Dissertations / Theses on the topic 'Dimers'

Controlling both energy and electron transfer processes is necessary for the performance of both light harvesting, and light emitting devices. The efficiency of both energy and electron transfer depends on the degree, or magnitude of the electronic coupling between molecules. In photosynthetic organisms such as plants and bacteria, long range energy transfer occurs between electronically coupled assemblies of chlorophyll molecules. The remarkable energy transfer efficiencies of light harvesting complexes employed in photosynthesis is intimately related to the orientation and intermolecular spacing between neighboring, identical chlorophyll molecules. In an attempt to develop inexpensive artificial light harvesting systems and devices researchers have synthesized an impressive catalogue of organic small molecules and polymers to generate free electrons and holes from absorbed light energy. The most commonly employed design principle for synthesizing both small molecule and polymer light absorbing systems is to link together an electron deficient (acceptor) molecule to an electron rich (donor) molecule through a conjugated linker. The conjugated bridge between donor and acceptor units enables charge transfer states that offer band-gap tuning and more easily separated electron/hole pairs. However, exciton migration in these synthetic donor-acceptor systems only partially mimics the assemblies of light harvesting molecules found in nature. As a result, exciton migration is limited to very short distances in synthetic light harvesting organic molecules. The following thesis presents a new approach for controlling the electronic coupling between neighboring identical molecules. By bridging two identical molecules about a sulfur atom, the electronic coupling between the two molecules can be increased by increasing the oxidation state of the “sulfur-bridge”. The broader implication of this work is that we may not need to use strongly coupled, conjugated donor-acceptor systems to achieve efficient charge separation and excitation migration. Instead, by using a non-conjugated linker, such as sulfur or perhaps even a saturated carbon atom, between molecules with identical electronic structure we may be able to enhance exciton transport in synthetic light harvesting assemblies.
Science, Faculty of
Chemistry, Department of
Graduate

We have obtained spectra of 10 vibrational bands of the Cs₂ (2)³∏_u ← ϰ³∑⁺₉ system. The molecules were formed in a supersonic free jet expansion, and were excited by light from a single mode CW dye laser. The total laser induced fluorescence was measured at 90° to the incident light and molecular beam, using a photomultiplier. Using a slit system to image a selected part of the interaction region, we have reduced the Doppler width to about 350MHz. We have been able to resolve the discrepancy between the different vibrational band positions given in two previous papers. Our vibrational bands show broad rotational contours, but we have not been able to resolve individual rotational lines. We have also obtained rotationally resolved spectra of the bandhead region of 22 vibrational bands of the Β¹∏₉ ← X¹∑⁺_u system. We found that the frequencies of the bandheads agreed with the bandhead positions deduced from the Dunham coefficients of a previous work. We have developed a theoretical model of the rotational structure and intensity distribution, taking into account optical pumping and the small solid angle subtended by the detector. By fitting this model to the experimental spectrum of the v' = 3,u∿ = 0 band using least squares optimization, we were able to extract rotational constants and line positions. We found that these line positions were in good agreement with those from the previous work. We have discussed how such spectroscopic data may be used in a determination of the s-wave scattering length of caesium, and we have reviewed the validity of the scattering length and other pararneterisations of low energy Cs-Cs interactions.

Thesis (M.S. in chemistry)--Washington State University, August 2009.
Title from PDF title page (viewed on Aug. 10, 2009). "Department of Chemistry." Includes bibliographical references (p. 53-55).

Thesis (Ph. D.)--University of Adelaide, Dept. of Physics and Mathematical Physics, 1997?
Includes copies of previously published articles. Includes bibliographical references (last 7 unnumbered leaves ).

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Sherrill, C. David; Committee Member: Bredas, Jean-Luc; Committee Member: Hud, Nicholas; Committee Member: Perry, Joseph.

