Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Optimization of trial wave functions for use in quantum Monte Carlo with application to LiH

Methods for both partial and full optimization of wavefunction parameters are explored, and these are applied to the LiH molecule. A partial optimization can be easily performed with little difficulty. But to perform a full optimization we must avoid a wrong minimum, and deal with linear-dependency, time step-dependency and ensemble-dependency problems. Five basis sets are examined. The optimized wavefunction with a 3-function set gives a variational energy of -7.998 + 0.005 a.u., which is comparable to that (-7.990 + 0.003) 1 of Reynold's unoptimized \fin ( a double-~ set of eight functions). The optimized wavefunction with a double~ plus 3dz2 set gives ari energy of -8.052 + 0.003 a.u., which is comparable with the fixed-node energy (-8.059 + 0.004)1 of the \fin. The optimized double-~ function itself gives an energy of -8.049 + 0.002 a.u. Each number above was obtained on a Bourrghs 7900 mainframe computer with 14 -15 hrs CPU time.

An analysis of the methyl rotation dynamics in the So (x' A) and T (a A) states of thioacetaldehyde from Pyrolysis jet spectra /

Jet-cooled, laser-induced phosphorescence excitation spectra (LIP) of thioacetaldehyde CH3CHS, CH3CDS, CD3CHS and CD3CDS have been observed over the region 15800 - 17300 cm"^ in a continuous pyrolysis jet. The vibronic band structure of the singlet-triplet n -* n* transition were attributed to the strong coupling of the methyl torsion and aldehydic hydrogen wagging modes . The vibronic peaks have been assigned in terms of two upper electronic state (T^) vibrations; the methyl torsion mode v^g, and the aldehydic hydrogen wagging mode v^^. The electronic origin O^a^ is unequivocally assigned as follows: CH3CHS (16294.9 cm"'' ), CH3CDS (16360.9 cm"'' ), CD3CHS (16299.7 cm"^ ), and CD3CDS (16367.2 cm"'' ). To obtain structural and dynamical information about the two electronic states, potential surfaces V(e,a) for the 6 (methyl torsion) and a (hydrogen wagging) motions were generated by ab initio quantum mechanical calculations with a 6-3 IG* basis in which the structural parameters were fully relaxed. The kinetic energy coefficients BQ(a,e) , B^(a,G) , and the cross coupling term B^(a,e) , were accurately represented as functions of the two active coordinates, a and 9. The calculations reveal that the molecule adopts an eclipsed conformation for the lower Sq electronic state (a=0°,e=0"') with a barrier height to internal rotation of 541.5 cm"^ which is to be compared to 549.8 cm"^ obtained from the microwave experiment. The conformation of the upper T^ electronic state was found to be staggered (a=24 . 68° ,e=-45. 66° ) . The saddle point in the path traced out by the aldehyde wagging motion was calculated to be 175 cm"^ above the equilibrium configuration. The corresponding maxima in the path taken by methyl torsion was found to be 322 cm'\ The small amplitude normal vibrational modes were also calculated to aid in the assignment of the spectra. Torsional-wagging energy manifolds for the two states were derived from the Hamiltonian H(a,e) which was solved variationally using an extended two dimensional Fourier expansion as a basis set. A torsionalinversion band spectrum was derived from the calculated energy levels and Franck-Condon factors, and was compared with the experimental supersonic-jet spectra. Most of the anomalies which were associated with the interpretation of the observed spectrum could be accounted for by the band profiles derived from ab initio SCF calculations. A model describing the jet spectra was derived by scaling the ab initio potential functions. The global least squares fitting generates a triplet state potential which has a minimum at (a=22.38° ,e=-41.08°) . The flatter potential in the scaled model yielded excellent agreement between the observed and calculated frequency intervals.

Individual differences in risk-taking and associated characteristics : a test of specificity in executive functions /

