O operon pst de Escherichia coli é formado pelos genes pstS, pstC, pstA, pstB e phoU. Os quatro primeiros genes codificam proteínas que compõem um sistema de transporte do tipo ABC denominado Pst. O sistema Pst, junto com a proteína PhoU participa da repressão dos genes do regulon PHO. A transcrição dos genes do operon pst é induzida pela carência de fosfato inorgânico (Pi). Neste trabalho, o padrão de transcrição do operon pst foi analisado. A existência de um transcrito primário instável foi comprovada através de uma nova técnica de RT-PCR. O papel da RNase E na degradação do mRNA de pst foi demonstrado. A análise das seqüências intergênicas do operon revelou a importância da seqüência REP (Repetitive Extragenic Palindrome) localizada na região intergênica entre pstS e pstC na estabilização da mensagem de pstS. As seqüências localizadas a 5\' dos genes pstC, pstB e phoU também foram analisadas e demonstraram uma atividade promotora fraca, que resulta na síntese de transcritos dos genes distais de pst. / The pst operon of Escherichia coli consists of the genes pstS, pstC, pstA, pstB and phoU. The four proximal genes of the operon encode the proteins of the ABC-type Pi phosphate (Pi) transporter Pst.The Pst system, together with the PhoU protein, also acts as a negative regulator of the PHO regulon. Transcription of the pst genes is induced by Pi starvation. The present study describes the transcription pattern of the pst operon. The existence of an unstable primary transcript was confirmed by using an improved RT-PCR protocol. The role of RNase E in pst transcript decay was demonstrated.Analysis of the operon intergenic regions revealed the role of a REP sequence (Repetitive Extragenic Palindrome) located between pstS and pstC in pst mRNA stability. The regions upstream of pstC, pstB and phoU displayed promoter activity. Transcription from these internal promoters resulted in a small amount of mRNAs corresponding to the pst distal genes.

L’activité RNase P est l'activité essentielle qui élimine les séquences 5' supplémentaires des précurseurs d'ARN de transfert. "PRORP" (PROteinaceous RNase P) définit une nouvelle catégorie de RNase P uniquement protéique. Avant la caractérisation de PRORP, on pensait que les enzymes RNase P étaient universellement conservées sous forme de ribonucléoprotéines (RNP). La caractérisation de PRORP a révélé une enzyme avec deux domaines principaux, un domaine N-terminal contenant plusieurs motifs PPR et un domaine NYN C-terminal portant l’activité catalytique. Nous avons utilisé une combinaison d'approches biochimiques et biophysiques pour caractériser le complexe PRORP / ARNt. La structure du complexe en solution a été déterminée par diffusion des rayons X aux petits angles (SAXS) et les Kd des interactions de différents mutants de PRORP avec l’ARNt ont été déterminées par ultracentrifugation analytique. Notre analyse révèle un cas intéressant d'évolution convergente. Il suggère que PRORP a développé un processus de reconnaissance de l'ARN similaire à celui des RNase P RNP. Par ailleurs, nous avons mis en place une approche de co-immunoprécipitation de PRORP avec l’ARN afin de définir le spectre de substrats des RNase P protéiques. / RNase P is the essential activity that removes 5'-leader sequences from transfer RNA precursors. “PRORP” (PROteinaceous RNase P) defines a novel category of protein only RNase P. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins (RNP). The characterization of PRORP revealed an enzyme with two main domains, an N-terminal domain containing multiple PPR motifs and a C-terminal NYN domain holding catalytic activity. We used a combination of biochemical and biophysical approaches to characterize the PRORP / tRNA complex. The structure of the complex in solution was determined by small angle X-ray scattering and Kd values of the PRORP / tRNA interaction were determined by analytical ultracentrifugation. We also analyzed direct interaction of a collection of PPR mutants with tRNA in order to determine the relative importance of individual PPR motifs for RNA binding. This reveals to what extent PRORP target recognition process conforms to the mode of action of PPR proteins interacting with linear RNA. Altogether, our analysis reveals an interesting case of convergent evolution. It suggests that PRORP has evolved an RNA recognition process similar to that of RNP RNase P. Moreover, we also implemented a PRORP-RNA co-immunoprecipitation approach to determine the full extent of PRORP substrates.