This thesis is devoted to the study of orientifolds and dynamical supersymmetry breaking in configurations of D-branes on toric Calabi-Yau singularities, through the lens of dimer models. We first review the basic ingredients of string theory that led to the formulation of gauge/gravity dualities in terms of dimers. Then, we discuss the non-abelian anomaly cancellation conditions for the supersymmetric gauge theories arising on D-branes and provide necessary geometric criteria to determine whether an orientifold projection can be safely introduced. We also find a new realization of orientifold projection without fixed loci in dimer models and expand on its physical features. We argue that it exhausts the possibilities of orientifolding dimer models. In the subsequent part of the thesis, we investigate dynamical supersymmetry breaking vacua in the same class of models and their typical instability along N=2 Coulomb branches. This leads us to formulate a no-go theorem against their stability based on geometrical features of the singularity, and then to establish a precise way to circumvent it. We eventually find the first instance of stable dynamical supersymmetry breaking vacuum in string theory from D-branes on a toric Calabi-Yau singularity, the Octagon.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Summary My PhD thesis is composed of two parts. A part deals with the characterization of alpha-synuclein (aS) dimers aggregation properties in respect to those of aS. The experimental work was conducted at CRIBI laboratory, at University of Padua, and constitutes the main project in which I was involved. During the third year of my PhD I spent six months at the Biopolymer Mass Spectrometry Laboratory of Imperial College in London. I conducted a glycomic analysis of mice tissues and a pilot study on expression and biosynthesis of mixed linked glucans emicellulose. Parkinson’s disease (PD) is a progressive, neurodegenerative disorder characterized by the loss of dopaminergic neurons in substantia nigra. The histological hallmarks of PD are intracellular inclusions, known as Lewy bodies (LBs), composed by filamentous and aggregated protein. The pathogenesis of the disease is still unclear, but a key step in the onset of PD is the aggregation of aS into amyloid fibrils, that deposit within LBs as the major component. Despite its importance in neurodegeneration, little is known about aS function, native physiological state and mechanism of aggregation. aS was recently described as a folded tetramer, but was generally considered a natively unfolded protein. aS is able to acquire alpha-helix conformation upon interaction with lipids and to convert to beta-structure in pathological processes. During the aggregation process, aS forms soluble oligomers, transient beta-structured intermediate between the physiological form of aS and amyloid fibrils. Dimerization of aS could represent a critical, rate-limiting step in the aggregation and amyloid formation of the protein. Therefore, we decided to study the aggregation of several different dimers of aS, produced through molecular biology techniques. A cysteine residue has been added at the N-terminal or at the C-terminal of aS, therefore producing a dimer N-N or C-C linked through a disulfide bond. A N-C dimer, formed two consecutive aS molecules, was obtained as a single polypeptide chain. During the project, another dimer, called DC dimer, was produced in order to further draw up the hydrophobic regions, and avoid the interferences of side chains within the molecule. DC is constituted by two consecutive central, highly amyloidogenic regions, containing aS residues from 1 to104 joined to residues from 29 to 140. The dimers represent a suitable tool for the study of intramolecular aS interaction pathway. Some remarkable differences define and limit the mobility freedom of the dimers respect to aS, hypothetically differentiating the fibrillation process of the four protein structures. The characterization of the dimers was performed using chemical and biophysical techniques in order to define their behaviour in solution as monomer. CD, IR and NMR spectroscopy studies show that all the dimers are unfolded. They undergo alpha-helical transition upon interaction with the detergent SDS. These results evidence that dimers strongly resemble aS conformational features. All the dimers were tested for the ability to form fibrils, by incubating the molecules under physiological buffer and at a protein concentration of 1 mg/ml. They show to be able to form fibrils, that are positive to Thioflavin T binding assay. Moreover, analysis of the structure of fibrils, conducted using circular dichroism (CD) and Fourier Transformed-IR (FTIR) spectroscopy, detects the structural transition from random to beta-sheet structure as attended for typical amyloid structure. Fibrils morphology was investigated by transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging. Fibrils derived from aS dimers are quite long, unbranched and formed by a single filament, a peculiar difference with aS fibril morphology. To identify which amino acids in the respective types of fibrils belong to the fibril core, proteolysis was performed. The rationale of this experiment reside in the fact that disordered regions of proteins are generally site of enzymatic attack and hydrolysis occurs at flexible chain region devoid of hydrogen-bonded secondary structure. Therefore, the prospects are to remove the flexible parts or tail from the amyloid core. Results showed that the core structures of the fibrils of the different molecules seems to be constituted by the same amino acidic region, which encompasses the segment 35-96, in analogy with previous studies. The kinetic of the process was analyzed by fluorescence techniques (ThT binding assay) and by evaluating the amount of protein present in fibrils on time. This calculation was indirectly performed measuring the absorbance of the supernatant obtained after centrifugation of each aliquot. NN and NC dimers show a slower kinetic of fibrillation than aS, while the rate of fibril formation of CC and DC dimers is faster than aS. Moreover, aggregation experiments on mixtures of aS in the presence of small amount of dimers were also conducted in order to check if the presence of dimer influence aS kinetic. Results evidenced the ability of CC dimer to affect the aggregation of aS. On the base of collected results, models of the dimer conformation within the fibrils are proposed. The research experience performed at Imperial College London gave me the possibility to learn and apply advanced techniques in mass spectrometry analysis of small organic compound, using GC-MS and MALDI-TOF spectrometers. N-acetylglucosaminyltransferase V (GlcNacT-V), encoded by the Mgat5 gene, is a medial Golgi enzyme which catalyzes the addiction of a beta-1,6-linked GlcNAc to the alpha-1,6 mannose of the trimannosyl N-glycan core. GlcNacT-V plays a pivotal role in the formation of tri- and tetra-antennary N-glycans on newly synthesized glycoprotein. This branch provides the preferred substrate for the enzymatic subsequent synthesis of polylactosamine chains and terminal modification including the Lewis antigens. In my study, glycomic analyses were performed to investigate possible changes in protein N-glycosylation in wild type conditions and in the absence of Mgat5 gene in C57B5 mice kidneys. In parallel, N-glycan profile of kidneys and spleens coming from mice treated with high fat diet GlcNAc supplementation were analyzed. Previous results demonstrate that the effects of GlcNAc salvage appear to increase flux to UDP-GlcNAc. Therefore we were interested to know whether this implementation affects N-glycan branching. Results show that Mgat5 deficient mouse kidney display less amount of tri-antennary and tetra-antennary structure compared to controls. However, GlcNAc dietary salvage has no apparent effect on N-linked glycosylation in the kidney and spleen, even if the experiments conducted on cell lines demonstrate that increased influx of UDP-GlcNAc resulted on increased N-glycan branching. Moreover, the performance of optimized glycome procedure allowed the identification of more tri-antennary glycan structures than the one reported on CFG (Consortium of Functional Glycomics) database.
Riassunto La mia tesi di dottorato è composta di due sezioni. Una sezione riguarda la caratterizzazione di dimeri di alpha-sinucleina (aS) in confronto con le proprietà di aS, sia in soluzione che in esperimenti di aggregazione. Il lavoro sperimentale è stato condotto nel laboratorio di Chimica delle Proteine (CRIBI Biotechnology Center), presso l’Università degli studi di Padova, e costituisce il progetto principale nel quale sono stata coinvolta. Durante il mio terzo anno di dottorato ho trascorso 6 mesi al laboratorio Biopolymer Mass Spectrometry Laboratory presso l’Imperial College a Londra. In questo laboratorio sono stata coinvolta in due progetti: uno studio di analisi glicomica di tessuti murini e un progetto pilota sulla biosintesi di emicellulosa mixed linked glucans (MLG). Il morbo di Parkinson è una malattia neurodegenerativa progressiva caratterizzata dalla perdita di neuroni dopaminergici nella substantia nigra. La principale caratteristica istologica della malattia è la presenza di inclusioni intracellulari, conosciute come corpi di Lewy, composti da aggregati proteici filamentosi. La patogenesi della malattia è ancora poco chiara, ma un passaggio chiave nello sviluppo della malattia è l’aggregazione di alpha-synuclein (aS) in fibrille amiloidi, che si accumulano dei corpi di Lewy e ne costituiscono il componente principale. Nonostante la sua importanza nella neurodegenerazione, si conoscono poco la funzione di aS, il suo stato nativo fisiologico e il meccanismo di aggregazione. aS è stata di recente descritta come un tetramero di proteine in alpha-elica, ma aS è stata generalmente descritta come una proteina natively unfolded. aS assume conformazione ad alpha-elica a seguito di interazione con lipidi e converte a struttura beta durante i processi patologici. Durante il processo di aggregazione, aS forma oligomeri solubili di struttura beta, transienti intermedi tra la forma fisiologica di aS e le fibrille amiloidi. La dimerizzazione di aS può rappresentare un fattore limitante nell’aggregazione e nella formazione di struttura amiloide. Pertanto, abbiamo deciso di studiare l’aggregazione di diversi dimeri di aS, prodotti mediante biologia molecolare. E’ stato aggiunto un residuo di cisteina all’ N- o al C- terminale di aS, producendo quindi dimeri NN o CC, legati attraverso un legame disolfuro. Un dimero NC, formato da due molecole consecutive di aS, è stato ottenuto come singola catena polipeptidica. Durante il progetto è stato prodotto un altro dimero, chiamato DC, disegnato in modo da avvicinare ulteriormente le regioni idrofobiche di aS, ed evitare le interferenze provocate dalle catene laterali, che vengono a trovarsi all’interno della molecola nei dimeri NN, CC ed NC. Il dimero DC contiene i residui 1-104 uniti al segmento 29-140 di aS, ed è quindi costituito da due regioni centrali di aS, altamente amilodoigeniche, disposte in modo consecutivo. I dimeri rappresentano uno strumento adatto per lo studio delle interazioni intramolecolari di aS. Alcune differenze sostanziali definiscono e limitano la libertà di movimento dei dimeri rispetto ad aS, ipoteticamente differenziando il processo di fibrillazione delle cinque strutture proteiche. La caratterizzazione dei dimeri è stata effettuata utilizzando tecniche biofisiche e chimiche al fine di definire il loro comportamento in soluzione come monomero. Studi di dicroismo circolare (CD), spettroscopia IR ed NMR hanno dimostrato che tutti i dimeri sono unfolded. Tutti effettuano transizione ad alpha-elica a seguito dell’interazione con il detergente SDS. Questi risultati provano che i dimeri hanno caratteristiche conformazionali simili ad aS. Successivamente, è stata esaminata la capacità dei dimeri di formare fibrille, incubando le molecole in tampone fisiologico alla concentrazione di 1 mg/ml. Tiutti sono in grado di formare fibrille, che sono positive al saggio di legame alla Tioflavina T (ThT), generalmente utilizzato per determinare la presenza di struttura amiloide. Inoltre, le analisi della struttura delle fibrille, condotte usando CD e spettroscopia IR in trasformata di Fourier (FT-IR), rilevano la presenza di transizione strutturale da random a struttura beta come ci si aspetta per fibrille amiloidi. La morfologia delle fibrille è stata studiata mediante microscopia elettronica a trasmissione (TEM) e microscopia di forza atomica (AFM). Le fibrille derivate dai dimeri di aS sono abbastanza lunghe, non ramificate e a singolo filamento, una differenza peculiare rispetto alle fibrille di aS, che si presentano twisted e formate da più filamenti. Per identificare quali amminoacidi di ciascun dimero fosse coinvolto nel core fibrillare sono sati eseguiti esperimenti di proteolisi. Il razionale di questo esperimento risiede nel fatto che le regioni non strutturate delle proteine sono in genere sito di attacco enzimatico, e l’idrolisi si verifica quindi in regioni flessibili, sprovviste di legani idrogeno intermolecolari che stabilizzano una struttura secondaria. Quindi lo scopo dell’esperimento è di rimuovere le parti flessibili dal core amyloide. I risultati hanno mostrato come le strutture core delle fibrille dei diversi dimeri sembrino essere costituite dalla stessa regione amminoacidica, che comprende il segmento 35-96, in analogia con studi precedenti su aS. La cinetica del processo è stata analizzata con tecniche di fluorescenza (saggio ThT) e valutando la quantità di proteine presenti nel tempo. Questo calcolo è stato effettuato indirettamente misurando l’assorbanza del surnatante ottenuto dopo ultracentrifugazione delle aliquote prelevate da miscele di aggregazione a diversi tempi. I dimeri NN ed NC hanno mostrato una cinetica di aggregazione più lenta rispetto ad aS, mentre il tasso di formazione delle fibrille di CC e DC è più veloce. Inoltre, esperimenti di aggregazione su miscele di aS in presenza di piccole quantità di dimeri sono stati condotti al fine di verificare se la presenza del dimero influenzasse la cinetica di aS. I risultati hanno evidenziato la capacità del dimero CC di influenzare l’aggregazione di aS. Sulla base dei risultati ottenuti, sono stati proposti dei modelli sulla conformazione dei dimeri all’interno delle fibrille. L’esperienza di ricerca svolta all’Imperial College London mi ha dato la possibilità di imparare e applicare tecniche avanzate di spettrometria di massa (MS) sull’analisi di composti organici, utilizzando gas cromatografia accoppiata ad MS (GC-MS) e spettrometri MALDI-TOF. L’enzima N-acetylglucosaminyltransferase V (GlcNAc-V), codificato dal gene Mgat 5, è un enzima del Golgi che catalizza l’addizione di un GlcNAc in posizione beta-1,6 a un mannosio alpha-1,6 della struttura di base degli zuccheri legati a residui amminici (N-glicani). GlcNAc-V svolge un ruolo fondamentale nella formazione di N-glicani a tre- e quattro-antenne su una proteina appena glicosilata. Queste ramificazioni forniscono il substrato favorito per la successiva sintesi enzimatica di catene poli-lactosamminiche e per le modificazioni terminali, compresi gli antigeni di Lewis. Ho svolto analisi glicomiche su tessuti renali murini per studiare possibili cambiamenti nella N-glicosilazione in topi wild type e knock out per il gene Mgat 5. In parallelo, è stato analizzato il profilo glicomico di tessuti renali e di milza di topi alimentati con una dieta ricca di GlcNAc. Risultati precedenti avevano dimostrato un aumento nel flussio di UDP-GlcNAc (substrato di GlcNAc-V), perciò eravamo interessati a determinare se il maggione apporto di zucchero influenzasse le glicosilazioni proteiche. I risultati hanno evidenziato come le glicoproteine dei topi ko per Mgat 5 hanno meno strutture a tre- e quattro-antenne nelle glicosilazioni rispetto ai controlli. L’apporto di GlcNAc nella dieta non ha alcun affetto apparente sulla struttura e composizione delle glicosilazioni dei tessuti analizzati, nonostante precedenti esperimenti condotti su linee cellulari abbiano avuto un diverso esito. Inoltre, le analisi che ho condotto hanno permesso di identificare glicosilazioni non ancora registrate nel database CFG (Consortium of Functional Glycomics) per i tessuti analizzati.

