Parmi les voies de signalisation, la voie hedgehog (HH) intervient dans la formation de la polarité segmentaire. Si elle est défectueuse, elle entraine plusieurs malformations. De nombreux cancers présentent une suractivation de cette voie. La voie HH activée par la fixation du ligand HH sur le récepteur Patched (hPtc) et fait intervenir plusieurs partenaires cytoplasmiques dont Supressor of Fused (SUFU).Peu de données moléculaires et structurales sont disponibles pour cette voie et pourtant, ces données sont nécessaires pour comprendre sans ambiguïté son fonctionnement. De plus, la voie HH a été proposée comme pouvant être la cible de traitements chimio thérapeutiques mais, la protéine hPtc est impliquée dans l’efflux des drogues anticancéreuses. Une inhibition de hPtc par la fixation de son ligand entraine l’inhibition de l’efflux de drogues. Néanmoins, le site de fixation de HH sur son récepteur n’a pas encore été déterminé.Durant cette thèse, les travaux effectués ont permis l’étude structurale de la protéine hPtc notamment la détermination du site de fixation de HH. Dans un deuxième volet de cette thèse, j’ai effectué des études structurales de certaines protéines SUFU.Dans un premier temps, je me suis concentrée sur les domaines extracellulaires de hPtc qui ont été décrits comme nécessaires pour la fixation du ligand HH. J’ai cloné une protéine chimère constituée de ces deux domaines liés par le lysozyme du phage T4 (hPtcD1D2). Cette construction a été exprimée dans la bactérie E.coli. Les conditions d’expression testées permettent d’obtenir la protéine sous forme de corps d’inclusion dans le cytoplasme de la bactérie. Dans un deuxième temps, j’ai cloné la protéine dans un vecteur d’expression en levure. De manière concomitante, j’ai exprimé la protéine hPtc tronquée de ses régions N et C terminales (hPtcΛNΛC). Ce sont des régions intrinsèquement désordonnées qui ne permettraient pas une bonne cristallisation de la protéine. L’expression a été effectuée dans la levure. La solubilisation de cette protéine membranaire est en cours d’expérimentation.Ce travail a permis de poser les bases de l’expression de hPtcD1D2 et de hPtcΛNΛC. Ceci va notamment permettre la surexpression de la protéine et sa cristallisation afin de déterminer sa structure 3D et de caractériser le site de fixation de son ligand.Enfin, j’ai entrepris des études structurales des protéines SUFU. Un nouveau site de fixation du Zn a été caractérisé. En effet, après purification de la protéine, j’ai effectué des mesures d’affinité à l’aide d’un composé colorimétrique, le PAR et des expériences de spectroscopie d’émission atomique dans lesquelles j’ai fait varier le pH et la concentration en Zn. Ainsi, j’ai pu déterminer que SUFU a une affinité nanomolaire pour le Zn meilleure à pH 8 qu’à pH 6,5. La fixation du Zn se ferait donc sur un site basique. La structure de SUFU a été publiée en 2013 par deux équipes, je me suis inspirée des conditions de cristallisation de ces deux articles, pour cristalliser SUFU en présence de Zn. Les expériences de dichroïsme circulaire ont permis d’affirmer que ces protéines sont organisées en hélices α et en feuillets β. De plus, grâce à la diffusion des rayons X aux petits angles, j’ai pu déterminer que dSUFU, hSUFU et zSUFU n’ont pas la même conformation en solution. Alors que SUFU de drosophile est un monomère globulaire, les protéines humaine et de poisson zèbre seraient plutôt allongées et dimériques. La région N-terminale potentiellement impliquée dans la dimérisation de hSUFU a été tronquée et hSUFUΛ30 présente des différences d’état d’oligomérisation. / The hedgehog (HH) signalling pathway is involved in the segmentary polarity formation. A dysfunction of this pathway is involved in several malformations. Many cancers are caused by an overactivation of this pathway. The HH signalling pathway is activated by the binding of HH on the receptor Patched (hPtc) and included many cytoplasmic partners such as Suppressor of Fused (SUFU). Few molecular and structural data are available on this pathway even if these data are important to fully understand the pathway functioning. Furthermore, the HH signalling pathway maybe be the target of chemotherapy