Chromosomal replication is a source of spontaneous DNA double-strand breaks (DSBs). In E. coli, DSBs are repaired by homologous recombination using an undamaged sister template. During repair, the RecA protein polymerizes on single-stranded DNA generated at the site of the DSB and catalyses the search for sequence homologies on the undamaged sister template. This study utilized fluorescence microscopy to investigate the spatial and temporal dynamics of the RecA protein at the site of a replication-dependent DSB generated at the lacZ locus of the E. coli chromosome. The DSB was generated by SbcCD-mediated cleavage of a hairpin DNA structure formed on the lagging strand template of the replication fork by a long palindromic sequence. The tandem insertion of a recA-mCherry gene with the endogenous recA gene at the natural chromosomal locus produced no detectable effect on cell viability in the presence of DSB formation. During repair, the fluorescently-labelled RecA protein formed a transient focus, which was inferred to be the RecA nucleoprotein filament at the site of the replication-dependent DSB. The duration of the RecA focus at the site of the DSB was modestly reduced in a ΔdinI mutant and modestly increased in a ΔuvrD or ΔrecX mutant. Most cells underwent a period of extended cohesion of the sister lacZ loci after disappearance of the RecA focus. Segregation of the sister lacZ loci was followed by cell division, with each daughter cell obtaining a copy of the fluorescently-labelled lacZ locus. The RecA focus at the site of the DSB was observed predominantly between the mid-cell and the 1⁄4 position. In the absence of DSB formation, the lacZ locus exhibited dynamic movement between the mid-cell and the 1⁄4 position until the onset of segregation. Formation of the DSB and initiation of repair occurred at the spatial localization for replication of the lacZ locus while the downstream repair events occurred very close to the mid-cell. Genomic analysis of RecA-DNA interactions by ChIP-seq was used to demonstrate that the RecA focus at the lacZ locus was generated by the repair of the palindrome-induced DSB and not the repair of one-ended DSBs emanating from stalled replication forks at the repressor-bound operator arrays. This study has shown that the repair of a replication-dependent DSB occurs exclusively during the period of cohesion of the sister loci and the repair is efficiently completed prior to segregation of the two sister loci.

Les cellules vivantes sont capables de réagir aux signaux mécaniques tels que la rigidité de la surface sur laquelle elles adhèrent, les forces de tractions ou compressions auxquelles elles sont soumises, le flux de liquide à la surface de leur membrane ou encore la géométrie de leurs adhésions ou de leur forme globale. Ces signaux influent sur des processus cellulaires tels que la prolifération, la différenciation, la migration et la mort cellulaire. Ces processus sont finement régulés par des réactions biochimiques qui forment un réseau de signalisation. La mécanotransduction est la traduction du signal mécanique en signal biochimique.C’est dans le but d’étudier la mécanotransduction que nous avons étudié l’utilisation d’ultrasons pour stimuler mécaniquement les cellules à des fréquences temporelles et spatiales relativement élevées. De nombreux montages expérimentaux et de nombreuses voies ont été considérées dans cette partie très exploratoire. Nous en retenons finalement des pistes prometteuses pour la continuation future de ce projet.Nous avons développé ce que nous nommons des substrats actifs, qui nous permettent de contrôler à la fois spatialement et temporellement la stimulation mécanique appliquée à des cellules vivantes. Ces substrats actifs consistent en des micropiliers de fer incrustés dans un élastomère peu rigide (PDMS) et manipulés par deux électroaimants. Nous pouvons contrôler dynamiquement le déplacement des piliers qui vont déformer localement et de manière continue la surface. Cette déformation va ensuite déformer en traction ou en compression les cellules vivantes étalées sur la surface à proximité. En employant des marqueurs fluorescents nous pouvons réaliser de la Microscopie de Forces de Traction et surveiller la contrainte appliquée par les piliers aux cellules à travers la surface de PDMS, et nous pouvons étudier la réponse mécanique des cellules. De plus, ces substrats sont compatibles avec la microscopie de fluorescence en cellule vivante, ce qui rend possible l’observation de la réponse cellulaire au niveau morphologique (forme des adhésions focales, activité protrusive, …) et surtout biochimique.En effet, pour étudier la réponse biochimique des cellules après une stimulation mécanique, nous observons par microscopie de fluorescence des biosenseurs portant des paires de fluorophores donneur/accepteur. Ces biosenseurs nous permettent d’observer l’activité de protéines impliquées dans la signalisation cellulaire en calculant l’efficacité de Transfert d’Énergie Résonnant de Förster (FRET) de ces biosenseurs. Pour ce faire, les échantillons sont illuminés alternativement aux longueurs d’ondes d’excitation des fluorophores donneurs puis accepteurs. Le signal de fluorescence est collecté simultanément dans un canal d’émission du donneur et un canal d’émission de l’accepteur. Une grande partie de ma thèse a été consacrée à la mise au point d’une méthode quantitative pour analyser les images de fluorescence afin de mesurer une efficacité de FRET qui ne dépende pas de facteurs expérimentaux ni de la quantité de biosenseurs