The first part of the work, described in Chapter One, concentrates on the attempted synthesis of discrete, water-soluble phthalocyanine dimers, which were of interest as model compounds for studies into the photochemical stability of phthalocyanine dyes. The phthalocyanines were linked together by two different bridges derived from diethyl 3,3-bis(4-(3,4- dicyanophenoxy)phenyl)pentanedioate and 1,2-di(4'-(3",4"-dicyano)phenoxy)-3,4,5,6- tetraphenyl-benzene. The formation of the dimers using 4-(2,6-di-/so- propylphenoxy)phthalonitrile was possible, as determined by mass spectrometry and visible absorption spectroscopy of the crude product mixture, but their isolation proved extremely difficult and so this work was abandoned to concentrate on the work described in chapters 3-4. After an introduction to the research area of organic microporous materials (Chapter 2) and a statement of the new aims and objectives, the second part of the work, described in Chapter 3, concerns the use of hexaphenylbenzene as a structural unit for the synthesis of polymers of intrinsic microporosity, PIMs. Even though hexaphenylbenzene does not have a site of contortion, typical of PIMs, it was thought that the rigidity and non-planarity of this unit due to the lack of rotational freedom of the benzene rings would hinder efficient packing of the polymer in the solid state and induce intrinsic microporosity. A number of different hexaphenylbenzene-based monomers suitable for use in the type of polymerisation reactions used to make PIMs due to their catechol units were synthesised. It was discovered that the position of the catechol substituents in relation to one another had a significant effect on the molecular mass and hence physical properties of the polymer, in particular its ability to form self-standing films. It can be concluded that placing the catechol units on opposite sides of the hexaphenylbenzene unit (para-substitution) suppressed cyclic oligomer formation and allowed the preparation of polymers of high molecular mass with good film-forming properties. In addition, the microporosity of this polymer was greater than that obtained from the monomer in which the catechol units were placed adjacent to one another (ortho-substitution). The Preface resulting self-standing films allowed the measurement of gas permeation through this polymer, which showed encouraging performance. The crystal packing properties of the monomers and a 2+2 cyclic oligomer isolated from the polymerisation reaction using the monomer with adjacent catechol units on the hexaphenylbenzene core are described in Chapter 4. The macrocyclic 2+2 cyclic oligomer is highly fluorescent and its binding with nitrobenzene, as a model for small nitrated aromatic compounds, was investigated in the context of its possible use as a sensor. However, studies offered no evidence of selective binding. Finally, Chapter 5 offers suggestions on the concept of using hexaphenylbenzene units in flow chemistry.

