This research examines the impact of formal and informal labeling on self-reported violent and non-violent delinquency. This longitudinal research design utilizes cohort 15 from the Project on Human Development in Chicago Neighborhoods (PHDCN) at two different points in time. This research not only evaluates the effect of formal labeling (arrest) but also determines the effect of informal labeling (warning and releasing) by police officers. Specifically, the hypotheses assess if labeling affects minority youth differently than white youth. Using nested ordinary least square re and logistic regression models, the results indicate that labeling only operates in simple models containing few variables. The apparent effects of labeling on non-violent delinquency are accounted for in the complete model by previous delinquent behavior, living in a two-parented home, and having a parent with a criminal record. Moreover, the apparent effects of labeling are diminished in the complete model predicting violent delinquency once controlling for the effects of race (Black), gender (male), lower income level, and having a parent with a criminal record.

Les molécules marquées par des isotopes de l’hydrogène possèdent de nombreuses applications dans divers domaines tels que la chimie, la biologie ou en science des matériaux. Dans le domaine de la recherche de nouveaux médicaments, les études liées à la pharmacocinétique nécessitent un accès rapide à des molécules marquées afin de ne pas impacter les coûts et les délais de développement. Le développement de la métabolomique a aussi entrainé une augmentation du besoin en molécules marquées isotopiquement. En effet, les molécules deuterées peuvent être utilisées en tant qu’étalons internes pour la quantification rapide des métabolites présents dans des tissus ou des fluides biologiques. La première partie de cette thèse concerne le développement d’une méthode générale de marquage de motifs de type thioéther dans des molécules complexes à l’aide d’une nouvelle réaction d’échange isotopique (catalysée par des nanoparticules de Ruthénium). D’un point de vue fondamental cette transformation représente le premier exemple de (Csp³)-H activation dirigée par un atome de soufre. En termes d’application, cette nouvelle réaction permet la synthèse rapide d’étalons internes pour la quantification LC-MS/MS et le marquage tritium de molécules complexes. La seconde partie de cette thèse relate le développement d’une nouvelle méthode d’homocouplage de phénylpyridines catalysée par Ru/C. Différents substrats comportant des substituants riches et pauvres en électron ont été couplés avec de bons rendements. Ces dimères ont ensuite été utilisés pour synthétiser de nouveaux complexes de bore dont les propriétés photophysiques ont été étudiées. Dans une troisième partie, la mise au point d’une réaction palladocatalysée permettant d’obtenir des molécules polycycliques contenant un motif de type pyridine est développée. / Deuterated and tritiated compounds are widely used in numerous applications in chemistry, biology and material science. In the drug discovery and development process, ADME studies require quick access to labelled molecules, otherwise the drug development costs and timeline are significantly impacted. The rapid development of metabolomics has also increased the need for isotopically labelled compounds. In particular, deuterated molecules are used as internal standards for quantitative LC-MS/MS analysis of metabolites in biological fluids and tissues. In this context, a general method allowing the deuterium and tritium labelling of bioactive thioethers using a HIE reaction is described in the first chapter. From a fundamental point of view, this transformation is the first example of (Csp³)-H activation directed by a sulfur atom. In terms of application, this new reaction has been proved to be useful for the preparation of deuterated LC-MS/MS reference materials and tritiated pharmaceuticals owning high specific activity.In the second chapter of this manuscript, the development of a method allowing the cross-dehydrogenative homocoupling of 2-arylpyridines catalyzed by Ru/C is developed. Various substrates with different substituents were efficiently coupled to give the desired dimers in good yield. In terms of application, a series of pyridine-boron complexes derived from the phenyl pyridine dimers were also synthesized and their photophysical properties were studied.In the third chapter, a regioselective palladium catalyzed intramolecular arylation reaction allowing the synthesis of pyridine containing polycyclic compounds is described.