présents dans les cellules. Nous évaluons alors les différentes méthodes pour déterminer les facteurs de correction répandus corrigeant le débordement de spectre du donneur dans le canal accepteur et l’excitation directe de l’accepteur à la longueur d’onde d’excitation du donneur. Pour obtenir des mesures plus quantitatives, nous avons mis au point une nouvelles méthode pour déterminer 2 facteurs de correction supplémentaires. Nous comparons cette méthode à la seule préexistante et évaluons l’influence des paramètres de traitement des images sur les valeurs d’efficacité de FRET mesurées. / Living cells can react to mechanical signals such as the rigidity of the surface they adhere on, the traction or compression forces applied on them, the liquid flow at their membrane surface or the geometry of their adhesions or of their overall shape. Those signals influence cellular processes such as proliferation, differentiation, migration or cell death. Those processes are tightly regulated by biochemical reactions that constitute a signaling network. Mechanotransduction is the translation of the mechanical signal into the biochemical one.In order to study mechanotransduction, we have considered the use of ultrasounds to mechanically stimulate cells at relatively high temporal and spatial frequencies. Numerous setups and options have been considered in this very exploratory project. Finally, we will retain some promising leads for the continuation of this project.We have developed what we call active substrates that allows us to control both spatially and temporally the mechanical stimulation on living cells. Those active substrates consist of iron micropillars embedded in a soft elastomer and actuated by 2 electromagnets. We can control dynamically the displacement of the pillar that will deform locally and continuously the surface. This deformation will then deform in traction or in compression the living cells spread on the surface nearby. Thanks to fluorescent trackers we can perform Traction Force Microscopy and monitor the stress applied by the pillars to the cells through the PDMS surface, and we can look at the mechanical response of the cells. Moreover, those substrates are compatible with live cell fluorescence microscopy, which makes possible the observation of the cellular response at the morphological level (focal adhesions, protrusive activity, …) and most importantly at the biochemical level.Indeed, in order to study the cellular biochemical response after a mechanical stimulation, we use fluorescence microscopy to observe biosensors containing pairs of donor/acceptor fluorophores. Those biosensors allow us to monitor the activity of proteins implied in cellular signaling by computing the Förster Resonance Energy Transfer (FRET) efficiency of those biosensors. To do so, samples are alternatively excited at donor and acceptor excitation wavelengths. The fluorescence signal is then simultaneously measured in donor and acceptor emission channels. A substantial part of my thesis has been dedicated to the development of a quantitative method to analyze fluorescence images in order to measure FRET efficiencies that do not depend on experimental factors or biosensors concentration in cells. We assess different methods to compute standard correction factors that account for spectral bleed-through and direct excitation of acceptors at donor excitation wavelength. To obtain more quantitative measurements, we have developed a new method to compute 2 additional correction factors. We compare this method with the only one preexisting, and we assess the influence of image processing parameters on FRET efficiency values.

Current single molecule localisation microscopy methods allow for multicolour imaging of macromolecules in cells, and for a degree quantification on molecule numbers in one colour. However, that has not yet been an attempt to develop tools capable of quantitative imaging with multiple colours in cells. This work addressed this challenge by designing linker peptides with chemospecific groups to allow attachment of activator and emitter dyes for STORM imaging, and a targeting module. The design ensured a stoichiometric ratio of targeting module to activator and emitter dyes. Peptides with HaloTag ligands attached were labelled with various activator and emitter pairs and used to label HaloTag fusions of S. pombe and mouse embryonic stem cells. These peptides were found to bind non-specifically to various areas of both cell types, and did not localise to HaloTag protein, whereas controls did. Another peptide was also labelled with activator-emitter pairs and attached to expressed anti-GFP and ant-mCherry nanobodies via native chemical ligation. The labelled anti-GFP nanobody was to demonstrate ensemble and single molecule imaging in S. pombe, as well as characterisation on single molecule surfaces in comparison to a conventional randomly labelled antibody. The stoichiometrically labelled nanobody had a more consistent number of photons detected per localisation, number of localisation per molecule and number of blinks per molecule, which implied that it could be more useful than randomly labelled nanobodies for counting experiments. It was also shown to be capable of specific laser activation for STORM imaging with both an Alexa405Cy5 and Cy3Cy5 pairs. These anti-GFP and anti-mCherry nanobodies and peptide linker are new tools for both counting and multicolour imaging in super-resolution, which could be widely applied to constructs that are already tagged with GFP or mCherry.

