Therapeutic viruses are a class of biotherapeutic which have enabled new treatments and medical advances in the areas of vaccines, cancer treatment, gene therapy, and more. In the production and purification of these products, the sterile filtration unit operation is known to have poor yields and contribute to the high cost of the final product, significantly hampering the large-scale production of some therapeutic viruses. Thus, this thesis seeks to explore various aspects of process development and fundamental understanding in the sterile filtration of therapeutic viruses. This thesis explores the mechanisms and membrane properties which govern how bacteria are retained during filtration, and applies these insights to improve the sterile filtration recovery of a therapeutic virus through proper membrane selection. To better understand the factors which cause membrane fouling and loss of virus during sterile filtration, the effect of host cell impurities on filtration performance was investigated. This revealed that small amounts of host cell protein are a major factor in both membrane fouling and reduced virus yield, and that there is a synergistic effect between the virus and the host cell protein adsorbing to the membrane surface. Recognizing that conventional polymeric membranes have many limitations, a novel ultrathin, isoporous, microfabricated silicon nitride membrane was tested for suitability as a sterile filter. Finally, the application of nanoparticles as model virus particles in filtration testing was examined, and a process was developed through which nanoparticles could be fused together to create controlled amounts of particle aggregates, similar to how viruses can be prone to aggregation. The work described here will help enable the development of next generation sterile filtration membranes and provides both insights and methodologies for improving sterile filtration performance. / Thesis / Doctor of Philosophy (PhD) / While many people are aware that viruses can be used in medicine as vaccines, there are even more new and developing ways they can be used, such as in fighting cancer or treating previously uncurable diseases. However, testing of and patient access to these new treatments is often limited due to the challenges in producing and purifying enough of the virus. Viruses are highly complex and large relative to other products, and so many of the common methods and manufacturing processes which are standard in the industry need to be significantly adapted or improved to suit the production of viruses. This study investigates one step of the purification process, sterile filtration, and considers how a variety of factors from the materials used to the properties of the virus solution can be optimized to improve performance. With a deeper understanding of the sterile filtration process, recommendations can be made to help improve the production of future virus-based therapies.

Le sélénium est reconnu comme un micronutriment important pour l'homme et les animaux. Plusieurs études ont montré qu'une supplementation en sélénium dans le régime alimentaire pourrait être bénéfique contre les cancers du foie, du colon, du pancréas et de la prostate. Le mécanisme anti-carcinogène du sélénium se produit au niveau systémique, cellulaire et nucléaire. Ces processus peuvent également impliquer le système immunitaire et ne doivent pas être interprétés par un seul mécanisme. Jusqu'à présent son mécanisme d'action est encore inconnu. L'objectif de cette étude était d'étudier l'effet des composés du sélénium, à faibles concentrations, sur la capacité de réparation de l'ADN dans les cellules du cancer de la prostate LNCaP (p53 compétentes). Ce travail est divisé en trois parties. La première partie du travail a été consacrée à étudier l'effet des deux composés du sélénium (SS et SM) sur les propriétés cytotoxiques et génotoxiques de différents stress oxydatifs et non oxydatifs. Les résultats ont montré qu'un prétraitement avec une faible dose en Se stimulait la synthèse des sélénoprotéines, et protègait contre la toxicité et