O ácido cis-11-metil-2-dodecenóico (DSF), substância utilizada como autoindutor nos sistemas de Quorum Sensing da bactéria Xanthomonas axonopodis pv. citri, foi utilizado como um modelo de estudos para a análise de moléculas secretadas por populações celulares utilizando técnicas eletroanalíticas. Cultivos celulares provenientes de três linhagens diferentes da bactéria Xanthomonas axonopodis pv. citri foram utilizados para os estudos. Uma linhagem selvagem (WT), que produzia DSF em quantidades naturalmente observadas, e duas linhagens geneticamente modificadas. Em uma linhagem (ΔC) a modificação genética resultava em elevada produção de DSF, para a segunda (ΔF) o resultado era a anulação da produção do autoindutor. As principais técnicas analíticas utilizadas foram: voltametria cíclica, eletroforese capilar e cromatografia líquida (com detecção UV-Vis e acoplada a espectrometria de massas), sendo a última para efeitos de comparação. Procedimentos de extração líquido-líquido com solventes orgânicos foram realizados como etapas de pré-tratamento de amostras, visando diminuir a quantidade de interferentes e realizar uma pré-concentração do analito. Três metodologias principais foram adotadas. Uma primeira voltada para a diferenciação dos cultivos celulares, analisando-se ou os sobrenadantes provenientes dos cultivos ou os extratos resultantes dos pré-tratamentos desses sobrenadantes, tendo como principal foco a identificação de um sinal diferencial entre os cultivos que correspondesse às quantidades de DSF, ou seja, inexistente para a linhagem ΔF, de alta intensidade para linhagem ΔC e de intensidade intermediária para a linhagem WT. A segunda metodologia foi baseada no uso de uma molécula modelo para realizar experimentos e obter dados como parâmetros físico-químicos, o ácido láurico (ácido dodecanóico) foi escolhido por sua semelhança estrutural e facilidade de obtenção, curvas de calibração e experimentos de separação de moléculas com estrutura semelhante foram realizados com a técnica de eletroforese capilar. Uma terceira metodologia foi desenvolvida utilizando o padrão do ácido cis-11-metil-2-dodecenóico (DSF), curvas de calibração foram obtidas com o padrão isolado apresentando ótimas linearidades, tanto com a técnica de eletroforese capilar quanto com cromatografia líquida com detecção UV-Vis, experimentos de adição de padrão às amostras também foram executados. Os experimentos realizados com as amostras não apresentaram os resultados esperados, os problemas foram associados principalmente à quantidade de interferentes na amostra e a baixa concentração do analito nos cultivos, sendo que os métodos de extração realizados não foram suficientes para solucioná-los. O insucesso da técnica de voltametria cíclica foi associado ao envenenamento do eletrodo, devido à alta quantidade de compostos orgânicos presentes na amostra, e dúvidas relacionadas à eletroatividade do DSF. As dificuldades encontradas com a técnica de eletroforese capilar foram associadas à alta força iônica da amostra, presença de interferentes e baixa concentração do analito nos cultivos. Mesmo os experimentos realizados com cromatografia líquida acoplada a detecção com