<p>The concept of light-activated "caged" metal ions was first introduced for Ca2+. These high affinity coordination complexes are activated by UV light to release calcium ions intracellularly and have found widespread use in understanding the many roles of calcium in biological processes. There is an unmet need for photocaging ligands for biologically relevant transition metal ions. Described here are the first examples of uncaging biologically important d-block metal ions using photoactive ligands. </p> <p>New nitrogen-donor ligands that contain a photoactive nitrophenyl group within the backbone have been prepared and evaluated for their metal binding affinity. Exposure of buffered aqueous solutions of apo-cage or metal-bound cage to UV light induces cleavage of the ligand backbone reducing the denticity of the ligands. Characterization of several caging compounds reveals that quantum efficiency and metal binding affinity can be tuned by modifications to the parent structure. The change in reactivity of caged vs. uncaged metal for promoting hydroxyl radical formation was demonstrated using the in vitro deoxyribose assay. The function of several of these compounds in vivo pre- and post-photolysis has been validated using MCF-7 cells. This strategy of caging transition metals ions is promising for applications where light can trigger the release of metal ions intracellularly to study metal trafficking and distribution, as well as, selectively impose oxidative stress and/or metal toxicity on malignant cells causing their demise.</p> / Dissertation

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Syftet med denna uppsats är att undersöka hur tre olika gitarrlärare upplever att gitarrens asymmetri- samt matrisegenskap kan påverka elevers förståelse för greppbrädan och musikteori. Syftet är också att undersöka hur lärarna relaterar musikteori till greppbrädan, hur de använder sig av olika mönster och visualiseringstekniker - så som CAGED-systemet, ett system som är baserat på de öppna ackorden C, A, G, E och D - och vilka verktyg de använder för att visa hur teoretiska kunskaper kan omsättas i praktiken. I den tidigare forskningen och gitarr-metodböckerna, som denna uppsats presenterar, avslöjas motsättningar om hur gitarrister bör visualisera mönster på greppbrädan, vad konsekvenserna är när tillförlitligheten till det motoriska minnet är för stort, om CAGED-systemet är bra eller dåligt för att visualisera ackord samt skalor över greppbrädan, hur mönster som t.ex. ackorddiagram ska användas etc. Gitarrlärarnas åsikter om uppsatsens ämne är erhållna via semi - strukturerade intervjuer. Lärarna har undervisat i elgitarr- samt akustisk gitarr på olika lärosäten som t.ex. kommunala musikskolan, studieförbund, gymnasium, högskola etc. Genom intervjuerna avslöjas det att ingen av de tre lärarna begrundar särskilt mycket över asymmetri- samt matrisegenskapen och hur egenskaperna möjligtvis kan påverka elevers förståelse för greppbrädan. Lärarnas musikteoriundervisning tar heller inte upp en stor del av deras lektionstimmar. Däremot bekräftar två av gitarrlärarna, samt den tidigare forskningen, att brukandet av mönster som t.ex. ackorddiagram, grepptabeller, CAGED-systemet m.m. uppmanar gitarrister att förlita sig, i alltför hög grad, på sitt motoriska minne. Detta kan undergräva gitarristers förståelse för greppbrädan. Gitarrlärare bör vara medvetna om detta problem för att deras elever skall kunna utvecklas som gitarrister och musiker. Kritik kan riktas mot några av de olika litterära texter som har använts i denna uppsats eftersom de inte har genomgått någon referentgranskning. Lärarnas expertis angående uppsatsens frågor kan kritiseras och varför lärarna valdes för denna studie kan därför ifrågasättas. Dock, när det gäller valet av informanterna, så finns det – som i all kvalitativ forskning – en fråga kring hur generella uppsatsens slutsatser är och därför, i framtiden kanske slumpmässiga val av informanter är nödvändiga. / The purpose of this essay is to investigate how three different guitar teachers think the asymmetry and matrix property of the guitar may affect students' understanding of the fretboard and ability to analyse music. The purpose is also to investigate how the teachers relate music theory to the fretboard, if they teach their students different visualisation techniques and patterns - like the CAGED system, a system which is based on the major chords C, A, G, E and D in the open position - and which tools they use to demonstrate how theoretical knowledge can be used in practice. In the previous studies and guitar method books, which this essay introduces, contradictions are revealed about how guitarists should learn, visualise, and play the instrument; what the consequences are when the dependence of the motor memory is too excessive; if the CAGED-system is good or bad for visualising chords and scales; how patterns like chord diagrams should be used etc. The guitar teachers' opinions about the issues, that this essay addresses, are obtained through semi - structured interviews. The teachers have taught guitar in different types of schools, such as municipal music school, adult school, high school, college etc. Through the interviews it is revealed that none of the three teachers contemplate very much on the asymmetry and matrix property, how the properties may affect students’ understanding of the fretboard, and the teachers’ music theory education doesn't take up a large part of their teaching hours. However, two of the guitar teachers, and the previous studies, confirm that the use of patterns exhorts guitarists to rely, to an excessive degree, on their motor memory. This may undermine guitar students’ understanding of the fretboard. Guitar teachers should be aware of this problem for their students to develop as guitarists and musicians. Criticism can be directed towards some of the various literary writings that have been used in the essay, because they have not been peer reviewed. The teachers’ expertness on this essay’s topic can be criticised and therefore, why the teachers were chosen for this study may also be questioned. However, with respect to the selection of the informants, there is – as in all qualitative research – a question of how general the essay’s conclusions are and in future research maybe a method of randomness in the selection of the informants should be used.

