Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and poses a global threat to human health. A third of the world’s population is infected with Mtb. Multi-drug resistant and extensively drug resistant Mtb strains are widespread and development of new drugs is urgently needed to treat drug resistant TB. This thesis focuses on the Mtb cytochrome P450 (P450) enzymes CYP126A1 and CYP143A1. P450s are heme-binding enzymes that catalyse activation of molecular oxygen and the oxidation of substrates bound close to the heme. CYP126A1 and CYP143A1 are “orphans” in terms of their functional characterization, but potential drug targets in view of ability of azole-based P450 inhibitors to inhibit growth and viability of Mtb. The CYP126A1 and CYP143A1 genes were cloned and expressed in Escherichia coli. Expression conditions and strains were optimised to maximise soluble protein production and methods were developed to purify the P450s using affinity, ion exchange and size exclusion chromatography. Both P450s were shown to bind heme b, and heme was shown to be axially coordinated by a cysteine thiolate and a water molecule in both cases using UV-visible and electron paramagnetic resonance (EPR) spectroscopy. Both P450s bound carbon monoxide (CO) in their reduced forms to produce heme Fe2+-CO complexes with absorption maxima at ~450 nm – characteristic of P450s. CYP126A1 and CYP143A1 bound avidly to a range of inhibitors, including several azole drugs. As examples, binding constant (Kd) values of 13.8 µM and 21.9 µM were determined for clotrimazole and econazole with CYP143A1; while ketoconazole bound CYP126A1 with a Kd of 0.20 µM. Each of these drugs is very effective in inhibiting Mtb growth. EPR confirmed inhibitory coordination of both P450s by azole drug nitrogen atoms; though indirect coordination via a retained axial water ligand may also occur in some cases. Extinction coefficients were determined as έ420 = 125 mM-1 cm-1 (CYP126A1) and έ415 = 105 mM-1 cm-1 (CYP143A1). CYP126A1’s heme iron redox potential was shown to be unusually positive (E°’ = -80 mV). Light scattering studies showed CYP126A1 to be a monodisperse, monomeric protein. CYP143A1 is also mainly a monomer, but with a small proportion of an oligomeric form. Despite its polydispersity, CYP143A1 was crystallized and its structure solved by X-ray diffraction to a resolution of 1.9 Å, using molecular replacement with the Mtb P450 CYP142A1. A limited compound screen of typical P450 substrates failed to provide “hits” to identify CYP143A1 substrate selectivity, but the presence of polyethylene glycol in the CYP143A1 active site in crystals suggests fatty acids as potential substrates. CYP126A1 was crystallized for studies to identify binding modes of small molecules (“fragments”) identified to interact with CYP126A1 by NMR. Crystal structures of CYP126A1 in complex with two such fragments (NMR401 and NMR343) were determined to ~2.0 Å resolution in ongoing research to build Mtb P450 isoform-specific inhibitors. Compounds identified as CYP126A1 substrates/inhibitors identified by high-throughput screening were validated by UV-visible titrations with the P450, and binding modes and affinity established. In conclusion, this thesis provides novel insights into the biochemical, biophysical and structural properties of two novel Mtb P450s that are potential targets for new anti-TB drugs.

The aim of my thesis is to evaluate whether on the Slovak market there is opportunity for expansion of the Czech company Pelmi spol., s.r.o. Based on analysis of the target market, the SWOT analysis, competition, and economic characteristics of Slovak market I will suggest my own recommendation what kind of form entry is the most convenient for company.

Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique. / Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach.

With the developments in computing and communication technologies, wireless sensor networks have become popular in wide range of application areas such as health, military, environment and habitant monitoring. Moreover, wireless acoustic sensor networks have been widely used for target tracking applications due to their passive nature, reliability and low cost. Traditionally, acoustic sensor arrays built in linear, circular or other regular shapes are used for tracking acoustic sources. The maintaining of relative geometry of the acoustic sensors in the array is vital for accurate target tracking, which greatly reduces the flexibility of the sensor network. To overcome this limitation, we propose using only a single acoustic sensor at each sensor node. This design greatly improves the flexibility of the sensor network and makes it possible to deploy the sensor network in remote or hostile regions through air-drop or other stealth approaches. Acoustic arrays are capable of performing the target localization or generating the bearing estimations on their own. However, with only a single acoustic sensor, the sensor nodes will not be able to generate such measurements. Thus, self-organization of sensor nodes into virtual arrays to perform the target localization is essential. We developed an energy-efficient and distributed self-organization algorithm for target tracking using wireless acoustic sensor networks. The major error sources of the localization process were studied, and an energy-aware node selection criterion was developed to minimize the target localization errors. Using this node selection criterion, the self-organization algorithm selects a near-optimal localization sensor group to minimize the target tracking errors. In addition, a message passing protocol was developed to implement the self-organization algorithm in a distributed manner. In order to achieve extended sensor network lifetime, energy conservation was incorporated into the self-organization algorithm by incorporating a sleep-wakeup management mechanism with a novel cross layer adaptive wakeup probability adjustment scheme. The simulation results confirm that the developed self-organization algorithm provides satisfactory target tracking performance. Moreover, the energy saving analysis confirms the effectiveness of the cross layer power management scheme in achieving extended sensor network lifetime without degrading the target tracking performance.