Multiple measures have been devised by clinicians and theorists from many different backgrounds for the purpose of assessing the influence of the frontal lobes on behaviour. Some utilize self-report measures to investigate behavioural characteristics such as risktaking, sensation seeking, impulsivity, and sensitivity to reward and punishment in an attempt to understand complex human decision making. Others rely more on neuroimaging and electrophysiological investigation involving experimental tasks thought to demonstrate executive functions in action, while other researchers prefer to study clinical populations with selective damage. Neuropsychological models of frontal lobe functioning have led to a greater appreciation of the dissociations among various aspects of prefrontal cortex function. This thesis involves (1) an examination of various psychometric and experimental indices of executive functions for coherence as one would predict on the basis of highly developed neurophysiological models of prefrontal function, particularly those aspects of executive function that involve predominantly cognitive abilities versus processes characterized by affect regulation; and (2) investigation of the relations between risk-taking, attentional abilties and their associated characteristics using a neurophysiological model of prefrontal functions addressed in (1). Late adolescence is a stage in which the prefrontal cortices undergo intensive structural and functional maturational changes; this period also involves increases in levels of risky and sensation driven behaviours, as well as a hypersensitivity to reward and a reduction in inhibition. Consequently, late adolescence spears to represent an ideal developmental period in which to examine these decision-making behaviours due to the maximum variability of behavioural characteristics of interest. Participants were 45 male undergraduate 18- to 19-year olds, who completed a battery of measures that included self-report, experimental and behavioural measures designed to assess particular aspects of prefrontal and executive functioning. As predicted, factor analysis supported the grouping of executive process by type (either primarily cognitive or affective), conforming to the orbitofrontal versus dorsolateral typology; risk-taking and associated characteristics were associated more with the orbitofrontal than the dorsolateral factor, whereas attentional and planning abilities tended to correlate more strongly with the dorsolateral factor. Results are discussed in light of future assessment, investigation and understanding of complex human decision-making and executive functions. Implications, applications and suggestions for future research are also proposed.

An analysis of the relationship between self-efficacy and performance in a continuous gymnastic routine

Research has shown a consistent correlation between efficacy and sport performance (Moritz, et aI., 2000). This relationship has been shown to be dynamic and reciprocal over seasons (e.g., Myers, Payment, et aI., 2004), within games (e.g., Butt, et aI., 2003), and across trials (e.g., Feltz, 1982). The purpose of the present study was to examine selfefficacy and performance simultaneously within one continuous routine. Forty-seven undergraduate students performed a gymnastic sequence while using an efficacy measure. Results indicated that the efficacy-performance relationship was not reciprocal; previous performance was a significant predictor of subsequent performance (p < .01; f3s ranged from .44 to .67). Results further revealed significant differences in efficacy beliefs between groups with high and low levels of performance [F (1,571) = 7.16,p < .01]. Findings suggest that high levels of performance within a continuous physical activity task result in higher performance scores and higher efficacy beliefs.

Prime Rational Functions and Integral Polynomials

Let f(x) be a complex rational function. In this work, we study conditions under which f(x) cannot be written as the composition of two rational functions which are not units under the operation of function composition. In this case, we say that f(x) is prime. We give sufficient conditions for complex rational functions to be prime in terms of their degrees and their critical values, and we derive some conditions for the case of complex polynomials. We consider also the divisibility of integral polynomials, and we present a generalization of a theorem of Nieto. We show that if f(x) and g(x) are integral polynomials such that the content of g divides the content of f and g(n) divides f(n) for an integer n whose absolute value is larger than a certain bound, then g(x) divides f(x) in Z[x]. In addition, given an integral polynomial f(x), we provide a method to determine if f is irreducible over Z, and if not, find one of its divisors in Z[x].

The Influence of Attentional Focus on the Self-Efficacy-Performance Relationship in a Continuous Running Task

The self-efficacy-performance relationship in continuous sport tasks has been shown to be significantly reciprocal yet unequal with stronger influences in the performance-to-self-efficacy pathway rather than self-efficacy-to-performance pathway (e.g., LaForge-MacKenzie & Sullivan, 2014b). Bandura (2012) suggested that sociocognitive variables may influence this relationship. Attention as a sociocognitve factor may bias the processing of performance and self-efficacy information (Bandura, 1982, 1997; Bandura & Jourden, 1991). As confidence and attention are important aspects of peak running performance (Brewer, Van Raalte, Linder, & VanRaalte, 1991), the primary purpose of the present study was to examine the self-efficacy-performance relationship under three conditions of attentional focus. The secondary purpose was to examine self-efficacy and performance as separate constructs under the same conditions of attention. Participants ran continuously for one kilometer in one of three randomly assigned attentional focus conditions: internal-focus (n = 51), external-focus (n = 50), and control (n = 49). Self-efficacy was assessed using a one-item measure every 200 meters. Path analyses examining the primary objective revealed significant self-efficacy-to-performance pathways in all conditions: external-focus (p < .05, βs ranging from -.17 to -.32), internal-focus (p < .05, βs ranging from -.26 to -.36), and control (p < .05, βs ranging from -.29 to -.42). Significant reciprocal relationships were absent in all conditions. ANOVAs examining the secondary objectives found significantly faster performance in the control condition at the start (F (2, 147) = 3.86, p < .05) and end of the task (F (2, 147) = 3.56, p < .05). Self-efficacy was significantly higher in the internal-focus condition at the end of the task (Self-Efficacy 4 (F (2, 147) = 3.21, p < .05) and Self-Efficacy 5 (F (2, 147) = 4.74, p < .05). In contrast to previous within-trial research (e.g., LaForge-MacKenzie & Sullivan, 2014b) self-efficacy-to-performance effects were more significant and robust than performance-to-self-efficacy effects. These results provided support for Bandura’s (2012) suggestion that sociocognitive factors may have the ability to alter the causal structure of the self-efficacy-performance relationship, proposing complexities in the self-efficacy-performance relationship (Sitzmann &Yeo, 2013). Results were discussed from both theoretical and applied perspectives.

