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Třepená fonace v reklamách: studie funkcí třepené fonace v audiovizuální prezentaci značky. / Creaky voice in commercials: a study of functions of vocal fry in audio-visual presentation of a brand.Nanić, Ada January 2021 (has links)
1 Abstract The present thesis is concerned with the study of creaky voice and its communicative functions in audio-visual presentation of a brand. This study analyzes nineteen videos that were part of the Sign On campaign produced by Greenpeace in 2009. The main purpose of this thesis is to measure the level of creaky voice in communicative functions. The communicative functions presented in this study are based on the model of Roman Jakobson (1960) and some new functions are proposed as addition to the model. One of the predictions of this thesis is that functions that are emotionally loaded will be comparatively more creaky and this prediction is partially met. Secondly, it was expected to see the same order of the communicative functions based on their level of creakiness among different groups of speakers. This expectation was not met and the possible reasons for the findings are discussed. This thesis uses only acoustic measurements for the comparison of the communicative functions based on their levels of creaky voice. Finally, this thesis discusses the possibilities and current limitations of acoustic methods used for detection of creaky phonation. Keywords: creaky voice, vocal fry, laryngealization, communicative functions, F0, HNR, H1-H2, antimode
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Integrating Chemical Looping Gasification for Hydrogen Generation and CO2 Capture in Pulp Mills / Integrering av Chemical Looping Gasification för Generering av Vätgas samt CO2 Infångning på MassabrukPalmér, Matilda January 2022 (has links)
Utsläpp av CO2 till atmosfären bidrar till ökningen av globala temperaturer. Industrisektorn står för 20 % av utsläppen och utav dessa kommer 6 % från pappers- och massaindustrin. För att lyckas minska den globala temperaturhöjningen till under 1,5 °C hjälper det inte bara att minska utsläppen. Även negativa utsläpp måste genereras. Syftet med denna studie är att undersöka implementeringen av CLG för att separera CO2 på ett energieffektivt sätt och samtidigt generera H2 och elektricitet. Processanalyser genomfördes för att undersöka möjligheten att implementera CLG-processen till ett typiskt massabruk. Processmodeller togs fram for att undersöka CLG, värmeåtervinning samt elektricitetsgenerering. Processmodellerna utvecklades med hjälp av Aspen Plus och Aspen HYSYS. De framtagna modellerna analyserades sedan med avseende på olika designparametrar inom CLG-processen. På ett typiskt massabruk som producerar 800 000 adt varje år kan 375 kg CO2/adt separeras och då uppnå negativa utsläpp, genom att byta ut multi-fuel forsrännaren med en CLG process. Den framtagna processmodellen skulle också kunna generera 360-504 kWh/adt av H2 beroende på de designparametrar som används för CLG-processen. Enligt modellen kan värme som återvinns från processen användas för att fånga upp ytterligare 13 % av CO2 från andra delar av bruket. Processanalys för olika designparametrar inom CLG systemet så som temperatur, luftflöde och flödet av syrgasbärare har presenterats. Nyckeltalen som undersöktes var den mängd CO2 som kunde fångas upp, mängd H2 genererad samt överskottet av elektricitet som produceras när multi-fuel förbränningen byts ut mot en CLG-process på ett typiskt massa bruk. / Emissions of CO2 to the atmosphere are contributing to the global temperature rise. The industrial sector contributed to 20 % of the emissions and out of that, 6 % are generated from the pulp and paper industry. To limit the temperature increase below 1,5 °C, the emissions not only need to be reduced but also negative emissions should be generated from different sectors. The purpose of this study is to realize the implementation of Chemical Looping Gasification (CLG) to separate CO2 (for permanent storage) in an energy-efficient way while co-generating H2 as well as electricity. Process analysis was carried out to investigate the possibility of substituting the multifuel boiler in a typical pulp mill with a CLG process. Process models for the CLG, heat recovery and electricity generation process were developed using AspenPlus and Aspen HYSYS. The process was analysed for different design conditions (temperature, autothermal condition, air flow, oxygen carrier flow) in the CLG process. It was found that in a typical pulp mill producing 800 000 adt per year, 375 kg- CO2/adt (14 % of total emissions from the process) can be inherently separated for storage to achieve negative emissions, if the multi-fuel boiler is replaced with a CLG unit. This process will also be able to generate 360-504 kWh/adt H2 depending on the design conditions in the CLG process. Heat recovered from the CLG unit can be utilized in capturing approximately 13 % additional CO2 from other sources in the pulp mill. Process analysis for different design conditions in CLG (temperature, airflow, oxygen carrier flow) have been presented. The key performance indicators were CO2 capture rates, H2 generated and net electrical output from the process.
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[en] H2 SYNTHESIS OF FINITE-DIMENSIONAL CONTROLLERS FOR STABLE, DISTRIBUTED-PARAMETER SYSTEMS / [pt] SINTESE H2 DE CONTROLADORES DE DIMENSAO FINITA PARA SISTEMAS ESTAVEIS DE PARAMETROS DISTRIBUIDOSALVARO GUSTAVO TALAVERA LOPEZ 10 March 2020 (has links)
[pt] Objetiva-se, nesse trabalho, formular e testar numericamente uma
perspectiva computacional, baseada em elementos de controle robusto e
indices de desempenho H2, para a sintese de controladores de dimensão
finita (DF) para sistemas lineares de dimensão infinita (DI) correspondentes
a certas equações de evolução parabólicas e, especialmente, a uma versão
simplificada da equação do calor. A abordagem aqui utilizada é a de usar
modelos aproximantes de DF (modelos nominais) e limitantes superiores
nas normas H (infinito) dos erros de aproximação correspondentes nas funções de transferência de DI em questão, de modo que o procedimento de síntese
baseie-se apenas em funções de transferência racionais e os controladores resultantes sejam de DF.Mais especificamente, uma classe de controladores que asseguram a estabilidade do sistema em malha fechada envolvendo o sistema de DI em questão e definida tomando-se as soluções ótimas de problemas
H2 (infinito) nos quais o funcional de custo (H2) nominal é minimizado sobre os
controladores nominalmente estabilizantes, sob uma restrição (H infinito) de margem
de estabilidade mínima definida por um parâmetro de projeto escalar denotado
por micro. A obtenção de um controlador é então feita pela escolha do valor de
micro de modo a minimizar um limitante superior (calculado apenas com base
em funçõess racionais) sobre o funcional de custo calculado no sistema de DI
original. Esse procedimento é ilustrado por exemplos numéricos envolvendo a
versão simplificada da equação do calor. / [en] A computational perspective based on robust control tools is presented
for the H2 synthesis of finite-dimensional controllers for linear, stable,
distributed-parameter systems corresponding to certain evolution equations.