A la lumière, l’activité carboxylase de la RuBisCO permet de fixer le CO2 inorganique en matière organique, sous forme de 3-phosphoglycérate (3-PGA), qui sera utilisé pour la biosynthèse de sucres, d’acides organiques et aminés, de la paroi végétale etc. Cependant, elle possède aussi une activité oxygénase qui produit du 3-PGA et du 2-phosphoglycolate. Ce dernier composé étant toxique, il est métabolisé en 3-PGA par le cycle photorespiratoire qui se déroule dans le chloroplaste, le peroxysome et la mitochondrie. Malgré une perte partielle en carbone et en azote, l’importance de la photorespiration pour les plantes est illustré par les phénotypes néfastes que les mutants d’enzymes photorespiratoires présentent dans l’air (comme un retard de croissance, la chlorose, et de la létalité) et qui sont absents en fort CO2. Ceci pourrait refléter des interactions étroites entre la photorespiration et le métabolisme primaire des plantes. Afin de mieux comprendre ces interactions et la mise en place des phénotypes photorespiratoires, des mutants pour la glycolate oxydase (GOX) et la glutamate:glyoxylate aminotransférase ont été caractérisés à travers plusieurs analyses complémentaires: des échanges gazeux, de la fluorescence chlorophyllienne, du marquage des métabolites avec du 13C, des dosages de métabolites, de cofacteurs, et de la RuBisCO. Les résultats montrent que, suite à un transfert de fort CO2 dans l’air, l’inhibition de la photosynthèse observée chez nos mutants est principalement due à un défaut du recyclage du carbone photorespiratoire qui diminue l’activité de la RuBisCO. Cette inhibition photosynthétique a un impact négatif sur la quantité de RuBisCO dans les feuilles de ces mutants par rapport aux plantes contrôles. De plus, lorsque l’inhibition de la photosynthèse est trop importante chez nos mutants photorespiratoires, la carence en carbone déclenche de la sénescence dans leurs feuilles âgées. En parallèle, une comparaison des paramètres cinétiques de la GOX d’A. thaliana (plante en C3) et de Z. mays (plante en C4) associée à la mesure d’effets isotopiques 13C et 2H a révélé que ces enzymes partageaient des paramètres Michaéliens équivalents pour le glycolate, ainsi qu’un mécanisme réactionnel identique mettant en jeu un transfert d’hydrure. / In the light, the RuBisCO carboxylase activity assimilates inorganic CO2 into organic compounds, via the production of 3-phosphoglycerate (3-PGA) that is used for the biosynthesis of sugars, organic and amino acids, plant cell walls etc. However, it also has an oxygenase activity that makes 3-PGA and 2-phosphoglycolate (2-PG). The toxic 2-PG is metabolized to 3-PGA by the photorespiratory cycle, which takes place in chloroplasts, peroxisomes and mitochondria. Despite a partial loss of carbon and nitrogen, the importance of photorespiration for growth can be seen by the negative phenotypes exhibited by photorespiratory enzyme mutants in air (i.e. slow growth, leaf chlorosis, and sometimes lethality), which are not observed under high CO2 conditions. This may reflect the metabolic interactions between photorespiration and plant primary metabolism. To better understand such interactions and the development of photorespiratory phenotypes, mutants for glycolate oxidase (GOX) and glutamate:glyoxylate aminotransferase have been characterized by several complementary methods: analysis of gas exchanges, chlorophyll fluorescence,13C-labeling of metabolites, measurements of metabolites, cofactors and RuBisCO levels. The results show that, after a high CO2-to-air transfer, the inhibition of photosynthesis in the mutants is mainly due to a defect in photorespiratory carbon recycling leading to a decreased RuBisCO activity. The inhibition of carbon assimilation negatively impacts mutant leaf RuBisCO content when compared to wild-type plants. In the mutants, when photosynthetic inhibition is too high, the resulting carbon starvation triggers the onset of senescence in their old leaves. In parallel to this work, a comparison of the kinetic parameters of GOX from A. thaliana (C3 plant) and Z. mays (C4 plant) coupled to measurements of 13C and 2H kinetic isotopic effects showed that these enzymes share similar Michaelian parameters for glycolate, and a similar hydride transfer reaction mechanism.

