Measurement of soil phytase activity (PA) and delineation of the impact of this important phosphomonoesterase on the P-cycling process in soil and sediments suffer from the lack of a reliable assay. A method for measuring PA in soil that promises to be accurate and reliable has been recently published. The method involves the use of a novel chromophoric analog of phytic acid, referred to as T(tethered)InsP5 (5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate). This study was conducted to measure PA in a Clymer forest soil, which contained over twice the amount of soil organic C as previously tested soils, using the TInsP5 PA assay. This investigation specifically addresses: (1) the development of a soil dilution technique for determining maximal PA, (2) identification of previously unsubstantiated soil-produced dephosphorylated intermediate probe species, (3) the impact of increasing assay buffer pH on soil PA and (4) testing stability of the probe's amide bond in a highly (bio)active forest soil. PA assays were conducted by measuring dephosphorylation of TInsP5 in citrate-acetate buffered (pH 4.2) active and autoclaved (Control) soil suspensions. Phosphorylated probe intermediates (i.e., TInsP4, TInsP3, TInsP2 and TInsP1) and T-myo-inositol were extracted from samples of soil suspension following incubation. Probe species were quantified using reversed phase high-performance liquid chromatography (RPHPLC) with UV detection. PA was calculated based on a mass balance approach. A soil dilution technique was developed to address the challenge of determining maximal PA in soils containing higher organic matter content. In the initial report on use of the TInsP5 method for measuring PA in soil, two "soil-generated" UV-adsorbing compounds (designated Y and Z) were observed, but never confirmed as probe species. The experimental evidence presented in this report supports inclusion of compound Y as a phosphorylated probe intermediate species (i.e. TInsPy), based primarily on its UV adsorption spectra (diode-array detection analysis). Compound Z could not be substantiated as a probe species based on the evidence presented in this study. PA of Claymer forest soil decreased with an increase in assay buffer pH. Further, the probe's amide bond linkage was stable in a forest soil exhibiting high PA. / Master of Science

Despite extensive preventative efforts, the problem of controlling infections associated with biomedical materials persists. Bacteria tend to colonize on biocompatible materials and form biofilms; thus, novel biomaterials with antibacterial properties are of great interest. In this thesis, titanium dioxide (TiO2)-associated photocatalysis under ultraviolet (UV) irradiation was investigated as a strategy for developing bioactivity and antibacterial properties on biomaterials. Although much of the work was specifically directed towards dental materials, the results presented are applicable to a wide range of biomaterial applications. Most of the experimental work in the thesis was based on a resin-TiO2 nanocomposite that was prepared by adding 20 wt% TiO2 nanoparticles to a resin-based polymer material. Tests showed that the addition of the nanoparticles endowed the adhesive material with photocatalytic activity without affecting the functional bonding strength. Subsequent studies indicated a number of additional beneficial properties associated with the nanocomposite that appear promising for biomaterial applications. For example, irradiation with UV light induced bioactivity on the otherwise non-bioactive nanocomposite; this was indicated by hydroxyapatite formation on the surface following soaking in Dulbecco’s phosphate-buffered saline. Under UV irradiation, the resin-TiO2 nanocomposite provided effective antibacterial action against both planktonic and biofilm bacteria. UV irradiation of the nanocomposite also provided a prolonged antibacterial effect that continued after removal of the UV light source. UV treatment also reduced bacterial adhesion to the resin-TiO2 surface. The mechanisms involved in the antibacterial effects of TiO2 photocatalysis were studied by investigating the specific contributions of the photocatalytic reaction products (the reactive oxygen species) and their disinfection kinetics. Methods of improving the viability analysis of bacteria subjected to photocatalysis were also developed.

<p>Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. </p><p>A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did.</p><p>Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. </p><p>Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.</p>

Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did. Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.

The herein presented Ph.D. dissertation describes kinetic and structural characterization of human glutamate carboxypeptidases II and III (GCPII and GCPIII) using a complete panel of their natural substrates. These enzymes hydrolyze C-terminal glutamate from their substrates. They share 67 % sequence identity and also similar enzymatic activities. This thesis quantitatively compares human GCPII and GCPIII in terms of their ability to hydrolyze the substrates N-acetyl-L-aspartyl-L-glutamate (NAAG), folyl-poly-γ-L-glutamic acids (FolGlun) and β-citryl-L-glutamate (BCG). We demonstrated that GCPIII hydrolyzes its substrates in a metal- dependent manner, that BCG is a specific substrate of GCPIII, and that NAAG and FolGlun are specific substrates of GCPII. We also provide indirect biochemical evidence that GCPIII might feature a heterometallic active-site cluster. Additionally, we characterized the relevance of a surface exosite of GCPII, the arene-binding site (ABS), for the hydrolysis of FolGlun substrates using mutagenesis and enzyme kinetics and showed that polymorphic His475Tyr variant of GCPII hydrolyzes FolGlun substrates with the same kinetic parameters as the wild-type enzyme. Furthermore, this thesis focuses on structural aspects of the substrate specificities of GCPII and GCPIII: we present...

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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N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.