The main aims and objectives of this project have been to (i) synthesise pregnane (C₂₁), dinorcholane (C₂₂) and cholane (C₂₄) steroidal monomers as precursors for the synthesis of steroid dimers, (ii) synthesis symmetrical and unsymmetrical steroid dimers via oxalate and simple ester linkages and (iii) perform flash vacuum pyrolysis (FVP) studies on synthesised oxalate steroid dimers in order to generate new steroidal derivatives. The chapter 1 of this thesis reviews the literature since the early part of 1997 on the synthesis of steroid dimers connected directly or via spacer groups in different ring systems as well as side chain through spacer groups. It also reviews the uses of synthetic dimers as molecular umbrellas and their potential applications in drug delivery in the lipid bilayer, and the occurrence of steroid dimers in various natural sources, such as plants and marine organisms, and their biological and pharmacological properties including anticancer activities. The synthesis of pregnane (C₂₁), dinorcholane (C₂₂) and cholane (C₂₄) derivatives by several classical methods has been described in the chapters 2 and 3. The difficulties for the synthesis of C₂₂ amino nitrile using Strecker synthesis and its corresponding acid have been explained. The synthetic routes for several new mixed C₁₈, C₂₁ and C₂₄ anhydrides via oxalate ester formation have also been presented. The chapter 4 depicts the synthesis of several symmetrical oxalate dimers from various natural steroidal hormones such as estrone (C₁₈), testosterone (C₁₉), androstane (C₁₉), pregnenolone (C₂₁), bile acid (C₂₄), cholesterol (C₂₇) and stigmasterol (C₂₉). The importance of the use of pyridine for the formation of oxalate dimers from primary alcohol has been evaluated experimentally. The synthesis of unsymmetrical dimers via side chain and ring A through simple ester linkage has also been presented in this chapter. Flash Vacuum Pyrolysis (FVP) technique and its use for the synthesis of dimers from oxalate dimers have been evaluated. It has been observed that none of the steroid oxalate dimers has produced any new dimers, instead yielded only monomeric olefins. The oximinyl oxalate dimers have been pyrolysed in similar fashion but the reaction has produced some volatile gas and no steroid derivative has been isolated. Interestingly, it has been noted that 5a-derivatives have produced 90% 2-end and 10% 3-ene compounds but 5b-derivatives have yielded solely 3-ene compounds. It has been easy to break 3-oxalate linkages at 600°C but 17-oxalate linkages have needed higher temperature. Possibly this has been because of the steric hindrance at 17-positon.