The approach pursued here relies on finite-dimensional approximations and
error bounds on the H (infinite) norms of the corresponding errors on transfer functions
so that the resulting synthesis procedure solely depends on rational transfer
functions, thereby yielding finite-dimensional controllers. More specifically, a
class of stabilizing controllers for a given infinite-dimensional system is defined
taking optimal solutions of H2 / H (infinite) problems - i.e., a nominal H2 cost is
minimized over controllers which satisfy a nominal stability margin defined
by a scalar parameter micro. A controller is then obtained by choosing micro in such a
way as to minimize an upper bound on the value taken by the cost functional on
the original infinite-dimensional system. This procedure is illustrated by simple
numerical examples involving the (simplified) heat equation in one dimension.
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[en] SYNTHESIS AND CHARACTERIZATION OF COPPER COATINGS ON POLYIMIDE MEMBRANES (PIR 003) / [pt] SÍNTESE E CARACTERIZAÇÃO DE RECOBRIMENTOS DE COBRE EM MEMBRANAS DE POLIIMIDA (PIR 003) E FITA KAPTON23 November 2023 (has links)
[pt] A formação de juntas metálicas / poliméricas estáveis é um enorme desafio nas
ciências dos materiais. A adesão requer uma interface que é capaz de interagir
especificamente com a fase metálica. As poliimidas apresentam grupos amino primários
altamente reativos às superfícies metálicas. Os recobrimentos metal/polímero se utilizam
principalmente como membranas de separação de gases (permeabilidade e permissividade)
e como material de baixa constante dielétrica para dispositivos microeletrônicos. Este
trabalho vem fornecer a síntese dos recobrimentos metal/polímero em poliimida e fita
kapton, por redução com H2, com posterior compreensão dos mecanismos de quimio-absorção a base de catalisadores da base de paládio, prata, hipoclorito de sodio e da adição
de solventes na poliimida PIR 003, que permite a adesão do cobre. Foram utilizados os
precursores de cobre em CuSO(4).5H(2)O e do CuCl(2), sintetizados a partir do CuO, para uma
posterior redução em atmosfera de H2 e obter cobre métalico no recobrimento, permitindo
o desenvolvimento de novas abordagens para metalização de materiais baseados naredução
por H2 em polímeros. A utilização de modelos matemáticos permitiu dar uma visualização
aproximada do ajuste dos parâmetros na redução do cobre por H2. As medidas do ângulo de
contato nas poliimidas funcionalizadas deram uma visualização da influenciada adesão com
o cobre e as medidas das caracterizações foram realizadas a fim de mostrar consistência dos
resultados dos diferentes tratamentos, entre as quais foram: FTIR, MEV-EDS, TGA, DRX.
O presente estudo demostra que com o precursor de CuSO(4).5H(2)O e a funcionalização com
o NaClO 50 ml/l na poliimida, apresentou o maior valor de tamanhode cristalito de 61.8
nm e também de maior espessura de recobrimento de 182 micrometros. Finalmente, os testes de
adesão para a poliimida PIR 003-Cu com o precursor de CuSO4.5H2O, no recobrimento
sem tratamento teve uma força de tração aproximada de 4MPa e no caso do tratamento
Sn/Pd (0.1/0.2 g/l) uma média aproximada de 10 MPa. / [en] The formation of stable metallic / polymeric joints is a huge challenge in materialsscience. Adhesion requires an interface that is capable of specifically interacting with themetallic phase. Polyimides have primary amino groups that are highly reactive to metalsurfaces. The coatings of these metal/polymer are mainly used as gas separationmembranes (permeability and permittivity) and as a low dielectric constant material formicroelectronic devices. This work provides the synthesis of metal/polymer coatings inpolyimide and kapton tape, by reduction with H2, with further understanding of the chemoabsorption mechanisms based on catalysts based on palladium, silver, sodium hypochloriteand the addition ofsolvents in the polyimide PIR 003, which allows the adhesion of copper.Copper precursors in CuSO(4).5H2O and CuCl(2), synthesized from CuO, were used forfurther reduction in H2 atmosphere and to obtain metallic copper in the coating, allowingthe development of new approaches for metallization of materials based on H2 reductionin polymers. The use of mathematical models allowed an approximate visualization of theadjustment of the parameters in the reduction of copper by H2. The contact anglemeasurements in the functionalized polyimides gave a visualization of the influence ofadhesion with copper and the characterization measurements were carried out in order toshow consistency of the results of the different treatments, among which were: FTIR,MEV-EDS, TGA, DRX. The present study demonstrates that with the CuSO4.5H2Oprecursor and the functionalization with 50 ml/l sodium hypochlorite in the polyimide, itpresented the largest crystallite size value of 61.8 nm and also the largest coating thicknessof 182 micrometers. Finally, the adhesion tests for the polyimide PIR 003-Cu with the precursor ofCuSO4.5H2O, in the untreated coating had a tensile strength of approximately 4MPa andin the case of the Sn/Pd treatment (0.1/0.2 g/l) a approximate average of 10 MPa.
