Au cours de la dernière décennie, les anticorps conjugués à des médicaments cytotoxiques ou des oligonucléotides ont acquis une grande attention dans la communauté scientifique en raison des propriétés uniques des anticorps, tels que leur long temps de circulation dans le sérum et leur sélectivité élevée par rapport à leur cible. Par exemple, les conjugués d'anticorps (ACs) sont de plus en plus appliqués en thérapie ciblée contre le cancer ou en bioimagerie. Par conséquent, le développement de méthodologies fiables pour la préparation des AC est actuellement en pleine expansion. Cependant, la conjugaison et la préparation contrôlables des ACs avec une structure définie rencontrent encore de nombreux obstacles en raison de l'excès élevé et de la variété des groupes réactifs dans la structure des anticorps, qui sont accessibles pour la conjugaison. En outre, les technologies de liaison actuelles sont basées sur la réaction de maléimide-thiol, produisant des adduits, qui sont instables dans le sang. Ce travail se concentre sur les approches chimiques pour la fonctionnalisation fiable des anticorps, qui permettent la préparation d'ACs stables présentant un ratio anticorps/principe actif bien défini. La première partie est consacrée à la conception et au développement du réactif maléimide-dioxane, solution auto-hydrolysable et stable dans le sérum, comme alternative à la chimie classique du maléimide. La deuxième partie est consacrée à l'évaluation de la réactivité sélective des différents acides aminés portés par les anticorps par spectrométrie de masse native à haute résolution. Finalement, une nouvelle technologie permettant d’obtenir des ACs stables avec un ratio anticorps/principe actif contrôlé est présentée au lecteur dans une 3ème partie. / Within the last decade, antibodies conjugated to cytotoxic drugs or oligonucleotides have gained a great attention in scientific community owing to the unique properties of the antibodies, such as their long circulation time in serum and high selectivity against their target. For instance, antibody conjugates (ACs) are increasingly applied for targeted cancer therapy or bioimaging. Consequently, the development of reliable methodologies for ACs preparation is currently of high demand. However, the controllable conjugation and preparation of ACs with defined structure are still challenging due to high excess and variety of reactive groups in antibody structure, which are accessible for conjugation. Moreover, current linker technologies are based on the maleimide-thiol reaction, yielding adducts, which are unstable during circulation in blood.This work is focused on chemical approaches for the reliable antibody functionalisation, which enable the preparation of stable ACs with well-defined payload to antibody ratios. The first part is devoted to design and development of maleimide-dioxane reagents as self-hydrolysable and serum-stable alternative to classical maleimide chemistry. The second part is dedicated to a screening approach for evaluation of residue-selective functionalities in reactions with an antibody using high resolution native mass spectrometry. Finally, in the third part the reader is introduced with a novel technology, which enables efficient preparation of stable ACs with a defined degree of conjugation and particularly mono-functionalisation of antibodies.

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Neste trabalho estudamos teórica e experimentalmente o efeito de Conjugação de Fase por Mistura Degenerada de Quatro Ondas (CFMD40) e Modulação Transversal de Fase em cristais dopados com Cr+3. Estudamos a CFMD40 em Al2O3:Cr+3 (rubi) e GdAlO3:Cr+3 com um laser de Ar (em λ=514nm) obtendo um bom acordo entre os resultados experimentais e os teóricos (nestes cálculos usamos os valores de n2 de um trabalho anterior [1]). O modelo teórico que fizemos explica muito bem o comportamento de saturação da eficiência da CFMD40 que não era compreendido em trabalhos anteriores [47,48.2]. Usando os mesmos valores de n2 obtivemos um bom acordo entre os resultados experimentais e teóricos para o efeito de Modulação Transversal de Fase. Também fizemos um modelo teórico para o efeito de Modulação Transversal de Fase em CFMD40 que explica nossas observações [2]. A não linearidade destes materiais foi investigada usando-se três técnicas experimentais diferentes [1.2] e por dois outros grupos [61.62] (para o rubi) através de mistura de duas ondas não degeneradas. Todas estas medidas estão em bom acordo. Na alexandrita (BeAl2O 4:Cr+3) estudamos o espectro de χ(3) (ou n2) em ressonância das linhas R. Nos atribuímos a forma assimétrica do espectro como sendo oriunda de duas contribuições para susceptibilidade, onde um termo é devido a interação ressonante com o sistema de dois níveis e o outro devido a mudança de polarizabilidade causada pela população do estado excitado (esta é a primeira vez que este efeito foi observado). / In this work we studied theoretical and experimentally the effects of Phase Conjugation by Degenerate Four Wave Mixing and Transversal Phase (PCD4WM) Modulation in Cr+3 doped crystals. We studied the PDC4WM in Al2O3:Cr+3 (ruby) and GdAlO3:Cr+3 with on Ar laser (at λ= 514 nm) and obtained a good agreement between our experimental and theoretical results (in these ca1culations we used the nonlinear refractive index n 2 values from a previous paper [1]. The theoretical model that we developed explains very well the saturation behaviour of the PDC4WM efficiency that was not understood in previous papers [47.48,2]. These values of n2 are also in good agreement with our results in Transverse Phase Modulation. We also developed a theoretical model for the effect of Transverse Phase Modulation in PCDFWM that explains our observations [2]. The nonlinearity ?n IND.2? from these materials was investigated by us using three different techniques [1.2], by other two groups [61.62] (for the ruby) in nondegenerate two-wave mixing and all those measurements are in good agreement. In alexandrite (BeAl2O4:Cr +3) we studied the χ(3) (or n2) spectrum in resonance with the R lines. We attributed the asymetric shape of the spectrum by the effect of two differents contributions, one term due to the resonant interaction of the two-level system and the other due to the polarizability change caused by excited state population (this is the first observation of this kind of effect.