The cell membrane is a recent target for the imaging and quantification of viscosity in light of evidence which suggests that abnormal membrane behaviour is related to various disease states. We propose the use of a novel technique involving a class of compounds termed ‘molecular rotors’, which can image cellular viscosity with high spatial and temporal resolution. This Thesis concerns one type of molecular rotors, conjugated porphyrin dimers, which have dual functionality: as viscosity sensors and as photosensitizers for photoinduced cell death during Photodynamic Therapy of cancer (PDT). First, we consider the photophysics of several conjugated dimers bearing various substituents and assess the effect of various parameters on dimer’s intermolecular rotation. We find that the photophysical properties of most dimers are primarily sensitive to the viscosity of their environment and are minimally sensitive to temperature and solution polarity. Secondly, using polymer solutions and melts, we establish the physical origin of the porphyrin dimer’s viscosity sensitivity as detection of solvent ‘free volume’, which is related to both macromolecular crowding and solvent hydrodynamic pressure. Thirdly, we establish conditions for the inclusion of a porphyrin dimer into model lipid membranes; demonstrating the detection of lipid phase as a function of temperature, sensitivity to free-volume occupancy of lipid tails according to saturation, and finally reporting viscosity values in close agreement with those from literature obtained using alternative methods. Finally we find that under conditions of imaging using fluorescence microscopy, the porphyrin dimer has compromised fluorescence signal due to limitations of solubility during sample preparation. However, by combining the detection from a poprhyrin dimer and a well-known molecular rotor BODIPY-C10 we were able to detect a viscosity increase in unsaturated lipid membranes upon increased exposure to light, replicating conditions of singlet oxygen production during PDT.

Conversion of 1-Methylpentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹]undecane- 8,11-dione into the corresponding mono(ethylene ketal) followed by Wolff-Kishner reduction resulted in a mixture of two isomers (i.e., 1- and 7-methyl-8-[2',-(1',3',dioxolano)]pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹] undecane. Hydrolysis of each isomer in turn resulted in 1- and 7- methyl pentacyclo[5.4.0.0²⋅⁶.0³⋅¹⁰.0⁵⋅⁹ ]undecan-8-ones (i.e.,"methylated PCU-8-ones"), respectively. "Titanium-promoted reductive dimerization of each of the methylated pentacycloundecane (PCU)-8-ones afforded mixtures of "methylated PCU alkene dimers". Individual isomers have been isolated from these mixtures via column chromatography by using silver nitrate impregnated silica gel as adsorbent followed by fractional recrystallizations of individual chromatography fractions. Structures of three isomerically pure methylated PCU alkene dimers (C₂₄H₂₈) have been established unequivocally by application of single crystal X-ray crystallographic methods.

In addition to the conserved active site cysteines that are responsible for the classical redox activity of thioredoxins (Trx’s), vertebrate Trx’s contain an additional three conserved cysteines at position 62, 69 and 73. These structural cysteines are known to be subjected to a variety of post translational modifications including dimerisation that are believed to contribute to the regulation and diversity of function of vertebrate Trx’s. Reports of the formation of “disulphide linked dimers” have been a long standing observation since the earliest studies on vertebrate Trx’s, however detailed studies on dimerisation have been limited in number and the extent of characterisation achieved. Despite the potential for a diversity of dimeric forms with different structural and functional properties there has been a common assumption arising from the literature (despite some evidence to the contrary) that all dimers so far described are much the same and all are redox inactive.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Science
Science, Environment, Engineering and Technology
Full Text

Over the past decade there has been a growing body of evidence, obtained from studies employing a wide variety of pharmacological, biochemical and biophysical techniques, suggesting that G protein coupled receptors (GPCRs) exist not as monomeric entities but rather as dimers or other higher order oligomeric arrays. To further the accumulation of knowledge pertaining to this research area, the work presented in this thesis has made use of one such particular biophysical technique called bioluminescence resonance energy transfer (BRET). In order to utilise this system GPCRs were modified at their carboxyl terminal tail with either the anthozoan enzyme Renilla luciferase or the fluorescent protein eYPP. Through this expedient, if the differentially tagged GPCRs are in close proximity when co-expressed within mammalian cells, upon addition of the bioluminescent molecule coelenterazine, there is a non-radiative exchange of energy between the Renilla and eYFP, resulting in a fluorescent emission from eYFP. The technique can be used to monitor interactions in real time, in living cells and does not require any biochemical manipulations/treatments such as are associated with more traditional approaches to this line of enquiry (e.g. co-immunoprecipitation). Using this technique, it was demonstrated that the β2-AR was closely associated when expressed within HEK 293T cells, as were the σ-opioid and K-opioid receptors since all gave robust signals in energy transfer experiments. Contrary to some previous reports however, it was not seen to be the case that the presence of ligand was capable of modulating the magnitude of the energy transfer signal. This indicated that for these GPCRs the binding of ligand did not result in any alteration in the dimerization status of the receptor. It was further shown, through monitoring the energy transfer at varying levels of receptor expression, that energy transfer was favourable between homomers of the K-opioid receptor. At similar receptor densities, energy transfer between coexpressed K-opioid receptor and TRHr was seen to be considerably less favourable, requiring far higher receptor expression levels to be achieved before meaningful levels of energy transfer could be detected. These results strongly indicated that closely related GPCR types had a greater propensity for mutual interaction than did more distantly related ones. Many of these results were confirmed using a modified version of BRET, designated BRET2, which conferred an additional sensitivity to detection of protein-protein interactions. Using BRET2 a previously ill-defined result obtained with traditional BRET, that suggested that the β2-AR might interact with the 6-opioid-receptor, was confirmed. An additional purpose of the work described herein was to explore the potential of GPCR dimerization as a means of providing a novel ligand detection assay suitable for application to industrial high-throughput screening programmes. Since the experiments concentrating on GPCR oligomerization failed to provide such an assay, it was decided that the ability of GPCRs to recruit β-arrestin as part of the process of desensitisation should be evaluated as a possible alternative. Using a cell line stably expressing the GPCR CCR2 the ability of this receptor to recruit various fluorescent proteins conjugated to β-arrestin2 (β-arrestin-red NFP and β-arrestin-cyan NFP) from the cytosol in response to receptor activation was demonstrated. The β-arrestin2 was localized into endocytic vesicles and remained tightly associated with the internalised receptors after sequestration had occurred. This behaviour was in accordance with other previous reports for GPCRs that, like CCR2, possessed serine and threonine clusters within their carboxyl terminal tails. If adapted to a FRET based format this particular protein-protein interaction could form the basis of a ligand screening method for agonists that would be equally applicable to most GPCRs. It was further shown that β-arrestin2-red NFP had a higher affinity for CCR2 than did β-arrestin1- GFP through the monitoring of the respective kinetics and extent of translocation when the constructs were co-expressed within the same cells. As an alternative strategy for the detection of ligands, a constitutively active mutant of β 2-AR (CAM β 2-AR) was modified C-terminally with the bioluminescent enzyme Renilla luciferase. This CAM 2-AR was structurally destabilized to a high degree so that only modest expression levels could be obtained upon expression of the Renilla modified receptor construct (CAM β 2-AR-Rluc) in HEK 293T cells. Upon prolonged exposure to various antagonist ligands, a two to three fold upregulation of the receptor construct could be detected via light output from the luciferase. From parallel competition binding experiments it was also demonstrated that, for each of these ligands, the EC50 for upregulation highly correlated with the dissociation equilibrium constant (Ki). This strongly indicated that it was the presence of the ligand within the receptor binding pocket that alone accounted for the observed upregulation effects. In a similar manner, it was demonstrated that agonist compounds were also capable of mediating a similar degree of upregulation. The increase in receptor density of CAM β2-AR in response to the presence of ligand was subsequently shown to be dependent on the constitutively active nature of the receptor. In an additional experiment co-transfection of CAM β 2-AR-Rluc along with a GFP conjugated version of the βib- adrenoceptor into HEK 293T cells and subsequent monitoring of the upregulation of either construct in response to selective ligands confirmed the necessity for pharmacological specificity in mediating the upregulatory effect. Finally, to show that this assay method would be suitable as a means of detecting ligands in a high- throughput screening format, the ability of β2-AR to be upregulated was assessed in the presence of a wide variety of compounds, only a proportion of which possessed pharmacological specificity for the β2-AR. When tested in this manner it was seen that only compounds that were specific for β2-AR were capable of mediating an upregulatory effect.