treatments. However, hPtc is involved in drugs efflux. Inhibition of hPtc by the binding of its ligand HH may lead to this efflux inhibition. Yet, the binding site of HH on its receptor hPtc is not yet determined.During this thesis, the structural study of hPtc have been engaged especially the study of the binding site of HH. On the second hand, I have structurally studied some SUFU proteins.First of all, I have expressed the extracellular domains of hPtc. These domains have been described as necessary for HH binding. I have cloned a chimeric protein made by the extracellular domains of hPtc associated with the lysozyme T4 (hPtcD1D2). This protein have been expressed in the E.Coli bacteria. The protein expressed in inclusion bodies in the cytoplasm of the bacteria. In the other hand, I have cloned the protein in a yeast expression vector. Part of this, I have also expressed the protein hPtc without its N and C terminus regions (hPtcNC). These regions are intrinsically disrupted. They may lead to crystallization problems. The protein has been expressed in yeast.This work permits to expressed hPtcD1D2 and hPtcΛNΛC. This will lead to the expression of the protein and its crystallisation in order to determine its 3D structure and to characterize its ligand binding site.Finally, I structurally studied the protein SUFU. A novel Zn binding site has been characterized. In fact, after the protein purification, I have made affinity measures using a colorimetric compound, PAR. I also performed spectroscopic experiments in which I varied the pH and the Zn concentration. I determined the SUFU has a nanomolar affinity for the Zn best at pH 8 than pH 6.5. Indeed, the Zn binding site may be basic.The SUFU 3D structure has been published in 2013 by two teams. Inspired by their crystallization conditions, I crystallized SUFU with Zn. Circular dichroism experiments permitted to know that the proteins are organized in  helices and  sheets. Moreover, small angles X ray spectroscopy experiments show that dSUFU, hSUFU and zSUFU did not have the same conformation in solution. Drosophila SUFU is globular and human and zebrafish SUFU are long and dimeric. The N-terminal region involved in hSUFU has been removed and hSUFUΛ30 is present in different oligomerization forms.

3D-skrivare baserade på FDM-teknik sprider sig snabbt på marknaden på grund av kommersiell tillgänglighet, låga ingångspris och allt mer  användarvänliga enheter. I takt med att additiv tillverkning kommer allt närmare hemmet är det viktigt att veta hur vår hälsa påverkas när vi sitter intill. Partikelbaserade ämnen är en av huvudkomponenterna av luftföroreningar som idag är världens största enskilda miljöhälsorisk. Framtiden för FDM som tillverkningsmetod ser ljus ut, men är tekniken hälsomässigt ljusare än luftföroreningarna dagens masstillverkande fabriker bolmar ut? Denna studie syftar att svara på den föregående frågan med en mängd kvantitativa tester. Partikelkoncentrationer i mikrostorlek jämförs mellan olika inställningar och miljöer med hjälp av två 3D-skrivare i syfte att öka förståelsen och förebygga dessa hälsorisker. Detta projekt bygger på en förstudie (se Bilaga 1) Denna förstudie talar mer detaljerat om vad luftföroreningar är, dess negativa hälsoeffekter samt går igenom konstruktionen av partikelmätaren, vilken i denna används för att mäta partikelkoncentrationer i klassificeringarna PM10 och PM2.5. Totalt genomfördes 19 tester, varav två tester var identiska med syftet att avgöra variansen. Från testerna som jämfört två skrivare kunde medelvärden av partikelkoncentrationer i PM10 eller PM2.5 för skrivaren Stratasys Uprint SE Plus  inte urskiljas från rummens grundvärden. Skrivaren Makerbot Replicator+ visade en snittökning av partikelkoncentrationer i både PM10 och PM2.5 med 60-70% jämfört med grundvärdet. Skillnaden mellan skrivare beror främst på att Stratasys Uprint har en innesluten byggplatta, vilket Makerbot Replicator+ saknar. Partikelkoncentrationen i luften steg markant efter dörren på Stratasys Uprint öppnats och nådde då i de flesta fall värden för PM10, vilka var större än ett hos Makerbot-skrivaren identiskt test. Filamentfärg, rumsstorlek samt ventilation har också en påverkan på rummets koncentration av mikropartiklar, där ett större välventilerat rum når lägre koncentrationer än ett litet rum utan ventilation. Tyvärr har denna typ av studie har ingen standardiserad mätmetod, vilket gör att resultaten blir situationsanpassade och kan ha en varierande precision vid jämförelser mellan studier. Slutligen rekommenderar jag, utifrån studiens resultat att det kan vara värt att satsa på en innesluten skrivare ifall man under längre perioder kommer vistas i närheten av en sådan. Alternativt bör skrivaren placeras i ett ventilerat rum där man inte vistas så ofta. / <p>Betyg 2020-08-21</p>