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Validation and standardization of a FRET-based whole-cell lysate RNase H2 activity assaySchulz, Marian Simon 20 February 2024 (has links)
Ribonucleotide excision repair (RER) is an RNase H2-dependent DNA-repair mechanism removing mis-incorporated ribonucleotides to maintain DNA stability. Decreased RNase H2 activity leads to an accumulation of ribonucleotides in the DNA, destabilizing and eventually damaging the DNA. This results in double-strand breaks, chromosome abberations, impaired segregation of defective chromosomes, and the formation of micronuclei with unstable nuclear membranes. Upon breakdown of the mironuclear envelope, the released chromatin triggers a cGAS-STING-dependent immune cascade that stimulates the production of type I interferons and cytokines. RNase H2 deficiency directly contributes to autoinflammation and autoimmunity and might further play a role in several types of cancer, aging and neurodegeneration. Therefore, RNase H2 activity is a promising diagnostic and prognostic marker. However, until today, no method for quantification of RNase H2 activity has been validated for a clinical use. Herein, a standard operating procedure for a high-throughput FRET-based whole cell lysate RNase H2 assay is implemented and validated delivering standard curves, statistical benchmarks and standardization to an externally validated control. Providing high sensitivity and strict linearity over a wide working range, the assay is applicable to various human cell or tissue samples with overall methodological assay variability from 8.6% to 16%. Human T cells were identified as a suitable cell type for the implementation of a clinical screening method, showing relatively small inter-individual variability when activity is normalized to cell number. Indeed, decreased RNase H2 activity was detected in T cells from one patient with systemic sclerosis and two patients with systemic lupus erythematosus who carried RNASEH2 mutations known to disrupt enzyme function in vitro compared with a control group of 24 healthy donors. With these findings, this dissertation provides fundamentals for the implementation of an RNase H2 assay screening method in the clinical setting. For the actual clinical application, however, the establishment of a significantly larger control group is necessary. This might allow identification of further inter-individual variables influencing RNase H2 activity and facilitate the determination of a threshold below which a reduction of RNase H2 activity is likely to become clinically relevant. Phenotypic effects of RNASEH2 mutations are often assessed in experiments with recombinant enzyme. However, this does not allow conclusions to be drawn about the extent to which the mutations affect enzyme activity through transcriptional, post-transcriptional, translational, or post-translational processes. In contrast, direct measurement of RNase H2 activity in cell lysates facilitates a more comprehensive assessment of the clinical relevance of genetic variants. To assess whether HeLa cell models are suitable for studying the intracellular effects of RNASEH2 mutations on enzyme activity, the well-studied RNASEH2B mutation A177T was inserted into HeLa cells using CRISPR/Cas9. However, due to high variability of RNase H2 activity in the HeLa cell clones after transfection and clonal selection, and low targeting efficiency, this approach has limited potential in HeLa cells. Direct use of immortalized cell lines derived from patient tissue, or a CRISPR/Cas approach in iPS cells might be more promising. As a secondary result, this study provides evidence that intracellular RNase H2 activity is increased in S, G2, and M phase of the cell cycle. In addition, increased RNase H2 activity was seen after stimulation with LPS and IL-2, and especially the mitogen PMA suggesting various pathways of RNase H2 activity regulation.:CONTENTS
CONTENTS 5
ABBREVIATIONS 8
ABSTRACT 13
ZUSAMMENFASSUNG 15
1 INTRODUCTION 17
1.1 RIBONUCLEOTIDE EXCISION REPAIR IN HEALTH AND DISEASE 17
1.1.1 Role and function of RER in mammals 17
1.1.2 Clinical relevance of dysfunctional RER 18
1.1.2.1 Aicardi-Goutières syndrome – a paradigm for autoimmunity 19
1.1.2.2 RNase H2 and malignancy 21
1.1.2.3 DNA-damage, aging and neurodegeneration 21
1.2 RIBONUCLEASE H2 21
1.2.1 Genetic and biochemical characteristics 21
1.2.2 Rnase H2 activity assays 23
1.2.3 Known mutations and their effects on enzymatic function 24
1.3 CRISPR/CAS: A TOOL FOR TARGETED MUTAGENESIS 27
1.4 AIM OF THIS THESIS 28
2 MATERIAL AND METHODS 29
2.1 MATERIAL 29
2.1.1 Chemicals and Reagents 29
2.2.1.1 Nucleic acids 29
2.2.1.2 Enzymes 30
2.2.1.3 Antibodies 31
2.2.1.4 Buffers and solutions 31
2.2.1.5 Cell growth media 32
2.2.1.6 Basic chemicals and reagents 32
2.1.2 Consumables 34
2.1.3 Kits 34
2.1.4 Devices 35
2.1.5 Cell lines 36
2.1.6 Animal clones 36
2.1.7 Software, databases and websites 37
2.2 METHODS 37
2.2.1 Cell methods 37
2.2.1.1 Isolation of primary cells 37
2.2.1.2 Cell culture 39
2.2.1.3 Cell analysis 41
2.2.2 Nucleic acid methods 42
2.2.2.1 DNA isolation via isopropanol precipitation 42
2.2.2.2 DNA isolation via ethanol precipitation in 96-well plates 42
2.2.2.3 DNA quantification 43
2.2.2.4 Primer design for PCR 43
2.2.2.5 Polymerase chain reaction (PCR) and gel electrophoresis 43
2.2.2.6 Purification of PCR products 44
2.2.2.7 Cloning methods 44
2.2.2.8 DNA Sequencing 45
2.2.3 Protein methods 46
2.2.3.1 Protein extraction from cells 46
2.2.3.2 Protein quantification 47
2.2.4 Statistics and informatics 47