Cyclobutane pyrimidine dimers (CPDs) are the major photo products that can occur in DNA after UV-light exposure. Detrimental effects of CPDs for organisms can be prevented by the DNA repair enzyme DNA photolyase. This protein repairs CPDs by a light induced (300–500 nm) electron transfer involving a non-covalently bound cofactor, FAD. The key role of FAD in the catalytic DNA repair can be investigated by replacing FAD in the enzyme with FAD analogues and compare the activity of wild-type enzyme to the mutant enzyme. As a part of this study, a stable expression system of photolyase was created and the enzyme was purified in concentrations in the uM range. The FAD analogue roseoFAD was biochemically synthesised from its precursor form roseoflavin. FAD was extracted from photolyase by unfolding the enzyme using the effects of denaturing agents or changes in buffer pH values. The protein was successfully refolded in the presence of roseoFAD and its integration into the proteins active site was confirmed by UV/Vis spectroscopy. Surface plasmon resonance spectroscopy (SPR) was used to monitor DNA repair by photolyase in real-time. For this purpose photolyase was overexpressed and purified. CPDs, the substrate of photolyase, were produced by UV irradiation of poly-T oligonucleotides. SPR signals were detected for steps of the repair process such as enzyme-substrate complex formation, DNA repair and release of product. Changes in SPR signals were used to obtain the kinetics of DNA repair by measuring the on and & off rates and calculating the rate constants for substrate binding and product release. According to the data obtained the dissociation constant KD was calculated and found to be in good agreement with published values obtained by different methods.

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010.
Committee Chair: El-Sayed, Mostafa; Committee Member: Perry, Joseph; Committee Member: Wang, Zhong; Committee Member: Whetten, Robert; Committee Member: Zhang, John. Part of the SMARTech Electronic Thesis and Dissertation Collection.

This bachelor thesis covers the initial development of a synthesis of fullerene dimers using two different types of linking reactions. Different setups for [3+2] cycloadditions to fullerenes (Prato reaction) were tested, and for that purpose, an N-alkylated amino acid was synthesised. Hydroarylation of fullerene using Rh-catalysis was also studied, using both MIDA protected and unprotected boronic acids, as well as by using cycloaddition products. A range of model compounds in form of fulleropyrrolidenes were synthesised. Products were puried with HPLC and analysed with MALDI-MS and 1H NMR. A range of new compounds were synthesised and characterisation of them was begun. With MALDI-MS, indications that the fullerene dimer had formed were found. Using synthesised model compounds, by-products of the hydroarylation reaction were identied.
Denna kandidatuppsats behandlar påborjandet av syntesutvecklingen för bildandet av fullerendimerer genom användandet av två olika sorters länkningskemi. Olika förhållanden och reagens for [3+2]-cykloaddition till fullerener (Pratoreaktionen) studerades, och i samband med det syntetiserades en N-alkylerad aminosyra. Hydroarylering av fullerener med hjälp utav rodiumkatalys studerades även, genom reaktioner med både skyddade och oskyddade borsyror, inklusive fulleropyrrolidiner. Produkter har renats upp med HPLC och analyserats med MALDI-MS och 1H NMR. En uppsättning nya substanser har syntetiserts, men karaktäriseringen av dessa har inte slutförts. Genom användning av MALDI-MS har indikationer att fullerendimer bildats framkommit. Genom att använda syntetiserade modellsubstanser har biprodukter från hydroaryleringsreaktionen identierats.