Doctor of Philosophy / Department of Chemistry / Sundeep Rayat / Research in our laboratory has focused on designing photoactivated DNA cleaving agents based on tetrazolethione scaffolds. The key step in the activation of these involves conversion of tetrazolethione moiety to carbodiimides upon irradiation. However, the mechanism of this reaction was not previously reported. Therefore, we undertook a study to elucidate the mechanism of photodecomposition of tetrazolethione as to identify reactive intermediates involved, that may interfere or aid with the activity of our synthesized DNA cleaving agents under physiological conditions. In Part 1 of this dissertation, we present mechanistic studies on this photodecomposition. Our results indicate the clean photoconversion of tetrazolethiones I to their respective carbodiimides IV via the expulsion of sulfur and dinitrogen. Photoirradiation in the presence of trapping agent (e.g. 1,4-cyclohexadiene) resulted into the formation of their corresponding thioureas. Thus, providing strong evidence for the intermediacy of a 1,3-biradical III, which is believed to be in its triplet spin multiplicity. Further investigations (triplet sensitization and quenching experiments) to determine the precursor of the biradical argued against the involvement of a triplet excited state (T[subscript]1). We believe that the mechanistic pathway that leads to the formation of a 1,3-triplet biradical III is a diradicaloid species II-II" generated directly from the singlet excited state of tetrazolethiones (S[subscript]1) after the expulsion of dinitrogen. Once formed, this diradicaloid species could be envisioned to undergo intersystem crossing to generate the 1,3 triplet biradical III which then undergoes desulfurization to form carbodiimides IV (Chapter 2). Bridgehead-nitrogen containing fused heterocycles are regarded as “privileged structure” in biology and have found widespread applications in pharmaceutical industry. These heterocycles have also been evaluated in electroluminescent devices and organic dyes. Part II of the dissertation present new, concise and low cost strategies to a unique class of bridgehead nitrogen-containing fused heterocyclic scaffolds which involves two sequential intramolecular cyclizations from heteroenyne-allenes in the presence of Lewis acids such as SnCl[subscript]4 and BF[subscript]3.OEt[subscript]2, and trace water. The starting heteroenyne-allenes VI can be prepared from commercially available substrates V in 4 – 5 steps following standard protocols (Chapter 3). Furthermore, we employed density functional theory to gain insights into the optoelectronic properties of select derivatives of phenanthridine-fused quinazoliniminiums (PNQs) VII and their free base in order to evaluate their scope in OLED technology. Our results show that the energies of the Highest Occupied Molecular Orbital (HOMO), Lowest Unoccupied Molecular Orbital (LUMO), the HOMO-LUMO energy gaps, the ionization potentials, electron affinities and the reorganization energies can be finely tuned by varying the substituents on these chromophores. In addition, we found that the introduction of an electron donating group (NMe[subscript]2) on the PNQs and their free base increases the energies of the HOMOs and decreases the ionization potentials, relative to its unsubstituted derivative, whereas substitution by an electron withdrawing group (NO[subscript]2) decreases the energies of the LUMOs and increases the electron affinities which in turn suggests an improvement in their hole and electron creating abilities, respectively (Chapter 4).