les dommages oxydatifs à l'ADN induites par les UVA ou H2O2, mais pas par MMS ou UVC. La deuxième partie a été consacrée à l'influence de la supplementation en sélénium sur la capacité de réparation de l'ADN. Notre travail a clairement montré l'augmentation de l'efficacité d'excision de certaines glycosylases que n'est pas nécessairement corrélée à une augmentation de l'expression génique et /ou protéiques. Enfin, la troisième partie de notre travail a été dédiée à l'optimisation de la technique Host Cell Reactivation (HCR) qui nous a permis d'étudier la capacité de réparation de l'ADN in cellulo, afin de cibler les partenaires impliqués dans la voie de signalisation affectées par la supplémentation en sélénium. En conclusion, nous pourront penser que le mécanisme d'action du sélénium est représenté par un délicat équilibre entre l'activation et la répression de l'activité de certaines protéines qui induit des changements conformationnels plus ou moins directement impliqués dans la réparation de l'ADN et la progression de la croissance cellulaire. / Selenium was recognized as an important micronutrient for both humans and animals. Several studies showed that increased selenium in the diet might be beneficial against liver, colon, pancreas and prostate cancer. The anticarcinogenic actions of Se occur at the systemic, cellular and nuclear level. These actions may also involve the immune system and thus cannot be interpreted by a single mechanism. Until now its mechanisms of action are not well understood. The objective of this study was to investigate the effect of selenocompounds at low doses on DNA repair capacity in the p53-proficient LNCaP prostate cancer cells. This work is divided into three parts. The first part of the work was devoted to study the effect of two selenocompounds (SS and SM) on the cytotoxic and genotoxic properties of various oxidative and non oxidative stresses. The results showed that low doses of Se pre-treatment stimulates selenoprotein synthesis, protects against toxicity and oxidative DNA damage induced by UVA or H2O2 but not by MMS or UVC. The second part of our investigation was devoted to the influence of selenium supplementation on DNA repair capacity. Our work clearly showed an increase in excision efficiency of the glycosylases activity that was not necessarily correlated with an increase of gene expression and/or protein levels. Finally, the third part of our work was devoted to the optimization of Host Cell Reactivation assay (HCR) to study the DNA repair capacity in cellulo, in order to target the partners involved in the signalling pathway affected by selenium supplementation. In conclusion, we could image that the mechanism of action of selenium is represented by a delicate balance between activation and repression of protein activity that induces conformational changes of several proteins more or less directly involved in DNA repair and progression of cell growth.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.

Introduction: Cryptosporidium parvum is a protozoan parasite that causes diarrhoea in many host species worldwide. CpCDPK1 appears to be essential for invasion and a promising drug target. Aim of the study: The aims of this study were to expand the knowledge of CpCDPK1. To achieve that, attempts were made to inhibit this gene by BKI-1294 in vitro and generate CpCDPK1 KO C. parvum. To maintain the genetically modified parasites, I studied the suitability of infection and propagation in a new animal model RAGgc × IFN-gamma mouse and in vitro model COLO - 680 N cell line. Animals, materials and methods: 4×106 freshly excysted C. parvum sporozoites were seeded into transfected GFP-MDBK cultures at the confluency of 70 – 80 % and simultaneously exposed to 500 nM of BKI-1294. IFA was applied to observe the invasion and host cell actin accumulation. Guide RNA (gRNA) for CRISPR-mediated transfection was designed and the Nluc-neoR repair cassette was flanked with 50 bp long 5’- and 3’UTR of CpCDPK1 by PCR. Transfection was performed by octaarginine transportation and compared to