2.2.5 CRISPR/Cas methods 47
2.2.5.1 sgRNA design 47
2.2.5.2 Cloning of plasmids containing sgRNA cassettes 48
2.2.5.3 Design of repair templates 49
2.2.5.4 Generating genetically modified HeLa cells using CRISPR/Cas9 49
2.2.5.5 Genotyping of cell clones generated by CRISPR/Cas9 50
2.2.6 RNase H2 activity 50
2.2.7 Control group inclusion criteria 50
3 RESULTS 51
3.1 ESTABLISHING THE RNASE H2 ASSAY 51
3.1.1 Method establishment 51
3.1.1.1 Methodological approach 51
3.1.1.2 Assay workflow and normalization 51
3.1.1.3 Establishing basic assay settings 54
3.1.1.4 Time-resolved measurement 54
3.1.1.5 Establishing controls 56
3.1.1.6 Fluorescence standard curves 62
3.1.1.7 Interpretation of the fluorescence progress curve 62
3.1.1.8 Steady-state kinetics: Definition of assay end-points 65
3.1.1.9 Standardization to externally validated controls 66
3.1.1.10 Ruggedness 68
3.1.1.11 Influence of cell cycle and stimulation on RNase H2 activity 70
3.1.2 Assay precision 70
3.1.2.1 Coefficient of variation 70
3.1.2.2 Experimental design 71
3.1.2.3 Error levels I – III: from linear regression to pipetting error 71
3.1.2.4 Error level IVa and IV: quantification error 75
3.1.2.5 Error levels V and VI: cell preparation errors 77
3.1.2.6 Calculation of individual CVs 79
3.1.2.7 Replication of individual assay steps and the effective CV 81
3.1.2.8 Inter-assay variability the use of standards 82
3.1.3 RNase H2 activity of different cell types 82
3.2 ESTABLISHING A SCREENING STRATEGY FOR RNASE H2 ACTIVITY 85
3.2.1 Choice of cell type and cell isolation 85
3.2.2 Recruitment of the control group 86
3.2.3 Biological variability of RNase H2 activity in B cells and T cells 86
3.2.4 Sample size and effect size 89
3.2.5 Reduced RNase H2 Activity in T Cells of Patients with Systemic Autoimmunity 91
3.3 GENERATION OF AN RNASEH2BA177T CELL MODEL 93
3.3.1 Experimental design 93
3.3.2 Genotyping results 94
3.3.3 Impact of the RNASEH2B A177T mutation on RNase H2 activity 95
4 DISCUSSION 98
4.1 RNASE H2 ASSAY 98
4.1.1 Qualitative validity 98
4.1.1.1 Assay end-points 98
4.1.1.2 Determination of RNase H2 activity from enzyme progress curves 100
4.1.1.3 Normalization 102
4.1.1.4 Validation and control of systematic errors 104
4.1.2 Quantitative considerations 107
4.1.2.1 Sensitivity, precision and replication 107
4.1.2.2 Applicability for high-throughput analysis 108
4.1.3 Perspective 108
4.2 RNASE H2 ACTIVITY SCREENING IN HUMAN CD3+ CELLS 109
4.3 CELL MODELS FOR PATHOGENIC RNASE H2 VARIANTS 112
4.4 RNASE H2 FUNCTION AND REGULATION 113
4.4.1 RNase H2 and transcription 113
4.4.2 RNase H2 kinetic parameters 115
4.4.3 RNase H2 activity during the cell cycle and induction by PMA 115
4.4.4 RNase H2 activity in different cell types 117
REFERENCES 119
APPENDIX 134
APP. 1: ASSAY SUBSTRATES 134
APP. 2: ANALYSIS OF ERROR SOURCES 134
Biological errors 134
Procedural errors 137
APP. 3: QUBITTM PROTEIN ASSAY PERFORMANCE CHARACTERISTICS 139
APP. 4: ‘ACCURACY’ AND RELATED TERMS 140
APP. 5: CHARACTERISTICS OF THE SYSTEMIC SCLEROSIS PATIENT SSC1 141
APP. 6: RNASE H2 SUBUNIT PROTEIN EXPRESSION IN DIFFERENT TISSUES 142
APP. 7: PARADIGM CALCULATION OF THE EFFECTIVE METHODOLOGICAL CV 143
APP. 8: GENOTYPING RESULTS OF CRISPR/CAS9-GENERATED HELA CLONES 144
APP. 9: RNASE H2 ASSAY STANDARD OPERATING PROCEDURE 146
SOP 1 cell preparation and lysis 146
SOP 1.1 Material and reagents 146
SOP 1.2 Assay planning 146
SOP 1.3 Prepare cell pellets 147
SOP 1.4 Lysis 147
SOP 2 Qubit™ protein assay 148
SOP 2.1 Material 148
SOP 2.1 Working procedure 148
SOP 3 RNase H2 assay 150
SOP 3.1 Material and reagents 150
SOP 3.2 Prepare a plate layout and a pipetting scheme 151
SOP 3.3 Prepare the reaction buffer and substrates 151
SOP 3.4 Prepare your lysate premix (volume B, 65 µl) 151
SOP 3.5 Prepare the photometer 152
SOP 3.6 Start the reaction by adding volume A (55 µl) to the reaction plate 152
SOP 3.9 Insert the plate, perform gain adjustment and start the test run 152
SOP 3.10 Data analysis 152
SOP 4 Figures and Charts 155
RNase H2 assay work flow 155
Assay substrates 156
Chart A. Corrected CVs of all error levels 157
Estimation of the effective CV for a planned experiment 158
Chart B: RNaseH2 assay working range for different cell types 159
Chart C: Approximate cell yield of biological material 159
Chart D: Plate layout 160
Chart E: Pipetting scheme 160
Pipetting work flow 161
Chart F: Fluorescence raw data table 161
Calculation of standard catalytic activity using standard curves 162
Inter-assay comparability 163
ACKNOWLEDGMENTS 164
DECLARATIONS 165 / Ribonukleotid-Exzisionsreparatur ist ein RNase-H2-abhängiger DNA-Reparaturmechanismus, der durch die Entfernung fälschlich eingebauter Ribonukleotide die Integrität und Stabilität der DNA erhält. Eine verminderte RNase-H2-Aktivität führt zu einer Anhäufung von Ribonukleotiden in der DNA, wodurch die DNA destabilisiert wird und schließlich Schaden nimmt. Das Resultat sind Doppelstrangbrüche, Chromosomenabberationen, eine gestörte Segregation der defekten Chromosomen und die Bildung von „Mikrokernen“ mit instabilen Kernmembranen. Bei Zerfall dieser Mikrokern-Hüllen löst das freiwerdende Chromatin eine cGAS-STING-abhängige Immunkaskade aus, welche die Bildung von Typ-I-Interferonen und Zytokinen stimuliert. Verminderte RNase-H2-Aktivität trägt dadurch direkt zur Entstehung von Autoinflammation und Autoimmunität bei und spielt wahrscheinlich auch als Malignitätsfaktor einiger Karzinome, sowie bei Alterungsprozessen und Neurodegeneration eine Rolle. Daher kann RNase-H2-Aktivität als ein vielversprechender diagnostischer