De par les centaines de milliers de données qui les caractérisent, les bases de données épigénétiques représentent actuellement un défi majeur. L’objectif principal de cette thèse est d’évaluer la performance d’outils statistiques développés pour les données de grande dimension, en explorant l’association entre facteurs alimentaires reliés au cancer du sein (CS) et méthylation de l’ADN dans la cohorte EPIC.Afin d’étudier les caractéristiques des données de méthylation, l’identification des sources systématiques de variabilité des mesures de méthylation a été effectuée par la méthode de la PC-PR2. Ainsi la performance de trois techniques de normalisation, très répandues pour corriger la part de variabilité non désirée, a été évaluée en quantifiant l’entendu de variabilité attribuée aux facteurs de laboratoire avant et après chaque méthode de correction.Une fois la méthode de normalisation la plus appropriée identifiée, la relation entre le folate, l’alcool et la méthylation de l’ADN a été analysée par le biais de trois approches : une analyse individuelle des sites CpG, une analyse de DMR et la régression fused lasso. Les deux dernières méthodes visent à identifier des régions spécifiques de l’épigénome grâce aux corrélations possibles entre les sites proches. La méthylation globale a aussi été utilisée pour étudier la relation entre méthylation et risque de CS.Grâce à une évaluation exhaustive d’outils statistiques révélant la complexité des données de méthylation de l’ADN, cette thèse offre un aperçu instructif de connaissances pour les études épigénétiques, avec une possibilité d’application de méthodologie similaire aux analyses d’autres types de données -omiques / Epigenetics data are challenging sets characterized by hundreds of thousands of features. The main objective of this thesis was to evaluate the performance of some of the existing statistical methods to handle sets of large dimension data, exploring the association between dietary factors related to breast cancer (BC) and DNA methylation within the EPIC study.In order to investigate the characteristics of epigenetics data, the identification of random and systematic sources of variability of methylation measurements was attempted, via the principal component partial R-square (PC-PR2) method. Using this technique, the performance of three popular normalization techniques to correct for unwanted sources of variability was evaluated by quantifying epigenetics variability attributed to laboratory factors before and after the application of each correction method.Once a suitable normalization procedure was identified, the association between alcohol intake, dietary folate and methylation levels was examined by means of three approaches: an analysis of individual CpG sites, of differentially methylated regions (DMRs) and using fused lasso regression. The last two methods aim at the identification of specific regions of the epigenome using the potential correlation between neighboring CpG sites. Global methylation levels were used to investigate the relationship between methylation and BC risk.By performing an exhaustive evaluation of the statistical tools used to disclose complexity of DNA methylation data, this thesis provides informative insights for studies focusing on epigenetics, with promising potentials to apply similar methodology to the analysis of other -omics data

By flavin's unique structure, nature predestined riboflavin and its derivatives to the participation in redox processes within the bodies of all the living organisms. These biomolecules draw attention with intriguing optical properties and photosensitising abilities. Nature-inspired flavin derivatives share these qualities, and there is also a possibility of fine-tuning for the particular application from the chemical point of view. The thesis deals with two main aims. The first aim handles the synthesis of the trimer heteroaromatic precursor and 1,2-diketone. These key intermediates are essential for the future synthesis of the central aromatic core of the novel NH-free non-fused flavin derivative. The thesis introduces and verifies three approaches, including oxidation of diarylalkynes, nucleophilic addition of a corresponding organolithium compound to a Weinreb amide and benzoin condensation. The second aim covers the properties customization of NH-free fused systems by implementation of linear and bulky alkyl side-chains on the nitrogen atoms N1 and N3 of the alloxazine dilactam. N,N-alkylation introduced an increase in solubility in common organic solvents dichloromethane and chloroform. For the derivatives with 2-(adamantan-1-yl)ethyl substituents, high thermal stability was observed via TGA.