electroporation. COLO - 680 N cells with the confluency of 70 – 80% were infected with 4×106 non-transfected and transfected sporozoites of C. parvum. To establish a laboratory animal model for propagation of C. parvum and drug screening RAGgc × IFN-gamma mice were infected with 500 (G2), 1000 (G3) and 5000 (G4) of oocysts. BALB/c WT mice were inoculated with 5000 (G1) oocysts as control. Faeces were sampled for C. parvum DNA extraction. Real time PCR was applied to calculate the oocyst yield. Results: In the presence of 500 nM BKI-1294, parasite-induced host cell actin accumulation was not observed at 24 and 48 h after inoculation in vitro pointing at altered infectivity of CDPK inhibited sporozoites. Extracellular noninvasive sporozoites were found at 24 h p.i., only one meront was observed in a host cell at 72 h p.i. CRISPR-mediated gene editing was applied to C. parvum to knock out CDPK1. Transfected C. parvum were found in COLO-680 N cells through 6 passages. However, no newly generated oocysts were harvested. RAGgc × IFN-gamma mice were tested suitable as an animal model for C. parvum infection studies and oocyst propagation. These crossbred mice are very sensitive to infection at doses as low as 500 oocysts. They displayed emaciation, rough fur and trembling. The survival percentage was 71.4 % (G2), 85.7 % (G3), 57.1 % (G4) and 100 % (G1) at the end of study. Oocyst yield of 108 OPG was calculated in the crossbred mice whereas only 104 OPG were counted in Balb/C mice. Yields did not differ significantly (P > 0.05) in crossbred mice infected with different oocysts doses. Conclusions: 1.The function of CpCDPK1 is obviously important to the invasion process including attachment and utilization of host cell actin to form PV. This assumption was confirmed by CDPK inhibition and genetic KO. However, methods that increase the transfection efficiency are needed to enhance the generation of KO C. parvum. 2. The transfection method mediated by octargninine is superior to electroporation in consideration of DNA consumption and requirement of device. 3. Due to the low required infection dose and clinical manifestation RAGgc × IFN-gamma mice appear very well suited to serve as an in vivo laboratory model of C. parvum infection and for propagation of particularly transgenic C. parvum strains. 4. COLO – 680 N cells appear suited to be an in vitro model for C. parvum infection and transfection study, however, not qualified for propagation.:Contents 1. Introduction 1 2. Literature Review 2 2.1 Biology 2 2.1.1 Systematics 2 2.1.2 Life cycle 2 2.1.3 Tenacity of oocysts 4 2.1.4 Excystation of oocysts and invasion of host cells 4 2.1.5 Formation of the PV 6 2.1.6 Nutrient supply by the host 7 2.2 Epidemiology 8 2.2.1 Human Cryptosporidiosis 8 2.2.2 Animal Cryptosporidiosis 9 2.3 Detection and Diagnosis 11 2.4 Treatment options 12 2.5 Hygiene 14 2.6 Vaccine 16 2.7 In vitro and vivo Models 16 2.8 Structure and function of Calcium-dependent protein kinases 18 3. Animals, materials and methods 21 3.1 Animals and materials 21 3.1.3 Mice 21 3.1.4 Cells 21 3.1.5 C. parvum oocysts 21 3.1.6 Reagents 21 3.1.7 Plasmids and oligonucleotides 23 3.1.7.1 Plasmids 23 3.1.7.2 Primers and probes 24 3.1.8 Kits 25 3.1.9 Instruments and software 25 3.2 Methods 26 3.2.1 Preparation of reagents 26 3.2.2 C. parvum oocysts maintaince 27 3.2.3 PCR 27 3.2.3.1 Amplification of NdeI and AatII flanked 5’CDPK1 27 3.2.3.2 Annealing of gRNA 27 3.2.3.3 Amplification of repair cassette via Touchdown PCR (TD-PCR) 28 3.2.3.4 Colony PCR 29 3.2.3.5 Real-time PCR for C. parvum hsp70 30 3.2.4 Restriction enzyme digestion 31 3.2.4.1 Restriction enzyme digestion of pA - pD 31 3.2.4.2 Enzyme digestion and dephosphorylation of p185 31 3.2.5 Agarose gel electrophoresis 32 3.2.6 Gel purification 32 3.2.7 Ligation 33 3.2.7.1 Ligation of CDPK1 KO plasmids 33 3.2.7.2 Ligation of gRNA and p185 33 3.2.8 Transformation 34 3.2.9 Plasmid extraction 34 3.2.10 C. parvum oocysts excystation 35 3.2.11 C. parvum infection 35 3.2.11.1 In vitro infection 35 3.2.11.2 C. parvum infection in mice 36 3.2.12 Transfection 