Les R-loops générés durant la transcription sont impliqués dans de nombreuse fonctions incluant la réplication, la recombinaison et l’expression génique tant chez les procaryotes que chez les eucaryotes. Plusieurs études ont montré qu’un excès de supertours négatifs et des séquences riches en bases G induisent la formation de R-loops. Jusqu’à maintenant, nos résultats nous ont permis d’établir un lien direct entre les topoisomérases, le niveau de surenroulement et la formation de R-loops. Cependant, le rôle physiologique des R-loops est encore largement inconnu. Dans le premier article, une étude détaillée du double mutant topA rnhA a montré qu’une déplétion de RNase HI induit une réponse cellulaire qui empêche la gyrase d’introduire des supertours. Il s’agit ici, de la plus forte évidence supportant les rôles majeurs de la RNase HI dans la régulation du surenroulement de l’ADN. Nos résultats ont également montré que les R-loops pouvaient inhiber l’expression génique. Cependant, les mécanismes exacts sont encore mal connus. L’accumulation d’ARNs courts au détriment d’ARNs pleine longueur peut être causée soit par des blocages durant l’élongation de la transcription soit par la dégradation des ARNs pleine longueur. Dans le deuxième article, nous montrons que l’hypersurenroulement négatif peut mener à la formation de R-loops non-spécifiques (indépendants de la séquence nucléotidique). La présence de ces derniers, engendre une dégradation massive des ARNs et ultimement à la formation de protéines tronquées. En conclusion, ces études montrent l’évidence d’un lien étroit entre la RNase HI, la formation des R-loops, la topologie de l’ADN et l’expression génique. De plus, elles attestent de la présence d’un nouvel inhibiteur de gyrase ou d’un mécanisme encore inconnu capable de réguler son activité. Cette surprenante découverte est élémentaire sachant que de nombreux antibiotiques ciblent la gyrase. Finalement, ces études pourront servir également de base à des recherches similaires chez les cellules eucaryotes. / R-loops generated during transcription elongation are implicated in many DNA reactions, including replication, recombination and gene expression both in prokaryotes and in eukaryotes. Many studies have shown that negative supercoils excess and G-rich sequences induce the formation of R-loops. Up to now, our results allow us to establish a direct link between topoisomerases, supercoiling level, and the formation of R-loops. However, what the physiological significance, if any, of R-loops is still largely unknown. In the first article, a detailed study on double topA rnhA mutants showed that the depletion of RNase HI activity induces a cellular response which renders gyrase unable to perform supercoils. This is the first evidence implicating RNase HI as a major player in DNA supercoiling regulation. Our results also show that R-loops formation can lead to the inhibition of gene expression. However, the exact mechanism(s) leading to the inhibition of gene expression are not yet understood. The accumulation of shorter than full length RNAs could be caused by road-blocks during transcription elongation or by the degradation of full length RNAs. In the second article, we show that hypernegative supercoiling can lead to sequence independent R-loop formation. The physiological consequence is extensive RNA degradation which ultimately culminates in the formation of truncated proteins. In conclusion, this study clearly shows a close link between RNase HI activity, R-loop formation, DNA topology and gene expression. In addition, this study also provides some evidence for the synthesis of a gyrase inhibitor that can regulate gyrase activity directly or indirectly via unidentified mechanisms. This surprising observation is still preliminary taking into consideration that many antibiotics target gyrase. Finally results from this study could open up avenues for research in eukaryotes.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

O operon pst de Escherichia coli é formado pelos genes pstS, pstC, pstA, pstB e phoU. Os quatro primeiros genes codificam proteínas que compõem um sistema de transporte do tipo ABC denominado Pst. O sistema Pst, junto com a proteína PhoU participa da repressão dos genes do regulon PHO. A transcrição dos genes do operon pst é induzida pela carência de fosfato inorgânico (Pi). Neste trabalho, o padrão de transcrição do operon pst foi analisado. A existência de um transcrito primário instável foi comprovada através de uma nova técnica de RT-PCR. O papel da RNase E na degradação do mRNA de pst foi demonstrado. A análise das seqüências intergênicas do operon revelou a importância da seqüência REP (Repetitive Extragenic Palindrome) localizada na região intergênica entre pstS e pstC na estabilização da mensagem de pstS. As seqüências localizadas a 5\' dos genes pstC, pstB e phoU também foram analisadas e demonstraram uma atividade promotora fraca, que resulta na síntese de transcritos dos genes distais de pst. / The pst operon of Escherichia coli consists of the genes pstS, pstC, pstA, pstB and phoU. The four proximal genes of the operon encode the proteins of the ABC-type Pi phosphate (Pi) transporter Pst.The Pst system, together with the PhoU protein, also acts as a negative regulator of the PHO regulon. Transcription of the pst genes is induced by Pi starvation. The present study describes the transcription pattern of the pst operon. The existence of an unstable primary transcript was confirmed by using an improved RT-PCR protocol. The role of RNase E in pst transcript decay was demonstrated.Analysis of the operon intergenic regions revealed the role of a REP sequence (Repetitive Extragenic Palindrome) located between pstS and pstC in pst mRNA stability. The regions upstream of pstC, pstB and phoU displayed promoter activity. Transcription from these internal promoters resulted in a small amount of mRNAs corresponding to the pst distal genes.