Introducción: El sobrepeso y la obesidad han alcanzado prevalencias epidémicas a nivel mundial. Una de las estrategias propuesta por la Organización Mundial de la Salud para revertir el sobrepeso y la obesidad, es utilizar el etiquetado nutricional frontal. Por ello, en el año 2018 en el Perú se aprobó el Manual de Advertencias Publicitarias (MAP), para que los productos ultra-procesados lleven en su empaque entre 1-3 Advertencias Octogonales (AO), según lo requieran. En el MAP se establecen dos parámetros. El primer parámetro entró en vigencia en enero del 2019 y el segundo entrará en vigencia en septiembre del 2021. Cabe resaltar que el presente estudio se inició en marzo del 2018, es decir, antes de que entrara en vigencia la implementación de las AO. Objetivo: Determinar la proporción de productos ultra-procesados que declararían al menos una advertencia octogonal para septiembre del 2021, en cumplimiento de la Ley N° 30021. Materiales y Métodos: Estudio exploratorio en el que se analizaron 786 fotografías de etiquetas de productos ultra-procesados. Según el perfil de nutrientes de la Organización Panamericana de la Salud, se clasificó en 10 categorías. El contenido nutricional (energía, azúcar, grasas totales, grasas saturadas y sodio) de los productos ultra-procesados se calculó en 100 gramos o mililitros de producto. Los parámetros finales establecidos en el MAP, se usaron para evaluar la cantidad de AO que declararían los productos ultra-procesados en caso de no modificar su formulación para septiembre del 2021. Resultados: Se encontró que 88,1% de los productos ultra-procesados llevarían al menos una AO para septiembre del 2021. El 46,2% llevaría una AO, el 38,2% llevarían dos AO, mientras que el 3,7% declararía las tres AO. De los productos ultra-procesados que llevarían una AO, el 80,7% declararía la frase “ALTO EN AZÚCAR”, mientras de los que llevarían dos AO, el 65,6% declararía las frases “ALTO EN GRASAS SATURADAS” y “ALTO EN AZÚCAR”. Conclusión: El 88,1% de los productos ultra-procesados llevarían al menos una AO para septiembre del 2021, en cumplimiento de la Ley N° 30021. / Introduction: Overweight and obesity have reached epidemic prevalences worldwide. One of the strategies proposed by the World Health Organization to reverse overweight and obesity, is to use frontal nutritional labeling. That is why, in 2018 in Peru the Manual of Advertising Warnings (MAW) was approved so that ultra-processed products carry in their packaging between 1-3 Octagonal Warnings (OW), as required. The MAP establishes two parameters. The first parameter entered into force in January 2019 and the second will enter into in September 2021. It should be noted that this study began in March 2018, that is, before the AO implementation went into effect. Objective: Determine the proportion of ultra-processed products that would declare at least one Octagonal Warning by September 2021, in compliance with Law N° 30021. Materials and Methods: Exploratory study in which 786 photographs of labels of ultra-processed products were analyzed. According to the nutrient profile of the Pan American Health Organization, it was classified into 10 categories. The nutritional content (energy, sugar, total fat, saturated fat and sodium) of the ultra-processed products was calculated in 100 grams or milliliters of product. The final parameters established in the MAP were used to evaluate the amount of OW that would be declared by ultra-processed products in case of not modifying their formulation for September 2021. Results: It was found that 88.1% of the ultra-processed products would carry at least one OW by September 2021. 46.2% would wear an OW, 38.2% would wear two OW, while 3.7% would declare the three OW. Of the ultra-processed products that would carry an OW, 80.7% would declare the phrase “HIGH IN SUGAR”, while of those that would take two OW, 65.6% would declare the phrases “HIGH IN SATURATED FATS” and “HIGH IN SUGAR”. Conclusions: The 88.1% of ultra-processed products would carry at least one OW by September 2021, in compliance with Law N°. 30021. / Tesis

Neurosteroids are an important group of substances that affect communication between neurons. They act as allosteric modulators of membrane receptors for neurotransmitters. One of the most important systems influenced by neurosteroids are NMDA receptors; however, a binding site(s) for their inhibition by steroids have not been found yet. This work is focused on the synthesis of fluorescently labeled photoaffinity probe, which may help explain the structure and location of binding site(s) and simplify the development of new neuroprotectives. A structural analogue of the endogenous neurosteroid, (20S)-20-Azido-5β-pregnan- 3α-yl N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-L-glutamyl 1-ester (8), was prepared. The structure of compound 8 includes photolabile azido group, as well as covalently bounded fluorescent NBD group. In addition, a photoaffinity probe with a modified steroid skeleton - pyridinium 17aα-azido-17α-methyl-17a-homo-5β-androstan-3α-yl 3-sulfate (29) - was synthesized. The ability of compound 8 and 29 to inhibit activated NMDA receptor has been verified for recombinant NR1-1a/NR2B receptors expressed in HEK293 cells using a patch-clamp technique. Additionally, the IC50 values of compounds 8 and 29 have been calculated. (In Czech) Key words: neuroactive steroid, NMDA receptor, photoffinity...