espectrometria de massas não atenderam às expectativas, dificultando a elucidação dos problemas encontrados até então e impossibilitando o desenvolvimento de uma metodologia que atendesse aos objetivos propostos. / Cis-11-methyl-2-dodecenoic acid, also known as DSF (autoinducer of the Quorum Sensing system used by the bacteria Xanthomonas axonopodis pv. citri) was explored as a study model for the cell secreted molecules analysis by electroanalytical techniques. Cell cultures from three different cell lines of the bacteria Xanthomonas axonopodis pv. citri were used for this study. A wild type (WT), which produced DSF in naturally observed quantities and two genetically modified ones. One of the lines (ΔC) had a genetic modification that resulted in an elevated production of DSF, and the other (ΔF) had a modification that stopped DSF production. Main analytical techniques used were: cyclic voltammetry, capillary electrophoresis and liquid chromatography (with UV-Vis detection and coupled with mass spectrometry), this last one was used with comparison purposes. Liquid-liquid extractions with organic solvents were realized as sample pre-treatment steps aiming the reduction of interfering species and analyte pre-concentration. Three main methodologies were adopted. One based in the differentiation of cell cultures analyzing or the cell culture supernatants or the resulting extracts from the pre-treatment steps. The main focus was the identification of a differential signal between the cultures that corresponded to the DSF quantities, in other words, null for the ΔF line, high intensity for the ΔC line and a an intermediary intensity for the WT line. The second methodology was based in experiments with a model molecule to obtain some data such as physical-chemistry parameters. Lauric acid (dodecanoic acid) was chosen for its structural similarity and easily acquisition. Calibration curves and similar structure molecules separation experiments were realized using capillary electrophoresis. Third and last methodology was developed using standard cis-11-methyl-2-dodecenoic acid (DSF). Calibration curves were obtained with the isolated standard showing great linearities using both capillary electrophoresis and liquid chromatography with UV-Vis detection. Experiments of standard addiction to the samples were also realized. Experiments using the samples did not show the expected results. Problems found were mainly associated with high amount of interfering species and low analyte concentration at the cultures, extraction methods used were not enough to solve this. The failures with the cyclic voltammetric techniques were associated to electrode poisoning, due to the high amount of organic compounds present at the sample, and DSF lack of electroactivity. Difficulties found with capillary electrophoresis were associated with the samples high ionic strength, presence of interfering species and low analyte concentration. Even liquid chromatography coupled with mass spectrometry experiments didn\'t attended the expectations. Making it difficult to elucidate the problems and not allowing the development of a methodology that could achieve the proposed objectives.