Ziel der Arbeit war es, die regulatorischen Mechanismen von Virulenz-assoziierten Genen in den Pathogenen Francisella novicida und Streptococcus pyogenes zu untersuchen. Kapitel eins befasst sich mit der Regulation des Virulenzfaktors Streptolysin S (SLS) von S. pyogenes. Wir untersuchten die Rolle der Ribonuklease (RNase) Y in der transkriptionellen und posttranstrikptionellen Regulation des Gens sagA. RNase Y begünstigte die Produktion einer kleinen RNA (sRNA) vom sagA Transkript, war jedoch nicht an der posttranskriptionellen Regulierung der sagA RNA beteiligt. Dennoch förderte RNase Y die Transkription von sagA indirekt. Wir konnten weiterhin zeigen, dass die 5′- untranslatierte Region (UTR) der sgaA RNA eine Sekundärstruktur besitzt, die möglicherweise einen Liganden bindet und damit die Zugänglichkeit der ribosomalen Bindungsstelle beeinflusst. Die Deletion einzelner Abschnitte der 5′ UTR hat einen negativen Effekt auf die sagA Expression. Wir haben eine Methode entwickelt um die Aktivität von Riboswitches, (u.a. die sagA 5‘ UTR) zu analysieren und konnten damit drei putative Riboswitches in S. pyogenes validieren. In Kapitel zwei charakterisierten wir den Mechanismus mit dem CRISPR-Cas9 aus F. novicida (FnoCas9) die Expression bakterieller Lipoproteine (BLPs) unterdrückt, um dem Immunsystem des Wirtes zu entgehen. Wir zeigen, dass FnoCas9 eine duale Funktion besitzt, die es dem Protein ermöglicht nicht nur DNA zu schneiden, sondern auch Transkription zu regulieren. In dieser erstmals beschriebenen Aktivität bindet FnoCas9 an den tracrRNA:scaRNA Duplex, wodurch der Protein-RNA Komplex an einen DNA Abschnitt hinter dem Promoter der blp Gene bindet und somit deren Transkription verhindert. Diese Bindungsstelle besitzt ein protospacer-adjacent motif (PAM) und eine scaRNA-komplementäre Sequenz, an die der FnoCas9-RNA Komplex bindet, allerdings nicht schneidet. Dieses System könnte in Zukunft das Repertoire an CRISPR-basierten Anwendungsmöglichkeiten erweitern. / The aim of this thesis was to study regulatory mechanisms of virulence-related genes in the bacterial pathogens Francicella novicida and Streptococcus pyogenes. Chapter one focuses on the regulation of the virulence factor streptolysin S (SLS) in S. pyogenes. First, we investigated the role of the ribonuclease (RNase) Y in the transcriptional and post-transcriptional regulation of SLS-coding gene, sagA. We found that RNase Y promotes the production of a small RNA (sRNA) from the sagA transcript but we observed no regulation at the post-transcriptional level. Yet, RNase Y promotes sagA transcription indirectly and affects hemolysis levels. We next showed that the sagA 5′ untranslated region (UTR) contains a secondary structure that is is possibly modulated by direct binding to a ligand and may affect the accessibility to the ribosomal binding site (RBS). Our results indicate that removing fragments of the 5′ UTR has a negative effect on sagA expression. We developed a method for testing the activity of putative riboswitches, including sagA 5′ UTR. Using this method, we validated three predicted riboswitches in S. pyogenes. In chapter two, we characterized the mechanism by which F. novicida CRISPR-Cas9 (FnoCas9) represses the expression of bacterial lipoproteins (BLPs), allowing evasion of the host immune system. We show that FnoCas9 is a dual-function protein that, in addition to its canonical DNA nuclease activity, evolved the ability to regulate transcription. In this newly-described mechanism, the non-canonical RNA duplex tracrRNA:scaRNA guides FnoCas9 to the DNA target located downstream of the promoter of the BLP-coding genes, causing transcriptional interference. The endogenous targets contain a protospacer-adjacent motif (PAM) and a sequence that is complementary to scaRNA, promoting FnoCas9 binding but not DNA cleavage. Engineering this system expands the toolbox of CRISPR applications by allowing repressing other genes of interest.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