Two-photon absorption is the process in which a molecule absorbs two photons simultaneously. The two key advantages of two-photon processes over one-photon processes are the possibility of excitation of materials with high three-dimensional spatial resolution and deep light-penetration into absorbing materials. Based on bond-cleavage reactions activated by photon-induced intramolecular electron transfer, two-photon activatable acid and radical initiators and two-photon removable protecting groups have been successfully designed and synthesized for photopolymerization and three-dimensional microfabrication and for biomedical photo-triggers. The optical and chemical properties of synthesized molecules, such as quantum yield of acid generation, initiation efficiency of photopolymerization, and photolysis efficiency, have been studied by using a variety of physical and analytical techniques under one-photon conditions. The two-photon characteristics and applications of these molecules are being investigated in collaboration with other groups.

Judicious addition of photolabile caging groups (PCGs) to protect biologically im- portant molecules, has enabled the development of many powerful chemical tools for the study of biological processes. These tools have the potential to be activated in a cellular setting by irradiation with light of appropriate wavelengths, restoring functionality, with excellent spatial and temporal control. This D. Phil. dissertation highlights two biologically relevant examples where PCGs can be applied: (i) 4,5-dimethoxynitrobenzyl (DMNB) caged derivatives of GSH and ESG, were synthesised and uncaging of the DMNB group at 350 nm to reveal the free α-carboxylic acid of the glycine residue was demonstrated. These molecules have the potential to probe the binding mode within the KefC KTN binding domain, a ligand-gated K+ efflux system, critical for bacterial response to electrophilic assault. The molecules are currently with collaborators awaiting further evaluation. (ii) The wavelength-dependent application of PCGs toward the study of protein post- translational modifications (PTMs) was developed. PTMs modulate protein function and have a ubiquitous role in a diverse range of cellular functions. A wavelength selective sequential pair of PCGs was developed and demonstrated in a tripeptide, using the diethylamino coumarin (DEACM), which was cleaved at 420 nm, and the DMNB caging group, which was subsequently cleaved at 350 nm. A chromatically orthogonal pair of PCGs, with possible applications in both organic synthesis and biological systems, incorporating the BODIPY-based and DEACM caging groups, which could be cleaved at 530 nm and 420 nm, respectively, was also developed. Photolysis was conducted on a tripeptide and hexapeptide and an in vitro application was demonstrated where when caged, the peptide was not susceptible to peptidolysis when incubated with enzymes. Following irradiation at 420 nm, the DEACM group was uncaged and the peptide underwent peptidolysis in the presence of Endoproteinase AspN, whereas following irradiation at 530 nm to uncage the BODIPY group, the peptide underwent peptidolysis in the presence of Endoproteinase LysC. These results not only provide conclusive evidence of the ability of this pair of caging groups to act in a wavelength orthogonal sense but also demonstrates the potential applicability of the pair to biochemical studies. This pair of PCGs represents the first example of an orthogonal pair of caging groups where both PCGs could be cleaved, at wavelengths longer than 400 nm, in a sequence independent manner.

