting of certain types of graph directed Markov systems (GDMS). For these systems the zeta function will be a type of Ruelle zeta function. We show that for a finitely generated primitive conformal GDMS S, which satisfies the strong separation condition (SSC) and the nestedness condition (NC), we have for each c>0 that the following holds, for each w \in\$C$ with Re(w)>-c, |\Im(w)|>1 and for all k \in\$N$ sufficiently large: log | zeta(w) | <<e {delta(S).log(Im|w|)} and card{w \in\ Q(k) | zeta(w)=0} << k {delta(S)}. Here, Q(k)\subset\%C$ denotes a certain box of height k, and delta(S) refers to the Hausdorff dimension of the limit set of S. In the second part of this thesis we show that in any dimension m \in\$N$ there are GDMSs for which the Hausdorff dimension of the uniformly radial limit set is equal to a given arbitrary number d \in\(0,m) and the Hausdorff dimension of the Jørgensen limit set is equal to a given arbitrary number j \in\ [0,m). Furthermore, we derive various relations between the exponents of convergence and the Hausdorff dimensions of certain different types of limit sets for iterated function systems (IFS), GDMSs, pseudo GDMSs and normal subsystems of finitely generated GDMSs. Finally, we apply our results to Kleinian groups and generalise a result of Patterson by showing that in any dimension m \in\$N$ there are Kleinian groups for which the Hausdorff dimension of their uniformly radial limit set is less than a given arbitrary number d \in\ (0,m) and the Hausdorff dimension of their Jørgensen limit set is equal to a given arbitrary number j \in\ [0,m).

La redéfinition de la profession infirmière, intervenue en Europe depuis 1990 afin de répondre aux nouveaux besoins en santé de la population, appelle les professionnelles à se centrer sur la prévention, le maintien et la promotion de la santé dans toutes les situations de soins (OMS, 1986). Cette réorientation, développée dans la formation initiale, touche le développement d’un rôle présenté dans les textes de lois et professionnels comme « autonome » et « indépendant » et pose problème quant à son intégration dans les pratiques de soins infirmiers. En effet, celles-ci, principalement déléguées par le corps médical et par l’institution, font apparaître l’injonction paradoxale au sein de laquelle les infirmières se situent : celle du développement d’une pratique autonome selon des objectifs donnés. A partir d’un échantillon (n=841) issu d’une enquête réalisée auprès de l’ensemble des infirmières d’un CHU en Suisse (n=1951), au travers d’une approche sociocognitive (BANDURA, 2003), la recherche des principaux facteurs favorisant le développement d’une « pratique de santé », identifiée comme la centration sur le malade et sa santé selon sa propre définition de la santé, amène à mettre en évidence une double dimension de l’agentivité: autonome et hétéronome. Ainsi, les résultats montrent que la valeur attribuée par l’infirmière à sa « pratique de santé » détermine son orientation au sein d’un contexte qui reste prioritairement dirigé vers une « pratique de soins », centrée sur la gestion de l’ensemble des soins auprès du patient selon la définition de l’institution. Dès lors, l’autonomie dans les pratiques infirmières se présente comme l’exercice de l’agentivité, soit la puissance personnelle d’agir de l’infirmière, selon sa propre orientation : santé ou soins. Elle se développe significativement chez les professionnelles ayant un haut niveau de formation, santé ou soins, par une approche centrée sur l’apprentissage tout au long de la vie. Cette thèse permet de tracer des perspectives relatives à la clarification des rôles et des missions des infirmières et de leurs développements, au sein des établissements hospitaliers universitaires en particulier. Elle pose la question de la construction d’un sentiment d’efficacité personnelle (SEP) qui ne correspondrait pas à l’attente prioritaire et explicite du contexte. Enfin, en distinguant l’agentivité de l’autonomie, elle contribue à la construction conceptuelle des questions liées à l’autoformation (CARRE, 2005). / The redefinition of the nursing profession, in effect in Europe since 1990 in order to meet the new health needs of the population, requires professionals to focus on the prevention, the maintenance and the promotion of health in all care situations (OMS, 1986). This reorientation, developed in initial training, concerns the development of a role depicted in the professional legal texts as "autonomous" and "independent", while raising the issue of its integration into nurses' care-giving practices. Indeed, these practices, which are mainly delegated by the medical corps and the institution, highlight the paradoxical injunction in which nurses find themselves: developing their autonomous practice in accordance with heteronomously-set objectives. Based on a sample (n=841) taken from a study which surveyed the nursing population of a Swiss university hospital (n=1951), via a social cognitive approach (BANDURA, 2003), the search for main factors that foster the development of "health practices", identified as a focus on the patient and the patient's health according to one's own definition of health, brings to the fore a double dimension of agency : autonomous and heteronomous. Thus, the results show that the value attributed by the nurse to her "health practices" determines their orientation within a context that remains first and foremost directed toward "treatment practices" centered on the management of patient care in all its forms as defined by the institution. Consequently, autonomy in nursing practices appears to be the exercise of agency or of the nurse's personal power to act, according to her own orientation, health or treatment. It develops significantly in highly-qualified professionals, according to an approach centered on lifelong learning. This thesis allows for a marking out of perspectives pertaining to the clarification of nurses' roles and missions as well as the development of these roles within the university hospital structure. It raises the question of the construction of a self-efficacy which would not correspond to the explicit, nor priority expectation of the context. Lastly, by distinguishing agency and autonomy, this thesis contributes to the conceptual construction of questions linked to self-directed learning (CARRE, 2005).