O ácido cis-11-metil-2-dodecenóico (DSF), substância utilizada como autoindutor nos sistemas de Quorum Sensing da bactéria Xanthomonas axonopodis pv. citri, foi utilizado como um modelo de estudos para a análise de moléculas secretadas por populações celulares utilizando técnicas eletroanalíticas. Cultivos celulares provenientes de três linhagens diferentes da bactéria Xanthomonas axonopodis pv. citri foram utilizados para os estudos. Uma linhagem selvagem (WT), que produzia DSF em quantidades naturalmente observadas, e duas linhagens geneticamente modificadas. Em uma linhagem (ΔC) a modificação genética resultava em elevada produção de DSF, para a segunda (ΔF) o resultado era a anulação da produção do autoindutor. As principais técnicas analíticas utilizadas foram: voltametria cíclica, eletroforese capilar e cromatografia líquida (com detecção UV-Vis e acoplada a espectrometria de massas), sendo a última para efeitos de comparação. Procedimentos de extração líquido-líquido com solventes orgânicos foram realizados como etapas de pré-tratamento de amostras, visando diminuir a quantidade de interferentes e realizar uma pré-concentração do analito. Três metodologias principais foram adotadas. Uma primeira voltada para a diferenciação dos cultivos celulares, analisando-se ou os sobrenadantes provenientes dos cultivos ou os extratos resultantes dos pré-tratamentos desses sobrenadantes, tendo como principal foco a identificação de um sinal diferencial entre os cultivos que correspondesse às quantidades de DSF, ou seja, inexistente para a linhagem ΔF, de alta intensidade para linhagem ΔC e de intensidade intermediária para a linhagem WT. A segunda metodologia foi baseada no uso de uma molécula modelo para realizar experimentos e obter dados como parâmetros físico-químicos, o ácido láurico (ácido dodecanóico) foi escolhido por sua semelhança estrutural e facilidade de obtenção, curvas de calibração e experimentos de separação de moléculas com estrutura semelhante foram realizados com a técnica de eletroforese capilar. Uma terceira metodologia foi desenvolvida utilizando o padrão do ácido cis-11-metil-2-dodecenóico (DSF), curvas de calibração foram obtidas com o padrão isolado apresentando ótimas linearidades, tanto com a técnica de eletroforese capilar quanto com cromatografia líquida com detecção UV-Vis, experimentos de adição de padrão às amostras também foram executados. Os experimentos realizados com as amostras não apresentaram os resultados esperados, os problemas foram associados principalmente à quantidade de interferentes na amostra e a baixa concentração do analito nos cultivos, sendo que os métodos de extração realizados não foram suficientes para solucioná-los. O insucesso da técnica de voltametria cíclica foi associado ao envenenamento do eletrodo, devido à alta quantidade de compostos orgânicos presentes na amostra, e dúvidas relacionadas à eletroatividade do DSF. As dificuldades encontradas com a técnica de eletroforese capilar foram associadas à alta força iônica da amostra, presença de interferentes e baixa concentração do analito nos cultivos. Mesmo os experimentos realizados com cromatografia líquida acoplada a detecção com espectrometria de massas não atenderam às expectativas, dificultando a elucidação dos problemas encontrados até então e impossibilitando o desenvolvimento de uma metodologia que atendesse aos objetivos propostos. / Cis-11-methyl-2-dodecenoic acid, also known as DSF (autoinducer of the Quorum Sensing system used by the bacteria Xanthomonas axonopodis pv. citri) was explored as a study model for the cell secreted molecules analysis by electroanalytical techniques. Cell cultures from three different cell lines of the bacteria Xanthomonas axonopodis pv. citri were used for this study. A wild type (WT), which produced DSF in naturally observed quantities and two genetically modified ones. One of the lines (ΔC) had a genetic modification that resulted in an elevated production of DSF, and the other (ΔF) had a modification that stopped DSF production. Main analytical techniques used were: cyclic voltammetry, capillary electrophoresis and liquid chromatography (with UV-Vis detection and coupled with mass spectrometry), this last one was used with comparison purposes. Liquid-liquid extractions with organic solvents were realized as sample pre-treatment steps aiming the reduction of interfering species and analyte pre-concentration. Three main methodologies were adopted. One based in the differentiation of cell cultures analyzing or the cell culture supernatants or the resulting extracts from the pre-treatment steps. The main focus was the identification of a differential signal between the cultures that corresponded to the DSF quantities, in other words, null for the ΔF line, high intensity for the ΔC line and a an intermediary intensity for the WT line. The second methodology was based in experiments with a model molecule to obtain some data such as physical-chemistry parameters. Lauric acid (dodecanoic acid) was chosen for its structural similarity and easily acquisition. Calibration curves and similar structure molecules separation experiments were realized using capillary electrophoresis. Third and last methodology was developed using standard cis-11-methyl-2-dodecenoic acid (DSF). Calibration curves were obtained with the isolated standard showing great linearities using both capillary electrophoresis and liquid chromatography with UV-Vis detection. Experiments of standard addiction to the samples were also realized. Experiments using the samples did not show the expected results. Problems found were mainly associated with high amount of interfering species and low analyte concentration at the cultures, extraction methods used were not enough to solve this. The failures with the cyclic voltammetric techniques were associated to electrode poisoning, due to the high amount of organic compounds present at the sample, and DSF lack of electroactivity. Difficulties found with capillary electrophoresis were associated with the samples high ionic strength, presence of interfering species and low analyte concentration. Even liquid chromatography coupled with mass spectrometry experiments didn\'t attended the expectations. Making it difficult to elucidate the problems and not allowing the development of a methodology that could achieve the proposed objectives.