This work is focused on the interactome study of 14-3-3ζ protein, a regulatory protein found in all eucaryotic cells. An important 14-3-3 protein feature is the ability to bind a number of structurally and functionally distinct protein ligands. This link is usually implemented through phosphorylated serine and threonine motifs. The first aim of this work is the preparation of sufficient amount of recombinant 14-3-3ζ protein with incorporated photoactivatable analogue of methionine (foto-Met, L-2-amino-5,5- azihexan acid). The four different conditions of recombinant expression in auxotrophic E. coli B834 (DE3) strain were tested to obtain a protein with a maximal rate of photoactivatable methionine analogue incorporation into the sequence 14-3-3 protein. The second aim is to study the methionine 121, 160 and 218 participation in the 14-3-3ζ protein binding groove and finding of potential covalent bond with the phosphorylated peptide 251-266 of Raf-1 kinase (phosphorylation on Ser259). The photo-initiated cross-linking method was used (photolysis), to form a reactive biradical of methionine analogue capable to attack any amino acid residues in close vicinity (till 5Å). Finally, the products of photo-initiated cross-linking were analyzed by cross-linking reactions using MALDI-TOF MS, LC-MS and...

Structural proteomic methods such as an ion mobility mass spectrometry, chemical cross- linking or covalent labeling have been established as powerful tools for structural studies of biomolecules in general. These methods have significantly contributed to the expansion of our knowledge about biomolecular functions, their dynamics and molecular interactions and therefore led to the understanding of important biological processes occurring in a cell. We decided to spread these methods and we developed a new radical labeling technique relaying on Fluor-alkyl radicals that does not require a laser dissociation pf hydrogen peroxide. We exploited the potential of Togni reagents to form Fluor-alkyl radicals by reducing agent under native conditions. The induction of Fluor-alkyl radicals was triggered by ascorbic acid and the labeling pulse was stopped by tryptophan. The modified proteins were analyzed by top down or bottom up approach using high resolution mass spectrometry. In case of top down approach, several fragmentation techniques (CID-ECD, ETD) were tested for protein analysis while in case of bottom up approach the analyzed proteins were digested by trypsin and separated on reverse phase column online coupled to mass spectrometer. As the model biomolecules we chose a 20 proteinogenic amino acids...