und prognostischer Marker angesehen werden. Bisher etablierte Methoden zur Messung der RNase-H2-Enzymaktivität verfügen jedoch nicht über die Standardisierung und Validierung, welche für den klinischen Einsatz notwendig sind. Diese Dissertation implementiert eine Standardvorgehensweise, Standardkurven und statistische Kenngrößen für einen FRET-basierten RNase-H2-Assay. Der Assay ist für die Anwendung mit Zelllysaten validiert, und liefert standardisierte Ergebnisse. Durch eine hohe Sensitivität und eine strikte Linearität über einen großen Arbeitsbereich kann der Assay in vielen verschiedenen Zell- oder Gewebetypen angewendet werden. Die Gesamt-Variabilität beträgt dabei zwischen 8,6 % bis 16 %. Aufgrund einer relativ niedrigen inter-individuellen Schwankung der zellulären RNase-H2-Aktivität sind menschliche T-Zellen ein geeigneter Zelltyp für klinische Vergleichsstudien. So konnte in T-Zellen einer Patientin mit Systemischer Sklerose und zweier Patientinnen mit Systemischem Lupus Erythematodes, welche bekannte heterozygote RNASEH2 Mutationen aufwiesen, eine verminderte RNase-H2-Aktivität im Vergleich zu einer Kontrollgruppe mit gesunden Probanden gefunden werden. Diese Dissertation liefert die Grundlagen für die Implementierung eines RNase-H2-Assays als klinisches Diagnostikum. Für eine tatsächliche klinische Anwendung ist jedoch die Etablierung einer deutlich größeren Kontrollgruppe notwendig. Dadurch könnten einerseits weitere interindividuelle Einflussgrößen auf die RNase-H2-Aktivität identifiziert werden. Andererseits könnte dies die Festlegung eines Schwellenwerts ermöglichen, unter welchem sich eine Reduktion der RNase-H2-Aktivität wahrscheinlich klinisch manifestiert. Zur Beurteilung der phänotypischen Auswirkungen von RNASEH2 Mutationen werden häufig Experimente mit rekombinanter RNase-H2-durchgeführt. Dies lässt allerdings keine Aussagen darüber zu, inwiefern die Mutationen die Enzymaktivität durch transkriptionelle, post-transkriptionelle, translationale oder post-translationale Prozesse beeinflussen. Die direkte Messung der RNase-H2-Aktivität in Zelllysaten ermöglicht eine umfassendere Bewertung der klinischen Relevanz genetischer Varianten. Zur Einschätzung ob HeLa-Zell-Modelle geeignet dafür sind, die intrazellulären Auswirkungen von RNASEH2-Mutationen auf die Enzymaktivität zu untersuchen, wurde mittels CRISPR/Cas9 die vielseits publizierte RNASEH2B-Mutation A177T in HeLa-Zellen eingefügt. Jedoch fand sich nach der Transfektion und Zellklonierung eine sehr hohe Variabilität der RNase-H2-Aktivität zwischen den HeLa-Zellklonen. Aufgrund der zudem relativ niedrigen Targeting-Effizienz scheinen HeLa-Zellen für diese Fragestellung ein wenig geeigneter Zelltyp zu sein. Die direkte Verwendung von immortalisierten Zelllinien aus Patientengewebe, oder die Anwendung von CRISPR/Cas in iPS-Zellen könnten vielversprechender sein. Als Nebenbefund fand sich in dieser Dissertation eine erhöhte RNase-H2-Aktivität in der S-, G2- und M-Phase des Zellzyklus, sowie nach der Stimulation mit LPS und IL-2, sowie insbesondere dem Mitogen PMA. Dies liefert Hinweise zu möglichen intrazellulären Regulationswegen der RNase-H2-Aktivität, über welche bisher wenig bekannt ist.:CONTENTS
CONTENTS 5
ABBREVIATIONS 8
ABSTRACT 13
ZUSAMMENFASSUNG 15
1 INTRODUCTION 17
1.1 RIBONUCLEOTIDE EXCISION REPAIR IN HEALTH AND DISEASE 17
1.1.1 Role and function of RER in mammals 17
1.1.2 Clinical relevance of dysfunctional RER 18
1.1.2.1 Aicardi-Goutières syndrome – a paradigm for autoimmunity 19
1.1.2.2 RNase H2 and malignancy 21
1.1.2.3 DNA-damage, aging and neurodegeneration 21
1.2 RIBONUCLEASE H2 21
1.2.1 Genetic and biochemical characteristics 21
1.2.2 Rnase H2 activity assays 23
1.2.3 Known mutations and their effects on enzymatic function 24
1.3 CRISPR/CAS: A TOOL FOR TARGETED MUTAGENESIS 27
1.4 AIM OF THIS THESIS 28
2 MATERIAL AND METHODS 29
2.1 MATERIAL 29
2.1.1 Chemicals and Reagents 29
2.2.1.1 Nucleic acids 29
2.2.1.2 Enzymes 30
2.2.1.3 Antibodies 31
2.2.1.4 Buffers and solutions 31
2.2.1.5 Cell growth media 32
2.2.1.6 Basic chemicals and reagents 32
2.1.2 Consumables 34
2.1.3 Kits 34
2.1.4 Devices 35
2.1.5 Cell lines 36
2.1.6 Animal clones 36
2.1.7 Software, databases and websites 37
2.2 METHODS 37
2.2.1 Cell methods 37
2.2.1.1 Isolation of primary cells 37
2.2.1.2 Cell culture 39
2.2.1.3 Cell analysis 41
2.2.2 Nucleic acid methods 42
2.2.2.1 DNA isolation via isopropanol precipitation 42
2.2.2.2 DNA isolation via ethanol precipitation in 96-well plates 42
2.2.2.3 DNA quantification 43
2.2.2.4 Primer design for PCR 43
2.2.2.5 Polymerase chain reaction (PCR) and gel electrophoresis 43
2.2.2.6 Purification of PCR products 44
2.2.2.7 Cloning methods 44
2.2.2.8 DNA Sequencing 45
2.2.3 Protein methods 46
2.2.3.1 Protein extraction from cells 46
2.2.3.2 Protein quantification 47
2.2.4 Statistics and informatics 47
2.2.5 CRISPR/Cas methods 47
2.2.5.1 sgRNA design 47
2.2.5.2 Cloning of plasmids containing sgRNA cassettes 48
2.2.5.3 Design of repair templates 49
2.2.5.4 Generating genetically modified HeLa cells using CRISPR/Cas9 49
2.2.5.5 Genotyping of cell clones generated by CRISPR/Cas9 50
2.2.6 RNase H2 activity 50
2.2.7 Control group inclusion criteria 50
3 RESULTS 51
3.1 ESTABLISHING THE RNASE H2 ASSAY 51
3.1.1 Method establishment 51
3.1.1.1 Methodological approach 51
3.1.1.2 Assay workflow and normalization 51
3.1.1.3 Establishing basic assay settings 54
3.1.1.4 Time-resolved measurement 54
3.1.1.5 Establishing controls 56
3.1.1.6 Fluorescence standard curves 62
3.1.1.7 Interpretation of the fluorescence progress curve 62