36 3.2.12.1 Electroporation for MDBK transfection 36 3.2.12.2 Electroporation for C. parvum transfection 37 3.2.12.3 CpCDPK1 knock out through Cell penetrating peptide (CPP) - octaarginine mediated transfection 38 3.2.13 Geneticin screening for GFP-MDBK cells 39 3.2.14 Indirect immunofluorescent assay (IFA) 39 3.2.15 Animal feeding and body conditioning score (BCS) monitoring 40 3.2.16 Faecal sample collection 43 3.2.17 DNA extraction and oocysts per gram (OPG) determination of fecal samples 43 3.2.18 Statistical analysis 44 4. Results 45 4.1 CDPK1 knockout by REMI 45 4.1.1 Construction of Knockout plasmid 45 4.1.2 Electroporation protocol and in vitro analysis 49 4.2 CDPK1 knockout by CRISPR/Cas 9-mediated gene editing 50 4.2.1 Constructing CRISPR/Cas9_CpCDPK1_7 plasmid 51 4.2.2 Amplification of CDPK1 flanked repair cassette 52 4.2.3 Knockout CDPK1 via CRISPR/cas 9 53 4.2.3.1 Electroporation and in vitro analysis 53 4.2.3.2 CPP transfection and in vitro analysis 55 4.2.3.3 Genetic assay of transfection 57 4.3 In vitro and in vivo model for infection and propagation 58 4.3.1 In vitro model - C. parvum cultivation in COLO - 680 N cells 58 4.3.2 In vivo model Infection pattern of C. parvum in RAGgc x IFN-g KO mice 60 4.3.2.1 Clinical symptoms 60 4.3.2.2 Oocysts excretion 63 4.4 In vitro inhibition of CDPK1 66 4.4.1 Generating bAct-GFP-MDBK cells 67 4.4.2 Influence of CDPK1 inhibition on infection 70 5. Discussion 73 5.1 Sub-cloning 73 5.2 Inhibition of CpCDPK1 delays the host cell actin accumulation in vitro 73 5.3 RAGgc x IFN-gamma KO mice for C. parvum propagation 76 5.4 CpCDPK1 knockout by CRISPR/cas 9 79 5.5 COLO-680 N cells are not suited to propagate C. parvum in vitro 82 6. Summary 85 7. Zusammenfassung 87 8. References 89

Neisseria meningitidis, a commensal β-proteobacterium residing exclusively in the human nasopharynx, is a leading cause of sepsis and epidemic meningitis worldwide. While comparative genome analysis was able to define hyperinvasive lineages that are responsible for most of the cases of invasive meningococcal disease (IMD), the genetic basis of their virulence remains unclear. Recent studies demonstrate that the type II C CRISPR/Cas system of meningococci is associated with carriage and less invasive lineages. CRISPR/Cas, an adaptive defence system against foreign DNA, was shown to be involved in gene regulation in Francisella novicida. This study shows that knockout strains of N. meningitidis lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells in a strain-dependant manner, which constitutes a central step in the pathogenesis of IMD. Consequently, this study indicates that the meningococcal CRISPR/Cas system fulfils functions beyond the defence of foreign DNA and is involved in the regulation of meningococcal virulence. / Neisseria meningitidis, ein ß-Proteobakterium, welches als Kommensale ausschließlich den humanen Nasopharynx besiedelt, ist ein weltweit führender Verursacher von Sepsis und epidemischer Meningitis. Auch wenn mittels vergleichender Genomanalysen hyperinvasive Stämme definiert werden konnten, welche für die meisten Fälle von invasiven Meningokokkenerkrankungen verantwortlich sind, bleibt die genetische Grundlage ihrer Virulenz ungeklärt. In vorangegangenen Studien konnte gezeigt werden, dass das Typ II-C CRISPR/Cas-System der Meningokokken assoziiert ist mit Trägerstämmen. CRISPR/Cas ist ein adaptives Verteidigungssystem der Bakterien gegen fremde DNA, das darüber hinaus Aufgaben in der Genregulation von Francisella novicida erfüllt. Diese Arbeit zeigt, dass knockout Stämme von N. meningitidis, denen das Cas9-Protein fehlt, in Abhängigkeit von ihrem genetischen Hintergrund die Fähigkeit verlieren an Zellen des menschlichen Nasopharynx zu adhärieren. Die Adhäsion an den Wirtszellen stellt einen zentralen Schritt in der Pathogenese der invasiven Meningokokkenerkrankungen dar. Die Ergebnisse dieser Arbeit deuten darauf hin, dass das CRISPR/Cas-System in Meningokokken neben seiner Funktion als bakterielles Immunsystem an der Regulation der bakteriellen Virulenz beteiligt sein könnte.