When provided with the opportunity to select their diet, most insect herbivores regulate their nutrient intake. However, in a nutritionally heterogeneous environment and with changing demands for growth, development and reproduction, obtaining the required amount and balance of nutrients is a challenge. This is especially true for social insects where the workers bring food into the colony to be shared by nestmates. The ability of insects to self-select their diet is an important trait related to fitness. In this study we investigated whether and how caged worker honeybees meet their nutritional requirements in response to the nutritional composition of the food they find. Using the ‘geometric framework’ we looked at the behavioural and physiological mechanisms used by caged worker honeybees in balancing their diet when provided with different pairs of complementary imbalanced foods. First, we investigated whether caged worker honeybees maintain their intake target by providing them with pairs of complementary imbalanced foods with varying protein to carbohydrate (P:C) ratios. Diets were formulated using different protein sources: casein, royal jelly and Feed-Bee®. Honeybees self-selected or balanced their diet by switching between the complementary foods in accordance with the composition of the food and the type of protein that they encountered. Honeybees selected average P:C ratios of 1:12, 1:14 and 1:11 on casein, royal jelly and Feed-Bee® diets respectively. The level of self-selection was confirmed using two performance measures: survival and ovarian activation. Both survival and ovarian activation differed depending on the type of protein source used. Second, we investigated if honeybees regulated their growth target, which is the amount of nutrients incorporated into growth and storage tissue, by measuring physiological parameters in honeybees confined on imbalanced complementary food combinations having different P:C ratios. Feed-Bee® was used as a protein source. The physiological parameters measured were head fresh mass, hypopharyngeal gland (HPG) development, and protein concentration in the haemolymph. The bees fed on different diet combinations with different P:C ratios maintained each of the performance measures to the same level, which supports the ability of worker honeybees to self-select their diet. The measured physiological parameters were compared with other studies to asses the appropriateness Feed-Bee® diet as a protein source for the bees. In the absence of brood the intake target is directly related to the physiological requirements of the worker bees. The behavior of these individual adult bees gives an insight in to the complex system; similar responses may be seen in nurse bees in the colony condition to obtain protein, carbohydrate and other nutrient requirements from stored pollen and nectar in the hive, either for their own nutritional requirements or for other colony members, especially larvae. / Dissertation (MScAgric)--University of Pretoria, 2010. / Zoology and Entomology / unrestricted

Mechanistische und kinetische Untersuchungen von komplexen zellulären Prozessen in situ sind in den vergangenen Jahren durch den Einsatz photoaktivierbarer Biomoleküle, sogenannter caged Verbindungen, möglich geworden. Bei den caged Verbindungen handelt es sich um photolabile inaktive Derivate von biologisch aktiven Molekülen, aus denen durch ultraviolettes Licht mit Hilfe einer photochemischen Reaktion die natürliche, biologisch aktive Substanz schnell freigesetzt werden kann. Im Rahmen der vorliegenden Arbeit wurden caged Verbindungen von den Neurotransmittern Octopamin und Dopamin, dem Octopamin-Antagonist Epinastin, den Proteinsyntheseinhibitoren Emetin und Anisomycin, dem Protonophor CCCP und dem Riechstoff Bourgeonal hergestellt. Zur Synthese dieser caged Verbindungen wurden sowohl bekannte als auch verschiedene im Rahmen dieser Arbeit neu entwickelte photolabile Schutzgruppen mit einem (Cumarin-4-yl)methyl- bzw. einem 2-Nitrobenzyl-Gerüst eingesetzt. Entsprechende Syntheseverfahren wurden erarbeitet. Anschließend erfolgte eine umfassende physikalisch-chemische sowie photochemische Charakterisierung der erhaltenen caged Verbindungen. Dabei wurde besonders auf gute Löslichkeit in Wasser bei physiologischer Ionenstärke, schnelle und effiziente Photoreaktivität, hohe Extinktion bei Wellenlängen von 350-430 nm und gute solvolytische Stabilität bei geringer Toxizität der freigesetzten Schutzgruppe geachtet. Ein Schwerpunkt bei der photochemischen Charakterisierung bildeten die Untersuchungen zur Quantifizierung der 2-Photonen-Anregung, uncaging action cross-sections, der Cumarinylmethyl-caged Verbindungen, aufgrund ihrer Bedeutung für die Photofreisetzung von Biomolekülen, da die gleichzeitige Absorption von 2 IR-Photonen eine höhere dreidimensionale Auflösung und eine wesentlich tiefere Gewebepenetration erlaubt. Mit Hilfe von Kooperationspartnern wurden zeitaufgelösten Fluoreszenz- und IR-Messungen an verschiedenen (Cumarin-4-yl)methoxycarbonyl-caged Modellverbindungen durchgeführt, mit denen die Geschwindigkeitskonstanten k1 und kdecarb des Photolysemechanismus ermittelt wurde. Am Ende folgten die Anwendungserprobungen ausgewählter caged Verbindungen in einem Translationsassay bzw. in Zelluntersuchungen. / In the last years mechanistic and kinetic in situ studies of complex cellular processes become possible by employing photoactivate Biomolecules, also called caged compounds, as a tool for these studies. Caged compounds are photolabile inactive derivates of biologic active molecules which are fast laid off the nature biologic active molecule by a photochemical reaction which was triggered by UV-light. In the present dissertation caged compounds of the neurotransmitters octopamine and dopamine, of the octopamine antagonist epinastine, of the proteine synthesis inhibitors emetine and anisomycine, of the ionophore CCCP and of the odorus substance Bourgeonal are synthesized. As precursors for the synthesis of that caged compounds some reported and several in these work newly developed photolabile protecting groups with (coumarin-4-yl)methyl- or 2-nitrobenzyl-scaffold were used. Corresponding Synthesis were designed. Afterwards the received caged compounds were global physical-chemical and photochemical characterised. In favour it was specifically valued for highly water solubility at pH 7,2, fast and efficient photo reactivity, high extinctions at wavelength 350-430 nm, well solvolytic stability and less toxicity of the redundant protecting groups. One key aspect of photochemical characterisation were the studies of uncaging action cross-sections of the coumarinylmethyl-caged molecules, because of their relevance for the photorelease of biomolecules in tissues. The simultaneous absorption of 2 IR-photons allowed highly three-dimensional release and a essentially deeper penetration in tissues. With the aid of co-operation partners were time-released fluorescence- and IR- measurements with several (coumarin-4-yl)methoxycarbonyl-caged molecules realised and therefore the rate constant k1 und kdecarb of the photolyse mechanismus were determined. At the end of the dissertation the achieved caged compounds were testet in translation assays and several cell cultures.

