Gatekeeping processes: grounded theory, young people and physical activity

This thesis has two purposes: firstly, to develop grounded theory methodology and secondly, to apply it in order to establish and further investigate those processes which structure young peoples' participation in physical activity. To satisfy the first of these aims, the Helix Model was created to provide a systematic framework to the grounded theory analysis. This Model was then employed to address the second aim, as it was used to analyse interviews conducted with a mixed sex sample of twenty nine very active and very inactive children and their parents. These young people were selected as a result of completing, on four occasions, a 24 hour self-report questionnaire specifically designed for them. The grounded theory analysis identified a series of interrelated 'gatekeeping processes' which construct those opportunities for young people to participate in physical activity. Several evolving processes, varying according to the context and nature of the physical activity, interrelate with one another to create a complex causal web. The gatekeeping processes are consciously, as well as unconsciously, manipulated relative to the social and physical context in which the young person and the other gatekeeping agents (parents, school, peers) exist and find themselves. The interrelationships between these agents, especially the young person and their parents, work through compromise and coercion to satisfy each of their personal agendas. The nature of each agenda is based on the definition associated with the three roles which gatekeepers adopt (guardian, facilitator, enforcer). The definition of each role affects the manner in which young people individually, as well as collectively with the gatekeepers, construct networks to accomplish an evolving combination of: independence, maximisation of the available resources, rewards, and care and control. The interrelationship between these factors and the extent to which participation in physical activity can achieve them, is what determines the likelihood of the young person's participation in that activity. However, physical activity has to compete with a myriad of the other activities the young person is involved in. These are activities, which for the more sedentary young person, are perceived to be more successful at providing the desired rewards

An intervention to facilitate 'high quality' physical education - from gymnastics to athletics

The summer edition of Research Matters included a short article on facilitating ‘high quality’ physical education and ‘high quality’ gymnastics in a city school (see Cale et al., 2011). The article provided a summary of the first phase of a research project which aimed to: i) facilitate high quality teaching and learning in physical education, and ii) identify key principles that contribute to high quality outcomes and which could be applied across the physical education curriculum. This article follows on and presents a summary of phase 2 of the research, which focused on athletics, as well as of the key findings and recommendations from the project overall

Development of a Micellar-Promoted Heck Reaction for the Synthesis of DNA-Encoded Libraries

The capability of DNA encoded libraries (DELs) as a method of small molecule hit identification is becoming widely established in drug discovery. While their selection method offers advantages over more traditional means, DELs are limited by the chemistry that can be utilized to construct them. Significant advances in DNA compatible chemistry have been made over the past five years; however such procedures are still often burdened by substrate specificity and/or incomplete conversions, reducing the fidelity of the resulting libraries. One such reaction is the Heck coupling, for which current DNA-compatible protocols are somewhat unreliable. Utilizing micellar technology, we have developed a highly efficient DNA-compatible Heck reaction that proceeds on average to 95% conversion to product across a broad variety of structurally significant building blocks and multiple DNA conjugates. This work continues the application of micellar catalysis to the development of widely applicable, effective DNA-compatible reactions for use in DELs

Micellar Buchwald–Hartwig Coupling of Aryl and Heteroarylamines for the Synthesis of DNA-Encoded Libraries

DNA-encoded libraries are a very efficient means of identifying ligands for protein targets in high throughput. To fully maximize their use, it is essential to be able to carry out efficient reactions on DNA-conjugated substrates. Arylamines are privileged motifs in druglike molecules, and methods for their incorporation into DNA-encoded libraries are highly desirable. One of the preferred methods for their preparation, the Buchwald–Hartwig coupling, does not perform well on DNA conjugates using current approaches. We report the application of our recently developed micellar technology for on-DNA chemistry to the Buchwald–Hartwig reaction. Optimization of conditions led to a robust, high-yielding method for the synthesis of DNA-conjugated aryl and heteroarylamines, which is broad in substrate scope for both the arylamine and the DNA-conjugated aryl halide and is fully compatible with DNA-encoding and decoding procedures. This method will enable the preparation of diverse, high-fidelity libraries of biarylamines

Development of a Micellar-Promoted Heck Reaction for the Synthesis of DNA-Encoded Libraries

The capability of DNA encoded libraries (DELs) as a method of small molecule hit identification is becoming widely established in drug discovery. While their selection method offers advantages over more traditional means, DELs are limited by the chemistry that can be utilized to construct them. Significant advances in DNA compatible chemistry have been made over the past five years; however such procedures are still often burdened by substrate specificity and/or incomplete conversions, reducing the fidelity of the resulting libraries. One such reaction is the Heck coupling, for which current DNA-compatible protocols are somewhat unreliable. Utilizing micellar technology, we have developed a highly efficient DNA-compatible Heck reaction that proceeds on average to 95% conversion to product across a broad variety of structurally significant building blocks and multiple DNA conjugates. This work continues the application of micellar catalysis to the development of widely applicable, effective DNA-compatible reactions for use in DELs

Binding of Daunomycin to Diaminopurine- and/or Inosine-Substituted DNA^†^,^‡

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5‘(A/T)GC and 5‘(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5‘IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5‘(A/T)GC and 5‘(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms

Mitomycin Dimers: Polyfunctional Cross-Linkers of DNA

The three dimers 3, 4, and 5 of mitomycin C (MC), a natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with −(CH2)4−, −(CH2)12−, and −(CH2)3N(CH3)(CH2)3− tethers, respectively. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied in direct comparison with MC, which is itself an ICL agent. Evidence is presented that 3−5 multifunctionally alkylate and cross-link extracellular DNA and form DNA ICLs more efficiently than MC. Reductive activation, required for these activities, is catalyzed by the same reductases and chemical reductants that activate MC. Dimer 5, but not MC, cross-linked DNA under activation by low pH also. Sequence specificities of cross-linking of a 162-bp DNA fragment (tyrT DNA) by MC, 3, and 5 were determined using DPAGE. The dimers and MC cross-linked DNA with the same apparent CpG sequence specificity, but 5 exhibited much greater cross-linking efficacy than MC. Greatly enhanced regioselectivity of cross-linking to G·C rich regions by 5 relative to MC was observed, for which a mechanism unique to dimeric MCs is proposed. Covalent dG adducts of 5 with DNA were isolated and characterized by their UV and mass spectra. Tri- and tetrafunctional DNA adducts of 5 were detected. Although the dimers were generally less cytotoxic than MC, dimer 5 was highly and uniformly cytotoxic to all 60 human tumor cell cultures of the NCI screen. Its cytotoxicity to EMT6 tumor cells was enhanced under hypoxic conditions. These findings together verify the expected features of the MC dimers and warrant further study of the biological effects of dimer 5

Sequential Double α-Arylation of <i>N</i>-Allylureas by Asymmetric Deprotonation and N→C Aryl Migration

On lithiation with lithium amides, N-allyl-N′-aryl ureas undergo rearrangement with transfer of the aryl ring from N to the allylic α carbon. From the α-arylated products, a further aryl transfer under the influence of a chiral lithium amide allows the enantioselective construction of 1,1-diarylallylamine derivatives. Stereoselectivity in these reactions results from the enantioselective formation of a planar chiral allyllithium under kinetic control

DNA Sequence Recognition by the Antitumor Drug Ditercalinium^†

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4−6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing nonconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N7-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium−DNA complexes whereas the major groove-oriented N7 of guanine does appear as a key element in the molecular recognition process. The loss of N7 at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N7 of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N7 with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N7 of guanines is not sufficient to explain the GC-selective drug−DNA association, and the implications of these findings are considered