Host cell reactivation (HCR) of viruses has been used in the past to assess the DNA repair capacities of various mammalian cell types. In this study, a PCR-based HCR technique was developed for determining DNA repair capacity of mammalian cells. Many DNA lesions, including UV photoproducts, block DNA amplification by Taq polymerase, and the exponential nature of PCR imparts a tremendous potential for quantifying the remaining non-adducted DNA templates from small samples. Ad5HCMVspl ZacZ is a recombinant nonreplicating adenovirus (Ad) containing the lacZ reporter gene under the control of the human cytomegalovirus (HCMV) immediate early promoter inserted into the deleted El region of the viral genome. This virus is unable to replicate, but it can efficiently express the reporter gene in many types of mammalian cells, including human fibroblasts. Using quantitative PCR, the induction and repair of UV photoproducts was measured in a 2.6 kb region of the lacZ reporter gene inserted into the deleted El region of Ad5HCMVspllacZ and in a 2.8 kb region of the endogenous E4 region of the virus. Primers flanking the regions were added to equal amounts of DNA extracted from cells infected with unirradiated or UV-irradiated Ad5HCMVspl lacZ and each sample was amplified by PCR using radiolabelled nucleotides as substrates. PCR products were separated by agarose gel electrophoresis and quantified using a phosphorimaging system. Results show a simple exponential decrease in PCR product with increasing UV fluence to the virus. There was a significant removal of UV photoproducts by 24 hours after infection of normal human fibroblasts. A reduced capacity for lesion removal was detected after infection of nucleotide excision repair deficient fibroblasts derived from patients with xeroderma pigmentosum (XP) and Cockayne syndrome (CS). Previous work from our lab using a P-gal reporter gene assay has shown that both UV light and heat shock treatment of cells prior to infection with UV-damaged Ad5HCMVspl lacZ enhances HCR. Application of the quantitative PCR technique to the study of inducible repair shows there is an enhancement in the rate of lesion removal from both regions of the vector in UV-irradiated normal lung fibroblast cells, compared to unirradiated cells. This demonstrates that previous reports of enhanced host cell reactivation are indicative of a genuine enhancement of DNA repair. Also, the P-gal reporter gene assay was used to investigate inducibility of UV lesion repair by ionising radiation; no significant increase in HCR of p-gal activity was found in cells treated with y-rays compared to untreated cells. / Thesis / Master of Science (MS)

No description available.