The purpose of this study was to investigate how a small selection of guitar teachers go about to teach chords on electric guitar, as well as how a small selection of guitar students have experienced the phenomena during their studies. To answer the research questions, two experienced guitar teacher and two guitar students were subjects to qualitative interviews. The theoretical perspective of the study was hermeneutic. The results showed that the methods employed by the teachers to a high degree related to how they have been taught and also learned by themselves. Another result is that the combination of systems, music theory and practical musical performance can help guitar students to reach a point of musical independence. The results provide an insight in how chordal playing on electric guitar can take place on a higher level, and also opens up for research within several areas of guitar education. / Syftet med denna studie var att undersöka hur ett mindre urval av gitarrlärare lär ut ackord på elgitarr, samt hur ett mindre urval gitarrelever upplevt att de undervisats i ackordspel. För att besvara forskningsfrågorna genomfördes kvalitativa intervjuer med två erfarna gitarrlärare, samt två gitarrelever som studerat elgitarr på musikgymnasium och folkhögskola. Studiens teoretiska perspektiv var hermeneutiskt. Resultaten visar att lärarna till stor del undervisar med metoder som fungerat för dem själva, samt att en kombination av system, musikteori och framförallt praktiskt musicerande kan ge gitarrelever de grundkunskaper som behövs för att vara självgående som elgitarrist. Resultat ger en inblick i hur ackordundervisning kan ta form på högre nivå, och öppnar upp för flera områden av framtida forskning i elgitarrmetodik.

Caged compounds are biologically active molecules that are rendered inert by masking an important functionality with a photolabile protecting, ‘caging’, group. The caging group can be removed by irradiation with light to reveal the active compound with restored pharmacological activity with high spatial and temporal control. This technology provides an ideal tool for the study of many chemical, physiological and biological systems. This DPhil dissertation highlights several projects in which caging technology has been employed to address biological problems and questions. The first example of spatially controlled mitochondrial inactivation is reported - a tool for the study of the role of mitochondria in Ca2+ signalling. Caged TRPV1 agonists and antagonists have been developed to probe TRPV1, specifically the location of the agonist-binding site. T cell activation has been controlled with light as a tool to gain insight into the adaptive immune response. Caged sodium channel blockers have been investigated. Wavelength-orthogonal photolysis in a neuronal system has been demonstrated using the neurotransmitters glutamate and GABA - this represents a significant advancement in caging technology. This dissertation also includes investigations into the development of novel caging groups.