(A) Photoregulation of DNA Functions by Cyclic Azobenzene-tethered Oligonucleotides (B) Site-specific Fluorescent Labeling of DNA using Inverse Electron Demand Diels-Alder Reaction between trans-Cyclooctene Derivatives and BODIPY-Tetrazine Adducts

(A) Most azobenzene-based photoswitches require UV light for photoisomerization, which limit their applications in biological systems due to possible photodamage. Cyclic azobenzene derivatives, on the other hand, can undergo cis-trans isomerization when exposed to visible light. A shortened synthetic scheme was developed for the preparation of a building block containing cyclic azobenzene and D-threoninol (cAB-Thr). trans-Cyclic azobenzene was found to thermally isomerize back to the cis-form in a temperature-dependent manner. cAB-Thr was transformed into the corresponding phosphoramidite and subsequently incorporated into oligonucleotides by solid phase synthesis. Melting temperature measurement suggested that incorporation of cis-cAB into oligonucleotides destabilizes DNA duplexes, these findings corroborate with circular dichroism measurement. Finally, Fluorescent Energy Resonance Transfer experiments indicated that trans-cAB can be accommodated in DNA duplexes. (B) Inverse Electron Demand Diels-Alder reactions (IEDDA) between trans-olefins and tetrazines provide a powerful alternative to existing ligation chemistries due to its fast reaction rate, bioorthogonality and mutual orthogonality with other click reactions. In this project, an attempt was pursued to synthesize trans-cyclooctene building blocks for oligonucleotide labeling by reacting with BODIPY-tetrazine. Rel-(1R-4E-pR)-cyclooct-4-enol and rel-(1R,8S,9S,4E)-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were synthesized and then transformed into the corresponding propargyl ether. Subsequent Sonogashira reactions between these propargylated compounds with DMT-protected 5-iododeoxyuridine failed to give the desired products. Finally a methodology was pursued for the synthesis of BODIPY-tetrazine conjugates that will be used in future IEDDA reactions with trans-cyclooctene modified oligonucleotides.

Rationalizability of Choice Functions on General Domains without Full Transitivity

The rationalizability of a choice function by means of a transitive relation has been analyzed thoroughly in the literature. However, not much seems to be known when transitivity is weakened to quasi-transitivity or acyclicity. We describe the logical relationships between the different notions of rationalizability involving, for example, the transitivity, quasi-transitivity, or acyclicity of the rationalizing relation. Furthermore, we discuss sufficient conditions and necessary conditions for rational choice on arbitrary domains. Transitive, quasi-transitive, and acyclical rationalizability are fully characterized for domains that contain all singletons and all two-element subsets of the universal set.

A Representation Theorem for Domains with Discrete and Continuous Variables.

This paper proves a new representation theorem for domains with both discrete and continuous variables. The result generalizes Debreu's well-known representation theorem on connected domains. A strengthening of the standard continuity axiom is used in order to guarantee the existence of a representation. A generalization of the main theorem and an application of the more general result are also presented.

Functional Forms and Educational Production Functions

Different Functional Forms Are Proposed and Applied in the Context of Educational Production Functions. Three Different Specifications - the Linerar, Logit and Inverse Power Transformation (Ipt) - Are Used to Explain First Grade Students' Results to a Mathematics Achievement Test. with Ipt Identified As the Best Functional Form to Explain the Data, the Assumption of Differential Impact of Explanatory Variables on Achievement Following the Status of the Student As a Low Or High Achiever Is Retained. Policy Implications of Such Result in Terms of School Interventions Are Discussed in the Paper.

Efficient Strategy-Proof Allocation Functions in Linear Production Economies

In a linear production model, we characterize the class of efficient and strategy-proof allocation functions, and the class of efficient and coalition strategy-proof allocation functions. In the former class, requiring equal treatment of equals allows us to identify a unique allocation function. This function is also the unique member of the latter class which satisfies uniform treatment of uniforms.