In this thesis, quantum dynamical simulations are performed in order to describe the vibrational motion of diatomic molecules in a highly quantum environment, so-called helium droplets. We aim to reproduce and explain experimental findings which were obtained from dimers on helium droplets. Nanometer-sized helium droplets contain several thousands of 4-He atoms. They serve as a host for embedded atoms or molecules and provide an ultracold “refrigerator” for them. Spectroscopy of molecules in or on these droplets reveals information on both the molecule and the helium environment. The droplets are known to be in the superfluid He II phase. Superfluidity in nanoscale systems is a steadily growing field of research. Spectra obtained from full quantum simulations for the unperturbed dimer show deviations from measurements with dimers on helium droplets. These deviations result from the influence of the helium environment on the dimer dynamics. In this work, a well-established quantum optical master equation is used in order to describe the dimer dynamics effectively. The master equation allows to describe damping fully quantum mechanically. By employing that equation in the quantum dynamical simulation, one can study the role of dissipation and decoherence in dimers on helium droplets. The effective description allows to explain experiments with Rb-2 dimers on helium droplets. Here, we identify vibrational damping and associated decoherence as the main explanation for the experimental results. The relation between decoherence and dissipation in Morse-like systems at zero temperature is studied in more detail. The dissipative model is also used to investigate experiments with K-2 dimers on helium droplets. However, by comparing numerical simulations with experimental data, one finds that further mechanisms are active. Here, a good agreement is obtained through accounting for rapid desorption of dimers. We find that decoherence occurs in the electronic manifold of the molecule. Finally, we are able to examine whether superfluidity of the host does play a role in these experiments
In dieser Dissertation werden quantendynamische Simulationen durchgeführt, um die Schwingungsbewegung zweiatomiger Moleküle in einer hochgradig quantenmechanischen Umgebung, sogenannten Heliumtröpfchen, zu beschreiben. Unser Ziel ist es, experimentelle Befunde zu reproduzieren und zu erklären, die von Dimeren auf Heliumtröpfchen erhalten wurden. Nanometergroße Heliumtröpfchen enthalten einige tausend 4-He Atome. Sie dienen als Wirt für eingebettete Atome oder Moleküle und stellen für dieseeinen ultrakalten „Kühlschrank“ bereit. Durch Spektroskopie mit Molekülen in oder auf diesen Tröpfchen erhält man Informationen sowohl über das Molekül selbst als auch über die Heliumumgebung. Man weiß, dass sich die Tröpfchen in der suprafluiden He II Phase befinden. Suprafluidität in Nanosystemen ist ein stetig wachsendes Forschungsgebiet. Spektren, die für das ungestörte Dimer durch voll quantenmechanische Simulationen erhalten werden, weichen von Messungen mit Dimeren auf Heliumtröpfchen ab. Diese Abweichungen lassen sich auf den Einfluss der Heliumumgebung auf die Dynamik des Dimers zurückführen. In dieser Arbeit wird eine etablierte quantenoptische Mastergleichung verwendet, um die Dynamik des Dimers effektiv zu beschreiben. Die Mastergleichung erlaubt es, Dämpfung voll quantenmechanisch zu beschreiben. Durch Verwendung dieser Gleichung in der Quantendynamik-Simulation lässt sich die Rolle von Dissipation und Dekohärenz in Dimeren auf Heliumtröpfchen untersuchen. Die effektive Beschreibung erlaubt es, Experimente mit Rb-2 Dimeren zu erklären. In diesen Untersuchungen wird Dissipation und die damit verbundene Dekohärenz im Schwingungsfreiheitsgrad als maßgebliche Erklärung für die experimentellen Resultate identifiziert. Die Beziehung zwischen Dekohärenz und Dissipation in Morse-artigen Systemen bei Temperatur Null wird genauer untersucht. Das Dissipationsmodell wird auch verwendet, um Experimente mit K-2 Dimeren auf Heliumtröpfchen zu untersuchen. Wie sich beim Vergleich von numerischen Simulationen mit experimentellen Daten allerdings herausstellt, treten weitere Mechanismen auf. Eine gute Übereinstimmung wird erzielt, wenn man eine schnelle Desorption der Dimere berücksichtigt. Wir stellen fest, dass ein Dekohärenzprozess im elektronischen Freiheitsgrad des Moleküls auftritt. Schlussendlich sind wir in der Lage herauszufinden, ob Suprafluidität des Wirts in diesen Experimenten eine Rolle spielt

The fullerene cage provides an ideal, isolated environment for trapping spin active atoms such as nitrogen or phosphorous. Alignment of these endohedral fullerenes in linear arrays would have applications in quantum computing as the interactions between spin-active molecules can be easily controlled. Self-assembled molecular networks such as block copolymers, Langmuir-Blodgett films, and self-assembled monolayers are ideal for this purpose as the spacing and geometry can be easily tuned. This dissertation will discuss using each of these methods to achieve alignment or orientation of fullerenes for application in quantum information processing.

Myosin binding to actin, and thus muscle contraction, is regulated by Tropomyosin (Tpm), Troponin (Tn) and calcium (Ca2+). Tpm, is an α-helical coiled-coil dimer, which exists as a homo- or heterodimer. Two major isoforms of Tpm are found in striated muscle, α and β. Though it is known that different dimers exist, the mechanism by which they form and exchange is not fully understood. The thermal stability and exchange between dimers was explored with the use of circular dichroism and SDS PAGE densitometry analysis. Homodimers showed little exchange to form heterodimers at temperatures up to 20 °C . Dimer stability at these temperatures reduces the need for chemical cross-linking samples. While extensive exchange was seen at 37 °C . Reverse exchange of WT and mutant (E54K - dilated cardiomyopathy mutant) containing heterodimers to form homodimers did not show the same extent of exchange, suggesting a dimer preference. Results showed the ability to determine dimer content of a solution with the use of polyacrylamide gels and chemical cross-linking. The thermal melting curves of Tpm highlighted a significant destabilisation of β Tpm against the α isoform. Tpm heterodimer containing E54K mutant showed a decreased thermal stability. Notable differences were seen not only for isoforms and homo- and heterodimers, but also for buffer conditions and protein tags. Increased salt concentrations led to an increase in thermal stability. Crosslinking dimers increased thermal stability, whereas addition of His-tags led to a decrease in thermal stability. Changes in thermal stability highlighted the need for caution when tagging or cross-linking the protein. Dimer exchange on actin provided conflicting results between SDS PAGE cosedimentation assays and pyrene fluorescence cosedimendation assays, which highlighted limitations of cross-linking Tpm and using fluorescent labels. The stiffness of Tpm dimers was explored using atomic force microscopy (AFM) to image Tpm particles. Significant differences were seen between the relatively stiff α Tpm and less stiff β isoform. Changes in stiffness of Tpm affect its ability to cooperatively activate the thin filament, and provides insight into the assembly of dimers in vivo.