[pt] Defensinas de planta, componentes inatos do sistema imune das plantas, são peptídeos antifúngicos, catiônicos, com estrutura primária rica em cisteína. Evidência dada pela literatura demonstrou que trechos de esfingolipídios complexos na membrana dos fungos, contendo manosildiinositolfosforilceramida e glicosilceramida, são sítios de ligação seletivos para as defensinas de planta isoladas de Dahlia merckii e Raphanus sativus, respectivamente. Entretanto, desconhece-se se as defensinas de planta interagem direta ou indiretamente com alvos intracelulares dos fungos. A fim de identificar interações físicas e diretas do tipo proteína- proteína, um sistema de duplo-híbrido, em levedura, baseado no fator de transcrição GAL4, foi construído utilizando-se como isca, a defensina da planta Pisum sativum, Psd1 (Pisum sativum defensin 1). Proteínas alvos, capazes de interagirem com o peptídeo Psd1, foram detectadas através do rastreamento de uma biblioteca de cDNA do fungo Neurospora crassa. Do resultado deste rastreamento, nove dentre quinze candidatos, selecionados pelo método do duplo-híbrido, foram identificados como proteínas nucleares da N. crassa. Um clone, detectado com alta freqüência neste rastreamento, apresentou homologia de seqüência com a proteína ciclina F, relacionada com o controle do ciclo celular. O ensaio de co-purificação utilizando a proteína conjugada a glutationa S-transferase (GST) validou in vitro o resultado obtido pelo sistema duplohíbrido. Análise por microscopia de fluorescência da Psd1, conjugada a FITC, e, dos núcleos do fungo Fusarium solani, marcados com DAPI, demonstrou in vivo a co-localização da defensina de planta Psd1 com os núcleos do fungo. Para pesquisar o modo de ação da Psd1 ao nível do ciclo celular, utilizou-se o modelo multicelular da retina de ratos neonatais, em desenvolvimento. Neste modelo, a migração nuclear intercinética, correlacionada com as transições de fase de S para M do ciclo celular, foi observada na presença da Psd1. Verificouse que Psd1 impediu a migração nuclear em neuroblastos, parando o ciclo celular na transição de S para G2. Estes resultados revelaram modos de ação da defensina de planta Psd1 sobre a fisiologia nuclear. / [en] Plant defensins, innate components of the plant immune system, are cationic, antifungal peptides, with a cysteine- rich primary structure. Evidence from the literature demonstrated that fungus membrane patches containing complex sphingolipids, mannosyldiinositolphosphorylceramide and glucosylceramides, are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. However, whether the plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify direct physical protein-protein interactions, a GAL4-based yeast two-hybrid system was constructed, using the plant peptide, Pisum sativum defensin 1 (Psd1), as the bait protein. Target proteins, capable of interacting with the bait Psd1, were detected by screening a Neurospora crassa cDNA library. In this screening, nine out of fifteen two-hybrid candidates were identified as N. crassa nuclear proteins. One clone, detected with high frequency in the screening, presented sequence similarity to a N. crassa cyclin F, related to the cell cycle control. The GST pull- down co purification assay corroborated this two-hybrid result in vitro. Fluorescence microscopy analysis of FITC- conjugated Psd1 and DAPI-stained Fusarium solani nuclei demonstrated in vivo the co-localization of the plant peptide Psd1 and the fungus nuclei. We used the developing retina of neonatal rats as a multicellular model to study Psd1 mode of action at the cell cycle level. In this model, we observed in vivo the interkinetic nuclear migration, correlated to the transitions from S to M-phase of the cell cycle, in the presence of the Psd1 peptide. It was shown that Psd1 impaired nuclear migration of neuroblasts by arresting the cell cycle at the S to G2- phase transition. These results revealed modes of action of the plant defensin Psd1 upon the nuclear physiology.