The aim of this diploma thesis is validation of numerical simulation of the ceramic core manufacturing in the investment casting proces. The first part submit the theoretical description of the manufacturing of the ceramic cores and a description of the basic principles of numerical simulations. The second part compares the video recording of filling the wax-ceramic mixture to the die-casting mold and simulation of the same process created in a simulation software Cadmould 3D-F.

Denna rapport detaljerar ett examensarbete där användbarheten på en lokalt skapad 3D-skrivare förbättrats i syfte av att tillåta skrivarens användning av studenter på KTH Södertälje. Skrivarens har ursprungligen byggts för att agera som ett pågående projekt som kan återupptas som examensarbete, varav detta är det tredje. Skrivaren i fråga är en FFF-skrivare i ”box” stil med utskriftsvolymen 400 x 400 x 375 mm. Vid projektstart är skrivaren i nominellt användbart skick men saknar dokumentation, processrutiner och användbara slicerprofiler. Skrivaren besitter även hårdvaran för att tillåta utskrifter med två material men denna funktion har aldrig implementerats. Framtagningen av slicerprofiler har tagit upp majoriteten av arbetet och har utförts genom iterativ testning där inställningar modifieras och testas, vartefter positiva ändringar implementeras för att gradvis eliminera defekter och minska haveririsk. Projektet har levererat en full slicerprofil som tillåter pålitliga utskrifter med två olika PLA-filament samt en användarmanual till skrivaren. Utöver användarverktygen så detaljeras även de reparationsarbeten och hårdvaruändringar som gjorts. Profil, manual, rådata och filer för utskrivna komponenter är tillgängliga i en projektmapp som kan hittas från en länk i början av kapitel 4. / This report details a thesis project for getting a 3D-printer internally built at KTH Södertälje into a state where it can be used by other students for unrelated projects. The printer acts as an ongoing project that can be used for thesis work, with this project in particular being the third in line. The printer in question is an FFF-printer with the ”box” configuration and a print volume of 400 x 400 x 375 mm. At project start, the printer is nominally in working condition but lacks any documentation for proper handling and slicer settings. Additionally, its multi-material function has never been successfully implemented. Adjustment of slicer settings is done using iterative testing, wherein settings are modified and tested, with positive changes being integrated and negative changes discarded with the intent of removing defects and minimizing risk of print failure. The project has delivered a slicer profile for Cura capable of reliably printing with two different PLA filaments as well as a user manual. Hardware upgrades and repair work has also been detailed. The profile, user manual, raw data and object files for upgrades and spares can be found through a link in the beginning of chapter 4.

Over the past decades, functional group manipulation of aromatic precursors has been a common strategy to access new aromatic compounds. However, these classical methods, such as Friedel-Crafts alkylations and electrophilic/nucleophilic aromatic substitutions, have shown lack of regioselectivity besides the use of activators in excess amounts. To this end, numerous benzannulations to form benzo-fused substrates via Diels-Alder (DA), ring-closing metathesis (RCM), cycloaddition, and transition-metal-promoted processes have been reported. Appending a benzene ring directly onto a pre-existing ring is preferable to many classical methods due to the likely reduction of reaction steps and superior regiocontrol. However, many of these benzannulation reactions require air- and/or moisture- sensitive reaction conditions, a last oxidation step, or the use of highly functionalized precursors. Here we disclose three ‘complementary’ intramolecular ring-opening benzannulations to access a large array of functionalized (hetero)aromatic scaffolds utilizing cyclopropenes-3,3-dicarbonyls, alkylidene cyclopropanes-1,1-diesters, and 2,3-dihydrofuran O,O- and N,O- acetals as building blocks. More than 70 benzo-fused aromatic compounds were synthesized using this complementary approach with yields up to 98% and low catalyst loadings. With these benzannulation reactions in hand, we aim to open the synthetic door to a handful of bioactive natural products.