Title: The affects of GDPR on companies: a study about the impact of GDPR on the collection of personal data for target marketing and data mining.  Keywords: GDPR, target marketing, data mining Subject: Bachelor's thesis, International Marketing, 15hp. Author: Denise Loponen Mejia & Viktoria Mirkovic Problem: How has GDPR affected companies when collecting personal data for target marketing and data mining? Purpose: The purpose of this study is to clarify the affects that GDPR has had on companies and what alterations the companies have had to make since the enforcement of the regulation regarding target marketing and data mining. Method: In this study a deductive method has been applied. Primary data has been obtained through qualitative semi-structured interviews, which were transcribed and analyzed in order to find a conclusion. Conclusion: The conclusion of this study shows that GDPR has had an impact on companies regarding their collection of personal data. The companies have had to change the way they have handled this in comparison to before. Furthermore, implementation of new strategies in order to be able to identify customer groups and lawfully be able to use data mining has had to be established. Moreover, the study shows that companies have had to introduce changes in order to comply with the regulation, as well as the fact that the change has been successful. / Titel: Påverkan av GDPR på företag: en studie om betydelsen av GDPR för insamling av personliga uppgifter till target marketing och data mining. Nyckelord: GDPR, target marketing, data mining.  Ämne: Kandidatuppsats i företagsekonomi, inriktning internationell marknadsföring, 15 hp Författare: Denise Loponen Mejia & Viktoria Mirkovic Problemformulering: Hur har GDPR påverkat företag vid insamling av personlig data till target marketing och data mining?  Syfte: Syftet med denna studie är att klargöra hur införandet av GDPR har påverkat företag och vilka ändringar de har varit tvungna till att införa vid  insamling av personliga uppgifter till target marketing och data mining. Metod: I denna studie har en deduktiv metod tillämpats. Primärdata har nhämtats genom kvalitativa semi-strukturerade intervjuer, vilka sedan transkriberat och analyseras i syfte att finna ett svar på frågeställningen.  Slutsats: Resultatet av denna studie påvisar att GDPR har haft en påverkan på hur företag hanterar insamling och hantering av personlig data. De har blivit tvungna till att förändra sättet de hanterar detta på. Även implementering av nya strategier för att kunna identifiera kundgrupper och lagligt använda data mining har krävts. Vidare visar resultatet att samtliga företag har fått införa förändringar för att efterfölja den nya regleringen, samt att den har varit framgångsrik.

Doctor Educationis / Independent Radio and Appliance retailers face very strong competition from chain groups, discounters and hypermarkets. These organisations rely on bulk-buying and negotiated deals to elicit favourable terms from suppl iers. Due to the volume of their turnover they can afford to work on lower profi t margins than the independent operator. Large marketing organisations also benefit from economi es of seal e in adverti si ng and promoti ons. In short the 1arge groups have a di fferenti al advantage over the small er i ndependents i n that thei r cost of sales and overhead structure is generally lower. The purpose of this study is to develop a marketing strategy for independent radio and appliance retailers which will enable them to survive, grow and trade profitably under current highly competitive conditions.