Coating colors are applied to the base paper in order to maximize the performance of the end product. Coating colors are complex colloidal systems, mainly consisting of water, binders, and pigments. To understand the behavior of colloidal suspensions, an understanding of the interactions between its components is essential.

Actin is one of the most abundant and highly conserved proteins in eukaryotic cells. The globular protein assembles into long filaments, which form a variety of different networks within the cytoskeleton. The dynamic reorganization of these networks - which is pivotal for cell motility, cell adhesion, and cell division - is based on cycles of polymerization (assembly) and depolymerization (disassembly) of actin filaments. Actin binds ATP and within the filament, actin-bound ATP is hydrolyzed into ADP on a time scale of a few minutes. As ADP-actin dissociates faster from the filament ends than ATP-actin, the filament becomes less stable as it grows older. Recent single filament experiments, where abrupt dynamical changes during filament depolymerization have been observed, suggest the opposite behavior, however, namely that the actin filaments become increasingly stable with time. Several mechanisms for this stabilization have been proposed, ranging from structural transitions of the whole filament to surface attachment of the filament ends. The key issue of this thesis is to elucidate the unexpected interruptions of depolymerization by a combination of experimental and theoretical studies. In new depolymerization experiments on single filaments, we confirm that filaments cease to shrink in an abrupt manner and determine the time from the initiation of depolymerization until the occurrence of the first interruption. This duration differs from filament to filament and represents a stochastic variable. We consider various hypothetical mechanisms that may cause the observed interruptions. These mechanisms cannot be distinguished directly, but they give rise to distinct distributions of the time until the first interruption, which we compute by modeling the underlying stochastic processes. A comparison with the measured distribution reveals that the sudden truncation of the shrinkage process neither arises from blocking of the ends nor from a collective transition of the whole filament. Instead, we predict a local transition process occurring at random sites within the filament. The combination of additional experimental findings and our theoretical approach confirms the notion of a local transition mechanism and identifies the transition as the photo-induced formation of an actin dimer within the filaments. Unlabeled actin filaments do not exhibit pauses, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis. This destabilization can be identified with an acceleration of the depolymerization prior to the interruption. In the final part of this thesis, we theoretically analyze this acceleration to infer the mechanism of ATP hydrolysis. We show that the rate of ATP hydrolysis is constant within the filament, corresponding to a random as opposed to a vectorial hydrolysis mechanism. / Aktin ist eines der am häufigsten vorkommenden und am stärksten konservierten Proteine in eukaryotischen Zellen. Dieses globuläre Protein bildet lange Filamente, die zu einer großen Vielfalt von Netzwerken innerhalb des Zellskeletts führen. Die dynamische Reorganisation dieser Netzwerke, die entscheidend für Zellbewegung, Zelladhäsion, und Zellteilung ist, basiert auf der Polymerisation (dem Aufbau) und der Depolymerisation (dem Abbau) von Aktinfilamenten. Aktin bindet ATP, welches innerhalb des Filaments auf einer Zeitskala von einigen Minuten in ADP hydrolysiert wird. Da ADP-Aktin schneller vom Filamentende dissoziiert als ATP-Aktin, sollte ein Filament mit der Zeit instabiler werden. Neuere Experimente, in denen abrupte dynamische Änderungen während der Filamentdepolymerisation beobachtet wurden, deuten jedoch auf ein gegenteiliges Verhalten hin: Die Aktinfilamente werden mit der Zeit zunehmend stabiler. Mehrere Mechanismen für diese Stabilisierung wurden bereits vorgeschlagen, von strukturellen Übergängen des gesamten Filaments bis zu Wechselwirkungen der Filamentenden mit dem experimentellen Aufbau. Das zentrale Thema der vorliegenden Dissertation ist die Aufklärung der unerwarteten Unterbrechungen der Depolymerisation. Dies geschieht durch eine Kombination von experimentellen und theoretischen Untersuchungen. Mit Hilfe neuer Depolymerisationexperimente mit einzelnen Filamenten bestätigen wir zunächst, dass die Filamente plötzlich aufhören zu schrumpfen und bestimmen die Zeit, die von der Einleitung der Depolymerisation bis zum Auftreten der ersten Unterbrechung vergeht. Diese Zeit unterscheidet sich von Filament zu Filament und stellt eine stochastische Größe dar. Wir untersuchen daraufhin verschiedene hypothetische Mechanismen, welche die beobachteten Unterbrechungen verursachen könnten. Die Mechanismen können experimentell nicht direkt unterschieden werden, haben jedoch verschiedene Verteilungen für die Zeit bis zur ersten Unterbrechung zur Folge. Wir berechnen die jeweiligen Verteilungen, indem wir die zugrundeliegenden stochastischen Prozesse