Neste trabalho estudamos teórica e experimentalmente o efeito de Conjugação de Fase por Mistura Degenerada de Quatro Ondas (CFMD40) e Modulação Transversal de Fase em cristais dopados com Cr+3. Estudamos a CFMD40 em Al2O3:Cr+3 (rubi) e GdAlO3:Cr+3 com um laser de Ar (em λ=514nm) obtendo um bom acordo entre os resultados experimentais e os teóricos (nestes cálculos usamos os valores de n2 de um trabalho anterior [1]). O modelo teórico que fizemos explica muito bem o comportamento de saturação da eficiência da CFMD40 que não era compreendido em trabalhos anteriores [47,48.2]. Usando os mesmos valores de n2 obtivemos um bom acordo entre os resultados experimentais e teóricos para o efeito de Modulação Transversal de Fase. Também fizemos um modelo teórico para o efeito de Modulação Transversal de Fase em CFMD40 que explica nossas observações [2]. A não linearidade destes materiais foi investigada usando-se três técnicas experimentais diferentes [1.2] e por dois outros grupos [61.62] (para o rubi) através de mistura de duas ondas não degeneradas. Todas estas medidas estão em bom acordo. Na alexandrita (BeAl2O 4:Cr+3) estudamos o espectro de χ(3) (ou n2) em ressonância das linhas R. Nos atribuímos a forma assimétrica do espectro como sendo oriunda de duas contribuições para susceptibilidade, onde um termo é devido a interação ressonante com o sistema de dois níveis e o outro devido a mudança de polarizabilidade causada pela população do estado excitado (esta é a primeira vez que este efeito foi observado). / In this work we studied theoretical and experimentally the effects of Phase Conjugation by Degenerate Four Wave Mixing and Transversal Phase (PCD4WM) Modulation in Cr+3 doped crystals. We studied the PDC4WM in Al2O3:Cr+3 (ruby) and GdAlO3:Cr+3 with on Ar laser (at λ= 514 nm) and obtained a good agreement between our experimental and theoretical results (in these ca1culations we used the nonlinear refractive index n 2 values from a previous paper [1]. The theoretical model that we developed explains very well the saturation behaviour of the PDC4WM efficiency that was not understood in previous papers [47.48,2]. These values of n2 are also in good agreement with our results in Transverse Phase Modulation. We also developed a theoretical model for the effect of Transverse Phase Modulation in PCDFWM that explains our observations [2]. The nonlinearity ?n IND.2? from these materials was investigated by us using three different techniques [1.2], by other two groups [61.62] (for the ruby) in nondegenerate two-wave mixing and all those measurements are in good agreement. In alexandrite (BeAl2O4:Cr +3) we studied the χ(3) (or n2) spectrum in resonance with the R lines. We attributed the asymetric shape of the spectrum by the effect of two differents contributions, one term due to the resonant interaction of the two-level system and the other due to the polarizability change caused by excited state population (this is the first observation of this kind of effect.