Preventing impairment of waterbodies requires control, reduction and interception of contaminant losses at the field and subwatershed level. Three specific research objectives were accomplished in this study: 1) compare the HSPF-predicted flow, sediment, total nitrogen (TN) and total phosphorus (TP) loads resulting from simulation of spatially distributed site-specific and county-level disaggregated land use data at subwatershed and watershed levels, 2) evaluate the effects of site-specific and county-level disaggregated BMP data on modeled BMP responses in HSPF-predicted flow, sediment, TN and TP loads at the watershed level, and 3) analyze the long-term effects of the two spatial BMP datasets on achieving the Chesapeake Bay Total Maximum Daily Load (TMDL) goals for sediment, TN and TP. Site-specific data are derived from the local watershed inventory while disaggregated data are based on county-level aggregated data that are distributed to portions of river segments that intersect each county. The study site was the Upper Opequon Watershed in northern Virginia. Results for each research objective are as follows: (1) HSPF-predicted flow, sediment, TN and TP were higher using disaggregated land use data in subwatersheds at monthly and annual time-steps. (2) Predicted load reductions were higher with site-specific BMP data than with disaggregated data for the study watershed. (3) Current levels of cost-shared BMP implementation in the Upper Opequon Watershed using either site-specific or county-level disaggregated BMP datasets do not meet the Chesapeake Bay TMDL goals. Increasing BMP implementation level to 100% of the available land also failed to meet TMDL target goals. Generally, use of disaggregated land use data does not accurately represent the existing watershed conditions. Further, for the study watershed, use of disaggregated county-level BMP data poorly represented actual watershed conditions, which resulted in higher pollutant yields and higher levels of BMPs needed to meet water quality goals. The study suggests that site-specific land use and BMP data must be used during TMDL implementation planning to maintain credibility with local stakeholders and improve the accuracy of the developed implementation plans. / Ph. D.

Who is 'the public'? The case of the EU's Public Diplomacy in the Eastern Partnership Master's Thesis - Kristin Aldag - Charles University, June 2021 Abstract For states and other international actors such as the European Union, public diplomacy is an important tool to achieve their political and economic interests abroad by communi- cating directly with foreign audiences. While the existing body of academic literature on pub- lic diplomacy is rich, few authors have thus far addressed the question of who actually consti- tutes the public, and which specific target groups can be distinguished. Using the EU PD prac- tices in the Eastern Partnership (EaP) from 2010-2020 as a case study, this thesis will thus attempt to fill this gap and contribute to the academic literature in the field by offering an overview of the various target groups within the European Union's public diplomacy. In a thorough content analysis of relevant primary sources, the thesis focuses on the policy objec- tives and practices of the European Union's outreach to different groups and audiences in the EaP region. The third chapter presents the results of this comprehensive research, which has shown that several distinct groups within the general public in the EaP can be identified, among them young people, media professional and...

Les leishmanioses sont des maladies tropicales négligées provoquées par un protozoaire parasite du genre Leishmania, et transmises par un insecte vecteur, le phlébotome. Les leishmanioses menacent 310 millions de personnes dans 98 pays à travers le monde. Les traitements antileishmaniens actuels sont limités et présentent des problèmes majeurs de toxicité et d'émergence de chimiorésistance. Dans ce contexte, il est nécessaire de développer de nouveaux agents antileishmaniens spécifiquement dirigés contre une cible thérapeutique chez le parasite. La GDP-Mannose Pyrophosphorylase (GDP-MP) est une cible thérapeutique essentielle à la survie du parasite à la fois in vitro et in vivo. Plusieurs différences ont été identifiées dans le site actif de GDP-MPs leishmaniennes par rapport à l'enzyme humaine, montrant ainsi l'intérêt de cette cible thérapeutique dans le développement de nouveaux traitements contre la leishmaniose. La GDP-MP catalyse la synthèse du GDP-mannose, la forme activée du mannose, brique moléculaire importante dans les processus de glycosylation et la synthèse de glycoconjugués essentiels à la reconnaissance hôte-parasite. Ce travail de thèse a consisté à produire et purifier les GDP-MPs de 3 espèces de parasites (Leishmania infantum, Leishmania donovani et Leishmania mexicana) ainsi que l'homologue humaine dans le but de comparer leurs propriétés enzymatiques. A partir d'inhibiteurs potentiels conçus et synthétisés sur la base de modèles moléculaires de GDP-MPs leishmaniennes et humaine, 100 composés ont été évalués sur les enzymes purifiées et sur les parasites in vitro. Cette analyse nous a permis de sélectionner des composés spécifiquement dirigés contre la cible du parasite et présentant une activité antileishmanienne. Nous avons également initié une étude de l'expression et de la localisation de la GDP-MP après traitement par les composés les plus intéressants. Ces composés pourront par la suite être utilisés comme outils pharmacologiques pour le développement de nouveaux agents antileishmaniens spécifiques. / Leishmaniases are Neglected Tropical Disease (NTD)caused by a protozoan parasite of the genus Leishmania and transmitted by an insect vector, the phlebotomine sandfly. Leishmaniases threaten 310 millions people in 98 countries around the world. Current antileishmanial treatments are limited and present major issues of toxicity and drug resistance emergence. In this context, it is necessary to develop new specific antileishmanial drugs specifically directed against a therapeutic target in the parasite.The GDP-Mannose Pyrophosphorylase (GDP-MP) is a therapeutic target which has been described to be essential for parasite survival both in vitro and in vivo. Several differences have been identified in the active site of leishmanial GDP-MPs compared to the human counterpart, showing the prominence of this therapeutic target in the development of new treatments against leishmaniasis. The GDP-MP catalyzes the synthesis of GDP-Mannose,the activated form of mannose, an important molecular constituent of the glycosylation processes involved in the biosynthesis of glycoconjugates which are essential for host-parasite recognition. My thesis work consisted in the production and purification of GDP-MPs from 3 Leishmania species (Leishmania donovani,Leishmania infantum and Leishmania mexicana)and from humanin order to compare their enzymatic properties. From potential inhibitors designed and synthesized on the basis of leishmanial and human GDP-MP molecular models, 100 compounds were evaluated on purified enzymes and on parasites in vitro. These analyses allowed us to select some compounds which are specifically directed against the parasite target and presenting antileishmanial activities. We have also initiated a study of expression and localization of GDP-MP after treatment with the most potent compounds. These compounds will be used as pharmacological tools for the development of new specific antileishmanial drugs.