Bakterien haben eine Vielzahl an Strategien entwickelt, um sich an ständig wechselnde Umweltbedingungen anzupassen, darunter auch post-transkriptionelle regulatorische Mechanismen. Die Genexpression kann hierbei durch gezielten Abbau oder Stabilisierung von RNA durch Ribonukleasen (RNasen) reguliert werden. RNasen weisen je nach Spezies allerdings unterschiedliche Effekte auf Genexpression und bakterielle Physiologie, sowie verschiedene Strategien der Substraterkennung auf. Dies zeigt, dass unser Verständnis des RNA-Abbaus bei weitem nicht vollständig ist. Ziel dieser Arbeit ist es, die Eigenschaften und Funktionen der endoRNase Y des humanpathogenen Bakteriums Streptococcus pyogenes zu studieren. Um Einblick in Funktion und Spezifität dieser RNase zu gewinnen, wurden deren genomweite Schnittpositionen (“targetome”) mit Hilfe von RNA-Sequenzierung identifiziert. Zur weiteren Analyse des RNase Y-abhängigen RNA-Abbaus wurde dieses Ergebnis mit dem “targetome” der drei 3′-5′-Exoribonukleasen (ExoRNasen) PNPase, YhaM und RNase R verglichen. Schließlich wurden die Anforderungen für die Prozessierung durch RNase Y und deren Rolle in der Regulation von Virulenzgenen in vivo anhand der speB mRNA, die einen wichtigen Virulenzfaktor codiert, untersucht. Wir konnten in dieser Arbeit zeigen, dass RNase Y Substrate bevorzugt nach einem Guanosin schneidet und dieses Nukleosid essenziell für die Prozessierung der speB mRNA in vivo ist. Obwohl RNase Y die speB mRNA schneidet, unterstützen die Daten ein Modell nach dem RNase Y die Expression von speB auf transkriptioneller Ebene reguliert. Mit Hilfe des “targetome”-Vergleichs konnten wir ferner zeigen, dass RNase Y den RNA-Abbau in S. pyogenes initiiert und die dabei generierten 3′-Enden der RNA hauptsächlich von den 3′-5′-exoRNasen PNPase und/oder YhaM prozessiert werden. Zusammenfassend erweitern diese Erkenntnisse unser Verständnis der Funktionalität von RNase Y und des RNA-Abbaus in Gram-positiven Bakterien. / Bacteria have developed a plethora of strategies to cope with constantly changing environmental conditions, including post-transcriptional regulatory mechanisms. With this regard, regulation of gene expression can be achieved by either the rapid removal or stabilization of RNA molecules by ribonucleases (RNases). RNases exhibit species-specific effects on gene expression, bacterial physiology and different strategies of target recognition, indicating that our understanding of the RNA degradation machinery is not yet complete. The aim of this thesis was to investigate the features and functions of endoRNase Y from the strict human pathogen Streptococcus pyogenes. To gain insight into the role and specificity of this RNase, we identified RNase Y cleavage positions (i.e. targetome) genome-wide by RNA sequencing. Next, to investigate the RNA degradation pathway depending on RNase Y, we compared the RNase Y targetome with the ones of the three 3′-to-5′ exoribonuclease (exoRNases), namely PNPase, YhaM and RNase R. Finally, to dissect the requirements for RNase Y processing and to decipher the role of RNase Y in virulence gene regulation, we studied the impact of RNase Y on speB mRNA, encoding a major virulence factor. This study reveals that RNase Y preferentially cleaves RNAs downstream of a guanosine and for the first time we were able to show that the presence of a guanosine residue is essential for the processing of speB mRNA, in vivo. Although RNase Y cleaves the speB mRNA, our data underpin a model in which RNase Y-mediated regulation of speB expression occurs at the transcriptional level. Using the targetome comparative approach, we demonstrated that RNase Y initiates RNA decay in S. pyogenes and that the RNase Y-generated RNA 3′ ends are usually further trimmed by PNPase and/or YhaM. Overall, these findings increase our understanding of RNase Y functionality and RNA degradation in Gram-positive bacteria.

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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