In the everlasting battle against cancer the development of drugs targeting new therapeutic pathways is of crucial importance. In the attempt to develop new anticancer agents we have synthesized a library of anthraquinone compounds that show selectivity against leukemia. Mechanistic evaluation of the lead compound reveal that this class of compounds achieve their effects through inhibition of MDM2-MDM4 heterodimer and upregulation of the tumor suppressor p53. Computer aided rational design resulted in the development of a number of compounds with activities in the nanomolar range against various cancer cells. Analysis of the physicochemical properties of selected compounds allowed for their evaluation as potential drug candidates. The successful development of non-toxic formulations permits for the further in vivo investigation of the compounds. Click reactions have found wide spread applications in sensing, materials chemistry, bioconjugation, and biolabeling. A number of very useful click reactions have been discovered, which allow for various applications. In bioconjugation applications, the ability to conduct a secondary conjugation will be very useful in, e.g., protein pull down and binding site identification. Along this line, we describe a neighboring group-assisted facile condensation between an aldehyde and a vicinal aminothiol moiety, leading to the formation of benzothiazoles. The conversion is completed within 5 minutes at low micromolar concentrations at ambient temperature. The facile reaction was attributed to the presence of a neighboring boronic acid, which functions as an intramolecular Lewis Acid in catalyzing the reaction. The boronic acid group is compatible with most functional groups in biomolecules and yet can also be used for further functionalization via a large number of well-known coupling reactions.

Horizontal gene transfer (HGT) is now indisputably the predominant driving force, if not the sole force, behind speciation and the evolution of novelty in bacteria. Of all mechanisms of horizontal gene transfer (HGT), conjugation, the contact-dependent plasmid-mediated transfer of DNA from a bacterial donor to a recipient cell, is probably the most universal. First observed between bacteria, conjugation also mediates gene transfer from bacteria to yeast, plant and even animal cells. The range of environments in which bacteria naturally exchange DNA has not been extensively explored. The interior of the animal cell represents a novel and potentially medically relevant environment for gene transfer. Since most antibiotics are ineffective inside mammalian cells, our cells may be a niche for the evolution of resistance and virulence in invasive pathogens. Invading bacteria accumulate in vacuoles inside human cells, protected from antibiotics. Herein, I demonstrate the ability of intracellular Salmonella typhimurium to meet and exchange plasmid DNA by conjugation within animal cells, revealing the animal intracellular milieu as a permissive environment for gene exchange. This finding evokes a model for the simultaneous dissemination of virulence and antibiotic resistance within a niche protected from both antibiotics and the immune system and extends the variety of environments in which bacteria are known to exchange genes. Unlike conjugation between bacteria, conjugation between bacteria and eukaryotic cells requires the import of transferred DNA into the nucleus before the transferred genes can be expressed and inherited. Plant-cell nuclear transformation by the conjugation system of the Agrobacterium tumefaciens Ti plasmid is believed to be mediated by nuclear localization sequences (NLSs) carried within the proteins that accompany the T-DNA during transfer. Whether NLSs are equally important for transmission of other conjugative plasmids to eukaryotic cells is unknown. Herein, I demonstrate nuclear localization potential within the putative conjugative escort protein TraI of the IncPa plasmid RP4. In contrast, MobA, the putative escort protein from the IncQ plasmid RSF1010, lacked any clear nuclear localization potential. It is therefore likely that specific nuclear localization signals within conjugative proteins are not essential for nuclear transformation per se, although they may assist in efficient plasmid transmission.

This paper presents the early-stage simulation results of linear dark field control (LDFC) as a new approach to maintaining a stable dark hole within a stellar post-coronagraphic PSF. In practice, conventional speckle nulling is used to create a dark hole in the PSF, and LDFC is then employed to maintain the dark field by using information from the bright speckle field. The concept exploits the linear response of the bright speckle intensity to wavefront variations in the pupil, and therefore has many advantages over conventional speckle nulling as a method for stabilizing the dark hole. In theory, LDFC is faster, more sensitive, and more robust than using conventional speckle nulling techniques, like electric field conjugation, to maintain the dark hole. In this paper, LDFC theory, linear bright speckle characterization, and first results in simulation are presented as an initial step toward the deployment of LDFC on the UA Wavefront Control testbed in the coming year.