3.1.1.8 Steady-state kinetics: Definition of assay end-points 65
3.1.1.9 Standardization to externally validated controls 66
3.1.1.10 Ruggedness 68
3.1.1.11 Influence of cell cycle and stimulation on RNase H2 activity 70
3.1.2 Assay precision 70
3.1.2.1 Coefficient of variation 70
3.1.2.2 Experimental design 71
3.1.2.3 Error levels I – III: from linear regression to pipetting error 71
3.1.2.4 Error level IVa and IV: quantification error 75
3.1.2.5 Error levels V and VI: cell preparation errors 77
3.1.2.6 Calculation of individual CVs 79
3.1.2.7 Replication of individual assay steps and the effective CV 81
3.1.2.8 Inter-assay variability the use of standards 82
3.1.3 RNase H2 activity of different cell types 82
3.2 ESTABLISHING A SCREENING STRATEGY FOR RNASE H2 ACTIVITY 85
3.2.1 Choice of cell type and cell isolation 85
3.2.2 Recruitment of the control group 86
3.2.3 Biological variability of RNase H2 activity in B cells and T cells 86
3.2.4 Sample size and effect size 89
3.2.5 Reduced RNase H2 Activity in T Cells of Patients with Systemic Autoimmunity 91
3.3 GENERATION OF AN RNASEH2BA177T CELL MODEL 93
3.3.1 Experimental design 93
3.3.2 Genotyping results 94
3.3.3 Impact of the RNASEH2B A177T mutation on RNase H2 activity 95
4 DISCUSSION 98
4.1 RNASE H2 ASSAY 98
4.1.1 Qualitative validity 98
4.1.1.1 Assay end-points 98
4.1.1.2 Determination of RNase H2 activity from enzyme progress curves 100
4.1.1.3 Normalization 102
4.1.1.4 Validation and control of systematic errors 104
4.1.2 Quantitative considerations 107
4.1.2.1 Sensitivity, precision and replication 107
4.1.2.2 Applicability for high-throughput analysis 108
4.1.3 Perspective 108
4.2 RNASE H2 ACTIVITY SCREENING IN HUMAN CD3+ CELLS 109
4.3 CELL MODELS FOR PATHOGENIC RNASE H2 VARIANTS 112
4.4 RNASE H2 FUNCTION AND REGULATION 113
4.4.1 RNase H2 and transcription 113
4.4.2 RNase H2 kinetic parameters 115
4.4.3 RNase H2 activity during the cell cycle and induction by PMA 115
4.4.4 RNase H2 activity in different cell types 117
REFERENCES 119
APPENDIX 134
APP. 1: ASSAY SUBSTRATES 134
APP. 2: ANALYSIS OF ERROR SOURCES 134
Biological errors 134
Procedural errors 137
APP. 3: QUBITTM PROTEIN ASSAY PERFORMANCE CHARACTERISTICS 139
APP. 4: ‘ACCURACY’ AND RELATED TERMS 140
APP. 5: CHARACTERISTICS OF THE SYSTEMIC SCLEROSIS PATIENT SSC1 141
APP. 6: RNASE H2 SUBUNIT PROTEIN EXPRESSION IN DIFFERENT TISSUES 142
APP. 7: PARADIGM CALCULATION OF THE EFFECTIVE METHODOLOGICAL CV 143
APP. 8: GENOTYPING RESULTS OF CRISPR/CAS9-GENERATED HELA CLONES 144
APP. 9: RNASE H2 ASSAY STANDARD OPERATING PROCEDURE 146
SOP 1 cell preparation and lysis 146
SOP 1.1 Material and reagents 146
SOP 1.2 Assay planning 146
SOP 1.3 Prepare cell pellets 147
SOP 1.4 Lysis 147
SOP 2 Qubit™ protein assay 148
SOP 2.1 Material 148
SOP 2.1 Working procedure 148
SOP 3 RNase H2 assay 150
SOP 3.1 Material and reagents 150
SOP 3.2 Prepare a plate layout and a pipetting scheme 151
SOP 3.3 Prepare the reaction buffer and substrates 151
SOP 3.4 Prepare your lysate premix (volume B, 65 µl) 151
SOP 3.5 Prepare the photometer 152
SOP 3.6 Start the reaction by adding volume A (55 µl) to the reaction plate 152
SOP 3.9 Insert the plate, perform gain adjustment and start the test run 152
SOP 3.10 Data analysis 152
SOP 4 Figures and Charts 155
RNase H2 assay work flow 155
Assay substrates 156
Chart A. Corrected CVs of all error levels 157
Estimation of the effective CV for a planned experiment 158
Chart B: RNaseH2 assay working range for different cell types 159
Chart C: Approximate cell yield of biological material 159
Chart D: Plate layout 160
Chart E: Pipetting scheme 160
Pipetting work flow 161
Chart F: Fluorescence raw data table 161
Calculation of standard catalytic activity using standard curves 162
Inter-assay comparability 163
ACKNOWLEDGMENTS 164
DECLARATIONS 165
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Integrating Chemical Looping Gasification for Hydrogen Generation and CO2 Capture in Pulp Mills / Integrering av Chemical Looping Gasification för Generering av Vätgas samt CO2 Infångning på MassabrukPamér, Matilda January 2022 (has links)
Utsläpp av CO2 till atmosfären bidrar till ökningen av globala temperaturer. Industrisektorn står för 20 % av utsläppen och utav dessa kommer 6 % från pappers- och massaindustrin. För att lyckas minska den globala temperaturhöjningen till under 1,5 °C hjälper det inte bara att minska utsläppen. Även negativa utsläpp måste genereras. Syftet med denna studie är att undersöka implementeringen av CLG för att separera CO2 på ett energieffektivt sätt och samtidigt generera H2 och elektricitet. Processanalyser genomfördes för att undersöka möjligheten att implementera CLG-processen till ett typiskt massabruk. Processmodeller togs fram for att undersöka CLG, värmeåtervinning samt elektricitetsgenerering. Processmodellerna utvecklades med hjälp av Aspen Plus och Aspen HYSYS. De framtagna modellerna analyserades sedan med avseende på olika designparametrar inom CLG-processen. På ett typiskt massabruk som producerar 800 000 adt varje ˚ar kan 375 kg CO2/adt separeras och då uppnå negativa utsläpp, genom att byta ut multi-fuel forsrännaren med en CLG process. Den framtagna processmodellen skulle också kunna generera 360-504 kWh/adt av H2 beroende på de designparametrar som används för CLG-processen. Enligt modellen kan värme som ˚återvinns från processen användas för att fånga upp ytterligare 13 % av CO2 från andra delar av bruket. Processanalys för olika designparametrar inom CLG systemet så som temperatur, luftflöde och flödet av syrgasbärare har