Over the past decades, functional group manipulation of aromatic precursors has been a common strategy to access new aromatic compounds. However, these classical methods, such as Friedel-Crafts alkylations and electrophilic/nucleophilic aromatic substitutions, have shown lack of regioselectivity besides the use of activators in excess amounts. To this end, numerous benzannulations to form benzo-fused substrates via Diels-Alder (DA), ring-closing metathesis (RCM), cycloaddition, and transition-metal-promoted processes have been reported. Appending a benzene ring directly onto a pre-existing ring is preferable to many classical methods due to the likely reduction of reaction steps and superior regiocontrol. However, many of these benzannulation reactions require air- and/or moisture- sensitive reaction conditions, a last oxidation step, or the use of highly functionalized precursors. Here we disclose three ‘complementary’ intramolecular ring-opening benzannulations to access a large array of functionalized (hetero)aromatic scaffolds utilizing cyclopropenes-3,3-dicarbonyls, alkylidene cyclopropanes-1,1-diesters, and 2,3-dihydrofuran O,O- and N,O- acetals as building blocks. More than 70 benzo-fused aromatic compounds were synthesized using this complementary approach with yields up to 98% and low catalyst loadings. With these benzannulation reactions in hand, we aim to open the synthetic door to a handful of bioactive natural products.

The IEEE standard of decimal floating-point arithmetic was officially released in 2008. The new decimal floating-point (DFP) format and arithmetic can be applied to remedy the conversion error caused by representing decimal floating-point numbers in binary floating-point format and to improve the computing performance of the decimal processing in commercial and financial applications. Nowadays, many architectures and algorithms of individual arithmetic functions for decimal floating-point numbers are proposed and investigated (e.g., addition, multiplication, division, and square root). However, because of the less efficiency of representing decimal number in binary devices, the area consumption and performance of the DFP arithmetic units are not comparable with the binary counterparts. IBM proposed a binary fused multiply-add (FMA) function in the POWER series of processors in order to improve the performance of floating-point computations and to reduce the complexity of hardware design in reduced instruction set computing (RISC) systems. Such an instruction also has been approved to be suitable for efficiently implementing not only stand-alone addition and multiplication, but also division, square root, and other transcendental functions. Additionally, unconventional number systems including digit sets and encodings have displayed advantages on performance and area efficiency in many applications of computer arithmetic. In this research, by analyzing the typical binary floating-point FMA designs and the design strategy of unconventional number systems, ``a high performance decimal floating-point fused multiply-add (DFMA) with redundant internal encodings" was proposed. First, the fixed-point components inside the DFMA (i.e., addition and multiplication) were studied and investigated as the basis of the FMA architecture. The specific number systems were also applied to improve the basic decimal fixed-point arithmetic. The superiority of redundant number systems in stand-alone decimal fixed-point addition and multiplication has been proved by the synthesis results. Afterwards, a new DFMA architecture which exploits the specific redundant internal operands was proposed. Overall, the specific number system improved, not only the efficiency of the fixed-point addition and multiplication inside the FMA, but also the architecture and algorithms to build up the FMA itself. The functional division, square root, reciprocal, reciprocal square root, and many other functions, which exploit the Newton's or other similar methods, can benefit from the proposed DFMA architecture. With few necessary on-chip memory devices (e.g., Look-up tables) or even only software routines, these functions can be implemented on the basis of the hardwired FMA function. Therefore, the proposed DFMA can be implemented on chip solely as a key component to reduce the hardware cost. Additionally, our research on the decimal arithmetic with unconventional number systems expands the way of performing other high-performance decimal arithmetic (e.g., stand-alone division and square root) upon the basic binary devices (i.e., AND gate, OR gate, and binary full adder). The proposed techniques are also expected to be helpful to other non-binary based applications.