The principal aim of this Thesis is the synthesis and characterisation of a range of novel liquid crystals designed to exhibit the twist-bend nematic phase (NTB), in order to enhance our understanding of the relationships between molecular structure and the observation of NTB behaviour. Moreover, the inclusion of chiral fragments allowed the effects of molecular chirality on the structure of the NTB phase, specifically the chiral twist-bend nematic phase, N*TB, to be studied. In Chapter 3, a series of non-symmetric odd-membered liquid crystal dimers are prepared and the terminal chain length m is varied. A change in the local molecular structure from intercalated to bilayer is seen on increasing m, but this has no apparent effect on the stability of both the nematic and twist-bend nematic phases, which show a regular dependence on m. A novel twist-bend smectic phase is reported. Chapter 4 investigates the effects of branching this terminal chain on phase behaviour. It is evident that in dimers with a shorter spacer, branching destabilises the N and NTB phases while stabilising smectic behaviour, but in longer homologues smectic behaviour is also destabilised. Chapter 5 explores the effect of molecular bulk on phase behaviour, specifically the stability of the NTB phase, by the incorporation of a pyrene moiety. This group supresses crystallisation such that stable, low temperature NTB phases are formed despite the bulky group. Chapters 6 and 7 study the inclusion of chiral moieties in bent-shaped, odd-membered dimers: specifically, 2-methylbutyl, 1-methylheptyl (6) and lactic esters (7). New examples of the rarely observed chiral twist-bend nematic phase are seen. Phase behaviour is investigated and compared to achiral and racemic analogues. Chapter 8 describes the mesogenic behaviour of molecular complexes assembled by hydrogen bonding between both achiral and chiral stilbazole-based and benzoic acid-based fragments. A selection of the complexes exhibit the N(*)TB phase despite only one or neither component being mesogenic.

Les complexes collecteurs de lumière des bactéries photosynthétiques absorbent l'énergie solaire, et transfèrent l'énergie avec grande efficacité aux centres réactionnels, siùge où elle est captée pour l'utilisation par la cellule. Nous savons peu des détails du transfert d'énergie entre les différents complexes collecteurs de lumière. Dans cette thèse, j'ai isolé différents complexes collecteurs de lumière à partir de plusieurs souches de bactéries pourpres. J'ai construit de modèles 3D par homologie et les structures possibles de dimères ont également été examinés. Les sites de pontage dans ces protéines montrent la possibilité de construire des dimères avec des structures biologiquement pertinentes. J'ai développé un protocole pour construire de dimères de protéines collectrices de lumière fortement oligomériques. Le protocole que j'ai mis en place contient trois grandes étapes : d'abord la réaction de lysines dans les complexes à un très faible degré de réaction, et la purification des protéines marquées. Ensuite, les groupes réactifs de dibenzocyclooctyne (DBCO) ou de l'azoture sont introduits au complexe. Finalement, la réaction sans cuivre de cycloaddition azoture-alcyne promue par distorsion a pour conséquence la synthèse de dimères
The light harvesting apparatus of photosynthetic bacteria absorb the energy from sunlight and transfer the energy with high efficiency to the reaction center, where it is captured for use by the cell. We know little about the details of energy transfer between different light-harvesting complexes. In this thesis I isolated several different types of light-harvesting complex from various stains of purple bacteria. 3D models were built, based on homology modeling, and possible dimer structures were examined. The cross linking sites in these protein shown the possiblity of forming biologically relevant dimer structures. I have developed a protocol to make dimers, from highly oligomeric light harvesting proteins. The protocol developed contains three main steps: first reaction of lysines in the complex at a very low degrees of reaction and purifying the labelled protein. Then coupling the reactive groups of dibenzylcyclootyne (DBCO) or of azide separately to the different complexes. Finally, the copper free strain promoted azide-alkyne cycloaddition reaction occurred to synthesize the dimer

GCN4 is a yeast transcriptional factor, which binds sequence selectively to DNA as a homodimer. Two domains are required to achieve this, an N-terminal basic domain, which interacts with the target site directly, and the C-terminal leucine zipper responsible for dimerisation. The latter can be replaced by chemically linking together two basic domains, to retain similar DNA binding. This work investigates using a ferrocene complex as the dimerization unit due to its flexibility and its characteristic electrochemical redox properties. A strategy was proposed which took advantage of coupling to the thiol side chains of two cysteine residues, and detailed studies on dimerising two equivalents of Cys, or the tripeptide glutathione, were performed. Design of five peptides based on GCN4, which were synthesized and dimerized with a ferrocene linker molecule, to combine the attractive redox electrochemistry of the stable ferrocene and the DNA binding ability of GCN4 dimerized basic domain. Molecular dynamic models were produced for four of the ferrocene peptide dimer complexes bound to target DNA over 10 ns. Their DNA binding ability was investigated by circular dichroism, and electrochemistry was studied in the absence and presence of non-specific and target DNA respectively.

The field of organic conductors and semiconductors has grown rapidly. The development of devices based on this technology, and the insights it provides into molecular electronics have led it to the forefront of materials research both industrially and academically. Electroactive organic species can have wildly varied properties and take on two main different forms: i. conjugated macromolecules, including polymers and metallopolymers and oligomers thereof; ii. molecular systems, such as small organic molecules and metal complexes with functional ligands. Compared to traditional inorganic materials, semiconducting polymer and oligomer systems have the potential to demonstrate major practical benefits, including tunability, cost effectiveness, ease of manipulation in processing and flexibility. Molecular systems tend to have very well defined electronic characteristics and careful design allows access to highly varied topologies and functionalities. Chapter 1 incorporates a description of band theory and the development of organic semiconducting technologies, particularly those based on oligothiophenes and small molecules, including characterisation and device structures. Chapter 2 describes the synthesis, design and characterisation of oligothiophene precursors for unique tetrathiafulvalene, metal dithiolene, and spirocyclic dimers, the synthesis and properties of which are discussed in chapters 3, 4, and 5 respectively.