This study is part of a project which aim is solving the structure of the catalytic domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is the preparation of new constructs of yeast Nth1 and optimization of their purification protocols, the selection of the ideal buffer for crystallization trials using the method of differential scanning fluorimetry (DSF) and at last the protein crystallization. Another part of the thesis is the measurement of the enzymatic activity of pNth1 WT in the presence of Bmh1 protein, verification of trehalose binding to the selected constructs of Nth1 using differential scanning fluorimetry (DSF), thermoforesis (MST) and further crystallization with trehalose. Neutral trehalase is highly conserved trehalase that has been found in a wide variety of organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and hydrolytically cleave trehalose into two glucose molecules. Trehalose is a naturally occurring non-reducing disaccharide serving in yeast cells a source of carbon and energy as well as protection against stress conditions such as a thermal shock. Trehalose hydrolysis is essential for flying insects, because it is present as the main sugar component of insect haemolymph, therefore trehalase inhibitors...

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

Den 25 maj 2018 kommer Personuppgiftslagen (PuL) att ersättas av EU-förordningen GeneralData Protection Regulation (GDPR). Denna studie har granskat vilka skillnader som finnsmellan GDPR och PuL och även hur företag planerar att hantera de förändringar som GDPRkommer att medföra. Även om GDPR i många avseende är lik PuL kommer den nyaförordningen att påverka allt ifrån den enskilda individen till de största organisationerna.Förordningen kommer innebära mer rättigheter och bättre skydd för den registrerade, vilketbetyder att det kommer ställas högre krav på de företag som behandlar personuppgifter. De nya kraven kommer innebära ett omfattande anpassningsarbete inom flera områden. Delskommer det behöva ske administrativa och juridiska förändringar inom verksamheten i form avnya eller uppdaterade avtal. Det kan även komma att krävas tekniska åtgärder för att hanterafrågor som “Rätten att bli glömd”, ”Dataportabilitet”, ”Registerföring” samt det förstärktaskyddet av personuppgifter. Det ställs även krav på att personuppgiftsincidenter måsterapporteras till tillsynsmyndigheten vid upptäckande. Då tillämpningsområdet utökas medfördetta att personuppgifter som lagras ostrukturerat kommer omfattas av lagstiftningen. För attuppnå efterlevnad av förordningen införs krav på att i vissa fall utse ett “Dataskyddsombud” iverksamheten; tillsynsmyndigheten får även befogenhet till att utdöma mycket kraftigasanktioner vid överträdelse av förordningen. Beroende på hur en verksamhets personuppgiftshantering ser ut i dagsläget kan dennaanpassning bli mer eller mindre omfattande. Anpassningsarbetets omfattning påverkas av ettantal faktorer exempelvis under vilka förutsättningar som behandlingen sker i nuläget och ivilken omfattning denna sker. Inställningen till förordningen bland företag är varierande, de som tar den på allvar har insettatt nya krav kommer att ställas på deras verksamheter och har påbörjat arbetet för att anpassasin verksamhet. Andra anser att de inte berörs i samma utsträckning och är därför inte likaoroliga. På det stora hela kommer förordningen göra att personuppgiftsbehandlingen inomEU/ESS blir säkrare även om resan dit kan kosta. / On the 25th of May 2018 the Swedish Personal Data Act (Personuppgiftslagen, PuL) will be replaced by the EU-regulation General Data Protection Regulation (GDPR). The aim of this study was to determine the big differences between PuL and the GDPR while also looking at how organisations plan to handle the changes that the new regulation will bring. PuL and GDPRare in many ways similar but there are some major changes that will affect every person and every organisation within the EU/EES. The regulation will mean better and morecomprehensive rights for the individual, which in turn will result in higher requirements beingput on the companies who process personal data. The new requirements will result in extensive work within several areas to adapt to the new regulation. The legal and administrative sections will need to review their current contracts & agreements and update them if necessary. There may also need to implement technical solutionsto manage the requirements concerning “the right to be forgotten”, “data portability”, “recordkeeping” and the improved protection of personal data. According to the regulationorganisations are required to notify the supervisory authority of any data breach concerningpersonal data. The change in material scope will result in personal data stored in an unstructuredway being covered by the GDPR. To make companies follow the new legislation thesupervisory authority gains the power to levy significant fines if organisations violate the newregulation; in addition some organisations will need to appoint a “Data protection officer”that’ll monitor the processing of personal data. There are several factors that will determine the amount of work required to reach compliance;for example the size of the corporation and the extent of their current processing of personaldata. The attitude to the new regulation vary, those that take the regulation seriously have realisedthat there will be new demands put on their business, and have already started to adapt theirbusiness to reach compliance with the regulation. There are others that deem that they will notbe affected to the same extent and are therefore not as worried. In the great scope of things, theregulation will make the processing of personal data more secure within the EU/EES eventhough the peregrination to reach compliance may turn out to be a costly one.

Master’s thesis deals with the generation of digital audio signals with band-limited frequency spectrum, i.e. without the aliasing distortion. Various methods of generating band-limited rectangular, triangular, and sawtooth waveforms are described in the theoretical part. The described methods are programmed in the Matlab programming environment and compared in terms of real-time parameter changes, such as duty cycle change of rectangular waveform or continuous change of frequency. The main part of the thesis describes implementation of methods of successive integration of band-limited impulse train and method of differentiated parabolic waveforms in C++ language. The implemented methods were integrated into a plug-in of VST technology that generates an audio signal in real time. The implemented methods are compared in terms of computational complexity and distortion of the generated signal.