modellieren. Ein Vergleich mit der gemessenen Verteilung zeigt, dass der plötzliche Abbruch des Depolymerisationsprozesses weder auf eine Blockade der Enden, noch auf einen kollektiven strukturellen Übergang des gesamten Filaments zurückzuführen ist. An Stelle dessen postulieren wir einen lokalen Übergangsprozess, der an zufälligen Stellen innerhalb des Filaments auftritt. Die Kombination von weiteren experimentellen Ergebnissen und unserem theoretischen Ansatz bestätigt die Vorstellung eines lokalen Übergangsmechanismus und identifiziert den Übergang als die photo-induzierte Bildung eines Aktindimers innerhalb des Filaments. Nicht fluoreszenzmarkierte Aktinfilamente zeigen keine Unterbrechungen, woraus folgt, dass ältere Filamente in vivo durch die ATP-Hydrolyse destabilisiert werden. Die Destabilisierung zeigt sich durch die Beschleunigung der Depolymerisation vor der Unterbrechung. Im letzten Teil der vorliegenden Arbeit untersuchen wir diese Beschleunigung mit theoretischen Methoden, um auf den Mechanismus der ATP-Hydrolyse zu schließen. Wir zeigen, dass die Hydrolyserate von ATP innerhalb des Filaments konstant ist, was dem sogenannten zufälligen Hydrolysemechanismus entspricht und im Gegensatz zum sogenannten vektoriellen Mechanismus steht.

Most urinary tract infections in humans and dogs are caused by uropathogenic strains of , and increasing antimicrobial resistance among these pathogens has created a need for a novel approach to therapy. Bacterial iron uptake and metabolism are potential targets for novel antimicrobial therapy, as iron is a limiting factor in . growth during infection. As a trivalent metal of similar atomic size to iron (III), gallium can interact with a wide variety of biomolecules that normally contain or interact with iron. Gallium compounds disrupt bacterial iron metabolism, are known to accumulate at sites of infection and inflammation in mammals, exert antimicrobial activity against multiple bacterial pathogens in vitro, and may be good candidates as novel antimicrobial drugs. We assessed the suitability of orally administered gallium maltolate as a potential new antimicrobial therapy for urinary tract infections by evaluating its distribution into the bladder, its activity against uropathogenic . in vitro, and its pharmacokinetics and efficacy in a mouse cystitis model. Using a novel application of synchrotron-based analytical methods, we confirmed the distribution of gallium to the bladder mucosa and characterized the relationship between iron and gallium distribution in the bladder. In vitro experiments with human and canine uropathogenic . isolates demonstrated that gallium maltolate exerts antimicrobial effects in a time-dependent, bacteriostatic manner. Minimum inhibitory concentrations ranged from 0.144 µmol/mL to >9.20 µmol/mL with a median of 1.15 µmol/mL. Isolates resistant to ampicillin, ciprofloxacin, or with decreased susceptibility to cephalothin were susceptible to the antimicrobial activity of gallium maltolate, suggesting that resistance to conventional antimicrobials does not predict resistance to gallium maltolate. Pharmacokinetic studies in healthy mice and in a mouse model of urinary tract infection confirmed that gallium is absorbed into systemic circulation after oral administration of gallium maltolate. Gallium is slowly eliminated from the body, with a trend toward longer terminal half-lives in blood and bladder for infected mice relative to healthy mice. This study did not reveal any statistically significant effect of infection status on maximum blood gallium concentrations (4.46 nmol/mL, 95% confidence interval 2.75 nmol/mL – 6.18 nmol/mL and 4.80 nmol/mL, 95% confidence interval 2.53 nmol/mL – 7.06 nmol/mL in healthy and infected mice, respectively) or total gallium exposure in blood and kidney as represented by area under the concentration vs. time curves. Gallium exposure in the bladder was significantly greater for mice with urinary tract infections than for healthy mice. The investigation of gallium distribution within tissues represented a novel application of synchrotron-based analytical techniques to antimicrobial pharmacokinetics. Prior to analysing tissue samples, a library of x-ray absorption spectra was assembled for gallium compounds in both the hard and soft x-ray ranges. The suitability of hard x-ray fluorescence imaging and scanning and transmission x-ray microscopy for localizing and speciating trace elements in tissues was subsequently assessed. Of these methods, only hard x-ray microprobe analysis was well-suited to the analysis and was successfully used for this application. This approach confirmed that gallium arrives at the bladder mucosa after oral administration of gallium maltolate. Furthermore, comparison of iron and gallium distribution within the bladder mucosa indicated that these elements are similarly but not identically distributed and that they do not significantly inhibit one another’s distribution. This finding suggests that gallium may be distributed in part via pathways that do not involve iron. Despite the favorable distribution characteristics of gallium and the persistence of gallium in target tissues following the oral administration of gallium