A non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the 13-galactosidase (l3-gal) reporter gene, was used to examine both constitutive and inducible repair of UVC-damaged DNA in Chinese hamster ovary (CHO) cells. Host cell reactivation (HCR) of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated nucleotide excision repair (NER) proficient parental CHO-AA8 and m mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of NER. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for 13-gal activity 24 h later. HCR of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-.UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UVC-damaged reporter gene in untreated CHO-AA8 cells utilizes TCR. Prior UVC-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UVC-damaged reporter gene in CHO-AA8 cells but not m TCR deficient CHO-UV61 cells. Pre-UVC-treatment of cells resulted also in an enhanced expression of 13 -gal for unirradiated Ad5MCMVlacZ in both CHO-AA8 and CHO-UV61 cells. However, compared to CHO-AA8 cells, the CHO-UV61 cells exhibited comparable levels of enhanced 13-gal activity following significantly lower UVC exposures to cells suggesting that persistent damage in active genes plays a direct role in enhancing 13-gal activity driven by the MCMV promoter in CHO cells. These results suggest that prior UVC treatment results in a transient enhancement in repair of UVC-damage DNA in the transcribed strand of the active reporter gene in CHO-AA8 cells through an enhancement of TCR or a mechanism that involves the TCR pathway and that the upregulation of reporter gene expression alone is not sufficient for enhanced repair of the reporter gene in CHO-UV61 cells. The HCR assay was used also to examine both constitutive and inducible repair of UVC-damaged DNA in mouse embryonic fibroblast (MEF) cells. HCR of B-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated NER proficient parental wild type MEF cells and in MEF cells with specific knockouts in the p53 (p53-/-), pRb (pRb-/-), and p107 (p107-/-) genes. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for ~-gal activity 24 h later. HCR of ~-gal activity for UVC-irradiated Ad5MCMVlacZ did not show a significant difference in non-irradiated cells for any of the MEF knockouts cells compared to the parental strain suggesting that p53, pRb and p107 does not play a role in repair of the UV -damaged reporter gene in untreated MEF cells. Prior UVC-irradiation of cells with low UVC fluences resulted in an enhancement of HCR for expression of the UV C-damaged reporter gene in MEF wild type cells, low passage pRb-/-and p 1 07 -I-MEF cells but not in p53-/-MEF cells or in high passage pRb-/-and p107-/-MEF cells. These results suggest that prior UVC treatment MEF cells results in an induced repair of UVC-damaged DNA that is dependent on p53. The presence of an enhancement of HCR for the UVC-damaged reporter gene in pre-UVC treated cells in low passage, but not in high passage, pRb-/-and p 1 07-I-cells suggests that the lack of pRb or pI 07 expression per-se does not result in a deficiency in inducible DNA repair. However, these results suggest that the lack of pRb or p 1 07 expression results in alterations in MEF cells at high passage number that abrogate inducible repair of UVC-damaged DNA. UVA produces predominantly single base damage that is repaired through base excision repair (BER), whereas UVC and UVB produce predominantly cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PP) that are repaired through NER. The colony survival following exposure to various UV sources was examined in cells proficient and deficient in (NER). The UV sources were a UVC source from a germicidal lamp emitting predominantly at 254 nm. and a UVA source from a lKW Hg-Xe arc lamp using either a Band pass filter (BPF) or a 335 Cut-off-filter (335COF). NER deficient CHO-UV5 and CHO-UV61 cells were more sensitive to UVC exposure compared to NER proficient CHO-AA8 cells, consistent with the production of UVC-induced DNA damage predominantly in the form of CPDs and 6-4PPs which are repaired through the NER pathway. NER deficient xeroderma pigmentosum cells from complementation group D (XPD) were more sensitive compared to NER proficient normal human cells following exposure to the UVA-BPF source. In addition XPDdenV cells, which express the denY gene from bacteriophage T4, were more resistant than XPD cells following exposure to the UVA-BPF source. Since the denY protein is specific for excision ofCPDs these results indicate a substantial proportion of the induced DNA damage resulting from the UV A-BPF is in the form of CPDs, presumably due to a significant UVB component in the beam. In contrast, the NER deficient CHO-UV5 and CHO-UV61 cells showed a similar sensitivity compared to the NER proficient CHO-AA8 cell line following UVA-335COF exposures up to 60 KJ/m2• However, for UVA-335COF exposures greater than 60 KJ/m2 the NER deficient cells were more sensitive compared to the NER proficient CHO-AA8 cells, although the difference in sensitivity between NER deficient and NER proficient cells was less than that detected following UV A-BPF exposure. These results suggest that the UVA-335COF exposure produces predominantly DNA damage of the single base type for exposures less 60 KJ/m2. This is consistent with the calculated spectral distribution, which showed a 5.62% UVB component for the UVA-BPF, but only 0.14% UVB component for the UVA-335COF. / Thesis / Master of Science (MS)