In passive target localization, source position is estimated by only using the source signal. In this thesis, position of a stationary target is estimated by using the data collected by a moving platform. Since the focus of the thesis is the location estimation, the parameters used for localization such as angle-of-arrival (AOA), time-difference-of-arrival (TDOA), Doppler frequency shift are assumed to be known. Different emitter localization methods are implemented in this thesis. Some of these methods are known in the literature and some are the modified or hybrid versions of these algorithms. Orthogonal Vector Estimator (OVE), Pseudolinear Estimator (PLE), Weighted Instrumental Variables Estimator (WIVE) and Maximum Likelihood Estimator (MLE) use only the AOA information. In MLE, Gauss Newton (GN) search algorithm is used to realize the search process effectively. AOA localization methods are also implemented together with the extended Kalman filter (EKF) realization. Doppler Shifted Frequency (DSF) based Least Squares (LS) and MLE are implemented which use Doppler frequency shift only. AOA-DSF combined hybrid algorithm is shown to perform better. LS and Maximum Likelihood (ML) TDOA localization methods are also implemented. AOA-DSF-TDOA combined hybrid algorithm is shown to perform better than the algorithms which use one type of parameter and AOA-DSF hybrid algorithm. Estimator performances are analyzed in this thesis. Error ellipsoid is a useful tool to evaluate an estimator

On May 25, 2018, the Directive, 95/46/EC, is superseded by the General Data Protection Regulation (GDPR), (EU) 2016/679. Companies and organizations will have to revise routines, restructure organizations' processes and rebuild IT systems. The purpose of this study is to identify the critical success factors for implementing GDPR in the Swedish banking and finance sector. The study carried out a literature study as a foundation for the qualitative interviews with which empirical was gathered. Subjects for interviews was data protection officers (DPO) at four banks, as well as the Swedish Data Protection Authority and Forum för dataskydd, a national forum for DPO:s. The study's results showed a number of significant success factors for implementation processes. Based on these success factors, three were identified as critical to the implementation of GDPR from a computer science perspective: data governance, privacy-by-design, and documentation. / Den 25 maj 2018 ersattes dataskyddsdirektivet, 95/46/EG, med EU-förordningen 2016/679, General Data Protection Regulation (GDPR). För företag och organisationer kommer detta bland annat innebära nya rutiner, omstrukturering av organisationers processer och ombyggnation av IT-system. Syftet med denna studie är att identifiera de kritiska framgångsfaktorerna för implementering av GDPR inom den svenska bank- och finanssektorn. I studien genomfördes litteraturstudie som låg till grund för insamlande av empiri genom kvalitativa intervjuer med dataskyddsombud vid fyra banker. Intervjuer genomfördes även med Datainspektionen och Forum för dataskydd. Studiens resultat visade på ett flertal betydande framgångsfaktorer för implementeringsprocesser. Utifrån dessa framgångsfaktorer identifierades tre som kritiska för implementation av GDPR ur ett datavetenskapligt perspektiv: data governance, privacy-by-design samt dokumentation.

Network reconstruction is the process of recovering a unique structured representation of some dynamic system using input-output data and some additional knowledge about the structure of the system. Many network reconstruction algorithms have been proposed in recent years, most dealing with the reconstruction of strictly proper networks (i.e., networks that require delays in all dynamics between measured variables). However, no reconstruction technique presently exists capable of recovering both the structure and dynamics of networks where links are proper (delays in dynamics are not required) and not necessarily strictly proper.The ultimate objective of this dissertation is to develop algorithms capable of reconstructing proper networks, and this objective will be addressed in three parts. The first part lays the foundation for the theory of mathematical representations of proper networks, including an exposition on when such networks are well-posed (i.e., physically realizable). The second part studies the notions of abstractions of a network, which are other networks that preserve certain properties of the original network but contain less structural information. As such, abstractions require less a priori information to reconstruct from data than the original network, which allows previously-unsolvable problems to become solvable. The third part addresses our original objective and presents reconstruction algorithms to recover proper networks in both the time domain and in the frequency domain.

Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.