maltolate, its efficacy in a mouse model of urinary tract infection was disappointing. In this study, no statistically significant difference was detected between gallium maltolate, ciprofloxacin and sham treatments in their ability to eliminate bacteria in the urinary tracts. The failure of ciprofloxacin treatment to render bladder tissue culture-negative for an organism that is classified as ciprofloxacin-susceptible in vitro is consistent with observations from other research groups. The similar lack of efficacy observed for gallium maltolate may be related to the large gap between minimum inhibitory concentrations observed in vitro and gallium concentrations observed in tissues from treated mice, but may also be related to the small study size if the effect size of gallium maltolate treatment is small. Given the magnitude of the difference between tissue concentrations and minimum inhibitory concentrations, it may not be possible to increase the dose sufficiently to achieve therapeutic concentrations without causing toxicity. While the results of these experiments suggest that orally administered gallium maltolate may not be a reasonable antimicrobial drug candidate for treating urinary tract infections caused by uropathogenic . , it may be useful for other applications. Other bacterial pathogens may be more susceptible to the antimicrobial effects of gallium maltolate, and local or topical administration could produce much higher concentrations than we observed following oral administration. Continued development of the synchrotron-based analytical techniques used in these experiments could provide new and important opportunities to investigate antimicrobial distribution and metabolism within cells and tissues, particularly for metal-based drugs.

No description available.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes the application of wide-field single-molecule fluorescence microscopy techniques to investigations of DNA-surface interactions on chemically graded organosilane surfaces. The adsorption and desorption behaviors of double-stranded (ds) plasmid DNA along the amino-trimethoxysilane and octyl-trichlorosilane gradients were explored as a function of solution pH, solution ionic strength and surface properties. The results provide an improved fundamental understanding of DNA interactions with different surfaces and are certain to aid in the development and advancement of DNA-based biological and biomedical devices. Three distinct experiments were performed in completion of the work for this dissertation. In the first study, total internal reflection fluorescence (TIRF) microscopy was employed to study DNA interactions with aminosilane gradient surfaces under relatively acidic and basic environments. Electrical potentials were applied to assist DNA adsorption and desorption. The single-molecule data clearly showed that DNA capture and release was achieved on the monolayer and submonolayer coated regions of the aminosilane gradient surface under relatively basic pH conditions, with the help of an electrical potential. Meanwhile, DNA adsorption was found to be quasi-reversible on the multilayers at the high aminosilane end of the gradient in the relatively acidic solution. The results of these studies demonstrate the importance of manipulating the electrostatic interactions of DNA with charged surfaces in order to achieve DNA capture and release. The fundamental knowledge of the DNA-surface interactions gained in these studies will be helpful in diverse fields ranging from the layer-by-layer assembly of polyelectrolyte-based thin films to the selective electronic sensing of charged biomolecules. In the second study, the local dielectric properties of the least polar environments in dsDNA were assessed by using the solvatochromic dye, nile red, as a polarity-sensitive probe. TIRF spectroscopic imaging methods were employed in these studies. Although the dielectric constant within the least polar regions of dsDNA was previously predicted by theoretical and computational methods, no experimental measurements of its value had been reported to date. The results provide important knowledge of the factors governing the polarity of DNA microenvironments to which intercalators bind, and provide vital experimental support for the values determined in computational studies. In the third study, TIRF microscopy and single molecule tracking methods were employed to study DNA interactions with an opposed two-component C8-silane and aminosilane gradient surface as a function of solution pH. The mobility of surface-adsorbed DNA molecules was explored and quantified in these studies. The preliminary results further demonstrated the importance of electrostatic interactions over hydrophobic interactions in governing the adsorption of DNA to surfaces. The mobility of surface-adsorbed DNA was found to be largely independent of position along the two-component gradient. These studies were originally undertaken as a route to observation of cooperative effects that are believed to govern DNA-surface binding. Unfortunately, no clear evidence of cooperative effects was observed at the mixed regions of the two-component gradient surface.

No description available.