presenterats. Nyckeltalen som undersöktes var den mängd CO2 som kunde fångas upp, mängd H2 genererad samt överskottet av elektricitet som produceras när multi-fuel förbränningen byts ut mot en CLG-process på ett typiskt massa bruk. / Emissions of CO2 to the atmosphere are contributing to the global temperature rise. The industrial sector contributed to 20 % of the emissions and out of that, 6 % are generated from the pulp and paper industry. To limit the temperature increase below 1,5 °C, the emissions not only need to be reduced but also negative emissions should be generated from different sectors. The purpose of this study is to realize the implementation of Chemical Looping Gasification (CLG) to separate CO2 (for permanent storage) in an energy-efficient way while co-generating H2 as well as electricity. Process analysis was carried out to investigate the possibility of substituting the multifuel boiler in a typical pulp mill with a CLG process. Process models for the CLG, heat recovery and electricity generation process were developed using Aspen Plus and Aspen HYSYS. The process was analysed for different design conditions (temperature, autothermal condition, air flow, oxygen carrier flow) in the CLG process. It was found that in a typical pulp mill producing 800 000 adt per year, 375 kg- CO2/adt (14 % of total emissions from the process) can be inherently separated for storage to achieve negative emissions, if the multi-fuel boiler is replaced with a CLG unit. This process will also be able to generate 360-504 kWh/adt H2 depending on the design conditions in the CLG process. Heat recovered from the CLG unit can be utilized in capturing approximately 13 % additional CO2 from other sources in the pulp mill. Process analysis for different design conditions in CLG (temperature, airflow, oxygen carrier flow) have been presented. The key performance indicators were CO2 capture rates, H2 generated and net electrical output from the process.
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Richard Cosin and the rehabilitation of the clerical estate in late Elizabethan EnglandHampson, James E. January 1997 (has links)
The royal supremacy established by Henry VIII was never fully defined or resolved. Was it an imperial kingship or a mixed polity - the king-in-parliament? Professor G.R Elton's theory of parliamentary supremacy has been accepted for many years, but more recently this theory has come under attack from Professors Peter Lake, John Guy, and Patrick Collinson. They have shown that it was not strictly the case that either the royal supremacy or the ecclesiastical polity of the Tudors was a settled issue; there was a tension and an uncertainty that underlay both the Henrician break with Rome in 1534 and the Elizabethan Settlement of 1559, yet this tension was not brought to surface of Tudor political debate until the latter part of Elizabeth I's reign. What brought the issue to the fore was the controversy between the puritans who opposed Archbishop John Whitgift's subscription campaign and the 'conformists' who sided with Whitgift's demand for order and congruity in the young Church of England. Part of this controversy was carried out in a literary battle between one of Whitgift's proteges, civil lawyer and high commissioner Richard Cosin, and puritan common lawyer James Morice. The debate focused on the legality of the High Commission's use of the ex officio oath and eventually came to hinge on the question of Elizabeth's authority to empower that commission to exact the oath by virtue of her letters patent. If the ex officio oath was strictly against the statutes and common laws of the realm, was the queen authorised to direct the commission to exact the oath anyway - over and above the law? To answer yes, as Cosin did, was to declare that the queen's royal supremacy was imperial and that her ecclesiastical polity was essentially theocratic. To answer no, as did Morice, was to assert that there were certain things that the queen could not do - namely that she was not empowered to direct the High Commission to contravene statute law, even in the name of ordering and reforming the church. Cosin's legal arguments for the imperial supremacy of the monarch were powerful, but his writings were steeped in a form of political rhetoric that was quickly coming into fashion in the late sixteenth century: the 'language of state'. The language of state was essentially an abandonment of the classical-humanist vocabulary of 'counseling the prince' for the sake of 'virtuous government' in pursuit of a 'happy republic'. This new political language used by Cosin and other conformists justified itself on the premise that the state was so thoroughly endangered by sedition and instability that an urgent corrective was needed: not wise or virtuous counsel but strict obedience to the laws that preserved civil and religious authority. With the threat of presbyterianism at the doorstep of the English Church, Cosin - protected and encouraged by the powerful Whitgift - was free to employ both his legal and his rhetorical skills in an effort to reinvigorate the English clergy by enhancing their jurisdictional status over the laity. By the time James VI and I began his systematic revitalisation of the clerical estate in 1604, the vocabulary that would justify it had already been created. The influence of Cosin demonstrably permeated the early years of the Stuart Church suggesting that Cosin might provide a link between the vague uncertainties of the Elizabethan Settlement and the stark realities of the Caroline Church.