The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. Results demonstrate the capture of a 30.8 kb SCCmec element on a conjugative plasmid for the first time, its transfer into both S. aureus and S. epidermidis recipients, and its excision from the plasmid with insertion in the orfX att site in recipients. The element was integrated into the plasmid by recombination between IS elements invariably present on all SCCmec types and pGO1/pSK41-like conjugative plasmids. These data explain the movement of SCCmec from reservoirs in commensal coagulase-negative staphylococci into different Staphylococcus aureus lineages using a ubiquitous conjugative plasmid that can transfer among staphylococci of different species and, thus, describes a mechanism for the environmental dissemination of methicillin resistance in nature.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / Antimicrobial resistance (AMR) has arisen as one of the most important public health concerns in the last 60 years. AMR results from pathogenic strains of bacteria adapting to antimicrobial-containing environments through mutations or through horizontal gene transfer (HGT) of genetic material containing resistance genes. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. This dissertation explores the factors, specifically catecholamines and antimicrobials that influence the conjugation frequencies of enteric bacteria including Salmonella, E. coli and Enterococcus. We found that the catecholamine norepinephrine (NE) at physiological concentrations enhanced conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an E. coli recipient in vitro. Additional experiments determined the influence of the antimicrobial concentrations above, equal to and below the minimum inhibitory concentration (MIC) under in vitro conditions on conjugation efficiencies using an Enterococcus to Enterococcus mating pair in addition to the Salmonella to E. coli mating pair. Conjugation occurred in all concentrations, but efficiencies of transfer were consistently low in 0 MIC and 1 MIC, with increased activity both above and below 1 MIC. These data were fit to a previously described mathematical model and the rate constant E that relates the rate of gene transfer to drug concentration was determined. The data showed highly similar patterns of conjugation efficiencies when compared to the rate constant E. A final study we measured conjugation frequencies when donor Salmonella Typhimurium and the E. coli recipient were exposed to both variable concentrations of oxytetracycline and NE. Conjugation was increased pre- and post- MIC, but conjugation frequencies were not enhanced further by the combination of the oxytetracycline and the NE. This dissertation defines the role of outside factors in conjugative gene transfer, and may provide future insight into better control of AMR.

Metabolomics is an emerging field that entails the detailed characterization of the ensemble of metabolites produced by living organisms; subfields include drug metabolism and natural environmental toxin production. The first part of the dissertation pursued metabolism of glyceollins, i.e., isoflavones produced by soybeans, that are potential cancer therapy agents. In vivo glyceollin metabolites produced in rats were investigated by on-line Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry. An odd-electron fragment ion at m/z 148, formed in violation of the even-electron rule, and diagnostic of the glyceollin backbone, was discovered. Based on this finding, a negative mode precursor ion scanning method was developed to screen for glyceollins and their metabolites from biological samples. Products of both Phase I and Phase II metabolism were identified, none of which have been previously reported. Sulfated metabolites were confirmed by accurate mass measurement, while glucuronide conjugation was confirmed by enzyme-assisted glucuronidation by rat liver microsomes. Intact GSH-glyceollin conjugates were not observed, but breakdown products of the GSH pathway, i.e., cysteinylglyceine, cysteine, and acetylated cysteine, were identified as conjugates of oxygenated glyceollins. The identification of GSH by-product conjugates was confirmed in product ion spectra acquired in the negative mode (where peptide anions, and glyceollin-bearing cleaved peptide portions were observed), as well as in the positive mode (where intact oxygenated glyceollin fragments appeared without the initially-present peptide portion). Mass spectral evidence strongly supports a metabolic pathway involving initial epoxidation of glyceollins followed by GSH addition at the epoxidation site. The second part of the dissertation undertook the investigation of secondary metabolites called microbial volatile organic compounds (MVOCs) produced by fungi (mold) that have been reported to have adverse human health effects. MVOCs were collected onto different sorbent materials and analyzed by Thermal Desorption Analysis coupled with on-line Gas Chromatography-Mass Spectrometry. Fungal MVOCs were characterized from various simulated flooding conditions (brackish, freshwater, and saltwater) and different substrates (nutrient rich vs. low nutrient) to determine diagnostic MVOCs. Ten fungi from simulated environments were identified by genetic sequencing. Cladosporium sp. and Chaetomium sp. were cultivated and their emitted MVOCs, 3-furaldehyde and 3-(4-hydroxy-3-methoxyphenyl)-2-propenal, were proposed as diagnostic indicators of these fungi.