archives@tulane.edu / Triple negative breast cancers (TNBCs) are clinically and biologically aggressive, with higher recurrence and metastasis rates compared to other subtypes. Acquisition of a mesenchymal and migratory cell phenotype is consequential process that promotes metastasis. There are no clinically approved small molecule targeted therapies for TNBC; kinases are effective drug targets in cancer research. Although some kinases are known to regulate the mesenchymal phenotype, a large subset of the human kinome is understudied. There are many approaches to discovering novel kinase targets in cancer. Here, a phenotypic screen approach is described to identify understudied kinases using the Published Kinase Inhibitor Set (PKIS). Initial screens using TNBC cell lines (MDA-MB-231, BT549 and MDA-MB-157) identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology. Our hits were further prioritized based on gene expression changes of the epithelial marker E-cadherin and migratory behavior. Active compounds were confirmed to reverse EMT on transcript and protein levels with qRT-PCR and Western blot. When pharmacologically similar compounds were more closely examined, different effects on cancer biology were observed (‘active’ versus ‘inactive’ compounds). Based on these observations, a kinase array was employed to compare both the active and inactive compounds to demonstrate how to identify candidate kinases responsible for the EMT reversal. Using this screening approach, small molecule inhibitors from the PKIS library (GSK346294A, GSK448459A, GSK237700A) were identified that were pharmacologically similar that reversed the mesenchymal phenotype in TNBC. These compounds have different biological effects in TNBC, despite having similar pharmacophores. Differential effects of the PKIS compounds on transwell migration, gene (qRT-PCR) and protein (Western blot) expressions, and mammosphere formation in TNBC cells was observed. In follow-up in vivo studies, our most active compound (GSK346294A) suppressed tumorigenesis and metastasis. RNA-sequencing confirmed downregulated pathways induced by GSK346294A treatment in TNBC cells included EMT, cytoskeletal rearrangement and cell cycle regulation. Because these compounds have different off-target activities, this approach can be used to identify candidate unique kinases responsible for the observed effects. NEK5 was one of these kinases candidates. NEK5 function remains understudied in cancer, and even more understudied in breast cancer. This study is the first, to our knowledge, to describe the function of NEK5 in breast cancer, specifically its roles in acquisition of a mesenchymal and migratory cell phenotype. Overexpression of NEK5 promotes a migratory and mesenchymal phenotype, and knockdown with a shRNA construct suppresses this migratory behavior. Data obtained using both qRT-PCR of the knockdown and overexpression cell lines, and follow-up RNA sequencing, revealed NEK5 regulates the PLAU/PAI-1/SRC axis. Furthermore, a role for NEK5 in resistance to SRC-targeting anticancer agents is demonstrated. The work described here demonstrates the utility of a novel approach to identify understudied kinases in cancer, and characterization of these kinases has potential impact in other metastatic diseases not limited to breast cancer. / 1 / Margarite Matossian