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Effects of radiolysis on the dynamics of UO2-dissolutionEkeroth, Ella January 2003 (has links)
No description available.
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Fuel Reforming for Hydrogen Production in Heavy-Duty Vehicle ApplicationsGranlund, Moa. Z. January 2015 (has links)
The depletion of fossil fuels together with growing environmental concerns have created incitement for developing a more energy-efficient and environmentally-friendly vehicle fleet. The development towards cleaner heavy-duty vehicles started already in the 80’s with the introduction of emission legislations. Initially, engine optimization was enough for reaching the legislated levels of emissions. However, at present engine optimization is not enough but exhaust aftertreatment has become an essential part of heavy-duty vehicles, in order to meet the emission standards. Today, the total emissions are targeted which means that there is an interest in decreasing the idling emissions as well as the emissions during operation. To reduce the overall emissions several states in the USA have introduced idling legislations. Due to the limitations in idling time alternative solutions for power generation during rests are requested. A possible alternative is a fuel cell auxiliary power unit, combining a fuel cell with a fuel reformer (FC-APU). The focus of this thesis is the development of the fuel reformer for an FC-APU, in which the hydrogen to the fuel cell is generated from diesel in a high-temperature catalytic process. The produced hydrogen can also be used in other heavy-duty vehicle applications i.e. selective catalytic reduction of NOx (HC-SCR), where addition of hydrogen is essential for reaching high conversion at low temperatures. The effect of using hydrogen from a fuel reformer in HC-SCR is included in this work. The catalytic material development is focused on developing promoted materials with lower rhodium content but with catalytic activity comparable to that of materials with higher rhodium content. This includes evaluation and extensive characterization of both fresh and aged promoted materials. The work also includes reactor design where a micro reactor with multiple air inlets is evaluated. This work has contributed to increased knowledge of catalytic materials suitable for reforming of diesel. By changing the support material from the traditionally used alumina to ceria-zirconia, increased H2 yield was achieved. In addition, the ceria-zirconia supported material was less prone to coke. By promoting the material with cobalt or lanthanum it was possible to decrease the rhodium content by 2/3 with enhanced catalytic performance. It was also discovered that promotion with lanthanum decreased the tendency for coking even further. Additionally, the lanthanum-promoted material had higher thermal stability as well as a stable highly dispersed rhodium phase. Furthermore, the work has contributed to an increased knowledge concerning the fuel reformer’s effect on HC-SCR. The work displays clear evidence of benefits with using hydrogen-rich gas from a fuel reformer instead of pure hydrogen. The benefits are derived from the content of low molecular weight hydrocarbons present in the hydrogen-rich gas, which are strong reducing agents increasing the NOx reduction. This finding proves that fuel reforming in combination with HC-SCR is a viable option for NOx abatement. / <p>QC 20150202</p>
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Sociala aspekter på en multikulturell byggarbetsplats : Arbetsmiljö & säkerhet / Social aspects in a multicultural construction-worksite : Work environment & safetyAnell, Nicklas, Haverstal, Albin January 2017 (has links)
Rapporten är skriven i samarbete med H2 Entreprenad AB och inriktad på arbetsmiljöarbetetmed utländska underentreprenörer på den svenska arbetsmarknaden inom byggbranschen. Enomfattande litteraturstudie har gjorts och kompletterats med en intervjustudie.Byggbranschens produktionstakt är på nivåer som jämställs med miljonprogrammets dagar på60- till 70-talet, det behövs 710 000 nya bostäder mellan åren 2015 och 2025 enligt Boverket.Med ett sådant tryck på byggbranschen har företagen svårt att få tag i arbetskraft, detta är enav anledningarna till att utstationerade arbetare ökat markant i Sverige de senaste åren.Den utländska arbetskraften ger tillgång till ny kompetent arbetskraft, som oftast är billigare.Kraven på ledningen att styra de utländska underentreprenörerna som tas in ökar dockmarkant och leder till ökade kostnader. Risken för en utländsk arbetare att skadas på ensvensk arbetsplats är också 36 % högre, och risken att dö är fyra gånger så stor som för ensvensk arbetare. Detta medför att ett större fokus på ett fungerande arbetsmiljöarbete behövssom fungerar trots språkförbristningarna.De sociala och kulturella skillnaderna tas upp och analyseras i rapporten, hur hierarkier ochstyrning för yrkesarbetare sker i jämförelse mellan svenska och utländska underentreprenörer. / The report is written in collaboration with H2 Entreprenad AB and focuses on the workenvironment with foreign subcontractors on the Swedish labor market in the constructionindustry. A comprehensive literature study has been done and completed with an interviewstudy.The construction industries production rate is at levels equal to the days of the millionprogram in the 60s and 70s. 710 000 new homes are needed between 2015 and 2025according to Boverket. With such pressure on the construction industry, companies havedifficulty in obtaining labor. This is one of the reasons why posted workers have increasedsignificantly in Sweden in recent years.The foreign labor force gives access to new skilled labor, which is usually cheaper. However,the requirements of management of foreign subcontractors taken in are increasingsignificantly, leading to enlarged costs. The risk of a foreign worker being injured in aSwedish workplace is also 36 % higher, and the risk of dying is four times the size of aSwedish. This means that a greater focus on a functioning work environment is needed, whichwill operate despite language barriers.The social and cultural differences are analyzed and processed in the report, how hierarchiesand guidance for professional workers are done in a comparison between Swedish and foreignsubcontractors.
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