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Tidigare forskning gällande insatsbeviljade familjer visar på att klienterna är stigmatiserade eller stämplade. Syftet med studien är att undersöka, bland socialarbetare inom barn- och familjeenheten, medvetenheten om och arbetet med stigmatisering och stämpling gällande de familjer de har kontakt med i sitt arbete inom socialtjänsten. Stigmateori och stämplingsteori utgjorde studiens teoretiska tolkningsram. Studien genomfördes med en kvalitativ ansats i form av sex semistrukturerade intervjuer som sedan analyserades med hjälp av en tematisk analys vilket härledde till två teman: bortprioriterat arbete och indirekt arbete. Studiens resultat visade på att medvetenheten hos socialarbetarna om risken att klienterna var stigmatiserade och stämplade var låg. Det saknades riktlinjer och rutiner gällande ämnet och att arbetet som gjordes för att stödja familjerna var indirekt och inte preciserat för bearbetning av stigmatisering och stämpling. Slutsatsen drogs att en ökad medvetenhet bland socialarbetarna som möter familjer inom socialtjänsten gällande risken att familjerna stigmatiseras och stämplas behövs. / Previous research has shown that families receiving interventions from the social services are stigmatized and labeled. The purpose of this study is to examine, among social workers within the family unit’s awareness of and work with stigmatization and labeling regarding the families they meet in their work. Stigma and labeling theory make up the theoretical framework for the study. It was conducted with a qualitative approach with six semi-structured interviews, the data was analyzed with a thematic analysis. Two themes emerged: de-prioritized work and indirect work. The results showed that the social workers awareness about family’s risk of being stigmatized and labeled was low. Guidelines and routines were missing and the work to support the families was indirect and not specified for processing stigma and labeling. It was concluded that there needed to be a greater awareness of the stigmatization and labeling of the clients among the social workers.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.:Abstract Table of contents 1 Introduction 1.1 The transforming principle 1.2 Chromatin 1.2.1 Nucleosomes 1.2.2 The 30 nm fibre: a mirage? 1.2.3 Histone code 1.3 Histone variant H2A.Z 1.3.1 H2A.Z and transcription 1.4 Single molecule studies of chromatin 1.4.1 Chromatin under tension 1.4.2 Open nucleosome 1.4.3 Twisted chromatin 1.5 Single molecule techniques 1.5.1 Atomic force microscopy 1.5.2 Foerster resonance energy transfer 1.5.3 Magnetic tweezers 1.5.4 Worm-like chain model 2 Aims of the project 3 Cut and paste method for internal DNA labelling 3.1 Introduction 3.2 Experimental design 3.3 Results 3.3.1 Sequence design and cloning 3.3.2 Labelling and religation efficiency 3.3.3 Structural modifications 3.3.4 Labelling of multiple loci 3.3.5 Opposite-strand labelling 3.4 Discussion 4 Reconstituting chromatin 4.1 Long array of NPSs 4.1.1 Polymer physics applied to molecular cloning 4.1.2 Preventing homologous recombination 4.2 Expression and purification of histone proteins 4.2.1 Protein expression 4.2.2 Inclusions bodies 4.2.3 Histone purification 4.2.4 Octamer reconstitution and isolation 4.2.5 H2AvD 4.3 Reconstitution of nucleosomal arrays and biochemical analysis 4.3.1 Reconstitution procedure 4.3.2 Biochemical analysis 4.4 Tweezers construct with nucleosomes 5 Eviction of nucleosomes 5.1 Nucleosome eviction 5.1.1 A two-stage process 5.1.2 Chromatin fibres 5.1.3 Reassembly of nucleosomes 5.1.4 Distinct populations within nucleosome eviction events 5.1.5 Nicked and supercoilable nucleosomal arrays 5.2 Eviction of H2AvD-nucleosomes 5.2.1 H2AvD-nucleosomes bind less inner turn DNA 5.2.2 H2AvD-nucleosomes evict at lower forces 5.2.3 Likelihood of nucleosome reassembly 5.2.4 Gradual weakening of nucleosomes 5.2.5 Analysis software NucleoStep 5.3 Towards a rotor-bead assay on chromatin 5.4 Discussion 5.4.1 Nucleosome eviction in two stages 5.4.2 The fate of dimers in single molecule experiments 5.4.3 Structural origin and biological relevance of the mechanical properties of H2AvD-nucleosomal core particles 5.4.4 Monolithic or dynamic nucleosomes 6 Conclusions Bibliography Appendix 6.1 Internal labelling Procedure 6.1.1 Cloning 6.1.2 Nicking & cutting 6.1.3 The replace reaction 6.1.4 Purification 6.1.5 Ligation (optional) 6.1.6 Opposite strand labelling 6.1.7 Assessing the results of the labelling reaction 6.2 Chromatin reconstitution 6.2.1 Long array of NPSs 6.2.2 Expression and purification of histone proteins 6.2.3 Reconstitution of nucleosomal arrays and biochemical characterization 6.2.4 Simple Phenol:chloroform isolation of DNA 6.3 Magnetic tweezers experiments 6.3.1 Flow cell assembly 6.3.2 Functionalization of flow cells 6.3.3 Magnetic tweezers and rotor bead measurements 6.3.4 Force calibration List of Figures List of Tables List of publications Acknowledgements Declaration of originality