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Investigation of Wafer-Level Electromagnetic Heating of Metallic Frames at Radio Frequencies: Analysis and Characterization of Standing Waves
No full textThe research provides concise insights into the thermal effects induced by standing waves. Through a combination of numerical FE simulations and experimental validations, the study comprehensively analyzes the distribution of EM fields, standing wave patterns, and resulting temperature profiles within the metallic frames. These findings offer crucial insights a lead to practical implications and limitations of the heating phenomena of several metallic structures at wafer-level, facilitating advancements in MEMS packaging processes. Further research regarding the coil, generator and process parameter optimization will be based on the presented results.:1. Introduction
2. Problem description
3. Preliminary results
4. Conclusions
5. References
6. Author
Einfluss von Cinacalcet auf den perioperativen Parathormonverlauf bei Patienten mit primärem Hyperparathyreoidismus
No full textAnfang 2020 prägte uns alle in Deutschland durch den Beginn der COVID-19-Pandemie. Die damals noch relativ neue Therapie von Cinacalcet als Kalzimimetikum bei Patienten mit primärem Hyperparathyreoidismus gewann dadurch noch mehr Aufmerksamkeit und es kam die Frage auf, welchen Einfluss dies auf die intraoperative Parathormon-Überwachung hat. Hyperparathyreoidismus beschreibt die Überfunktion der Nebenschilddrüsen bzw. die der Epithelkörperchen, das heißt einen Parathormonexzess durch vermehrt aktive Neben- schilddrüsen. Grundsätzlich differenziert man beim Hyperparathyreoidismus allgemein den primären, sekundären (renalen) und den tertiären Typ. Der primäre Hyperparathyreo- idismus ist eine Regulationsstörung der Nebenschilddrüsenzellen mit vermehrter Para- thormonsekretion ohne einen erkennbaren physiologischen Stimulus. Zu 90% liegt ein solitäres Adenom, zu ca. 10% Mehrdrüsenerkrankungen und zu 1% ein NSD-Karzinom vor. Prinzipiell werden beim primären Hyperparathyreoidismus der symptomatische und asym- ptomatische Typ unterschieden. Insbesondere früher, als die mittlerweile standardmäßigen Screenings zur exakten Kalzium und Parathormon- Bestimmung noch nicht möglich waren, wurde die Erkrankung anhand von Organmanifestationen diagnostiziert. So galt damals als klassische Symptomtrias: „Stein-, Bein- und Magenpein“ in den Kliniken. Heutzutage kann durch biochemisches Labor (erhöhtes PTH und Kalzium) bedeutend frühzeitiger die richtige Diagnose gestellt werden, man spricht von sicherer Diagnosestellung ganze zehn Jahren vor klinischer Symptomatik des Patienten. Im Oberlausitz-Kliniken gGmbH - Krankenhaus Bautzen werden zunehmend Patienten mit pHPT präoperativ mit Kalzimimetika, mit dem Wirkstoff Cinacalcet, Handelsnahme Mimpara®, bei Hyperkalzämie therapiert. Der sonst enorme Abfall des Parathormons postoperativ erscheint in den meisten Fällen dadurch deutlich geringer. Bisher galt, dass nach erfolgreicher Entnahme des Nebenschilddrüsenadenoms ein Parathormonabfall auf etwa 50% des ursprünglichen gemessenen Ausgangswertes zu erwarten war. Doch durch die Gabe von Cinacalcet erhält man präoperativ einen geringeren pathologischen PTH- Wert, dementsprechend keinen so großen Abfall nach der OP. Dieser Qualitätsindikator für die vollständige Entfernung des pathologisch veränderten Gewebes erscheint damit nicht mehr so hoch und eindeutig wie ohne präoperative Mimpara® - Therapie. Hintergrund des neuartigen Kalzimimetikums ist es, das Risiko einer hyperkalzämischen Krise deutlich zu minimieren bzw. solch einen akuten Notfallzustand zu verhindern. Dabei stellte sich die Frage, ob das IOPTH Monitoring weiterhin einen zentralen Stellenwert als operativer Qualitätsindikator hat. Für die Studie wurde eine Kohorte von 72 Patienten ausschließlich aus dem Oberlausitz -Kliniken gGmbH-Krankenhaus Bautzen zwischen 2018 und 2021 mit diagnostiziertem pri- märem Hyperparathyreoidismus, davon 22 mit präoperativer Cinacalcet Medikation und 50 Patienten als Nicht-Cinacalcet-Gruppe, analysiert. Aufgrund der sehr kurzen Halbwertszeit des Parathormons von nur zwei bis vier Minuten gehört die intraoperative Parathormonbe- stimmung (IOPTH), zu einer der wichtigsten Indikatoren für die erfolgreiche Entfernung der Nebenschilddrüsen, bzw. NSD-Adenomen. In acht bis zwanzig Minuten können an- fangs durch immunoradiologische Assays, später durch Chemilumineszens-Assays die Laborergebnisse erfasst und somit intraoperative resektionsstrategische Entscheidungen getroffen werden. Die Ergebnisse der PTH-Dynamik zeigten eine Wertabnahme im Laufe der Zeit ohne signifikanten Unterschied zwischen beiden Studienkollektiven. Die Kalziumspiegel waren nach der Cinacalcet-Therapie deutlich niedriger bis normwertig, fielen jedoch postoperativ weiter ab. Zusammenfassend kommt man zu dem Ergebnis, dass die Alternativ- oder Überbrückungs- therapie mit Kalzimimetika keinen Einfluss auf die IOPTH-Überwachung und die Höhe des intraoperativen PTH-Abfalls hat. Die Kalziumspiegel haben keinen Einfluss auf Morbidität und Krankenhausaufenthalt. Geschlecht und Alter zeigten ebenfalls keinen Einfluss auf die Behandlungswirksamkeit. Kalzimimetika, wie Cinacalcet, können in Fällen von verschobe- nen Operationen wie zur Zeit der COVID-19-Pandemie eingesetzt werden,ohne den peri- und postoperativen Verlauf der Parathyreoidektomie zu beeinflussen. Weitere Studien mit größeren Patientenkohorten sollten durchgeführt werden, um diese Schlussfolgerung zu bestätigen oder zu widerlegen. Eine Parathyreoidektomie führt bei fast allen Patienten mit pHPT zur Heilung und sollte immer als Primärtherapie in Betracht gezogen werden. Chirurgen sollten die biochemischen Veränderungen berücksichtigen, die in solchen Fällen auftreten und die IOPTH-Überwachung beeinflussen können.:1 Einleitung
1.1 Anatomie der Nebenschilddrüse
1.2 Physiologie und Pathophysiologie der Nebenschilddrüse
1.3 Studie und Forschungsziel
2 Hintergrund
2.1 Definition
2.1.1 Primärer Hyperparathyreoidismus
2.1.2 Sekundärer Hyperparathyreoidismus
2.1.3 Tertiärer Hyperparathyreoidismus
2.2 Diagnostik
2.2.1 Bildgebende Diagnostik
2.2.2 Interpretationder Laborparameter
2.3 Klinische Symptomatik
2.3.1 Symptomatischer pHPT und Differentialdiagnosen
2.3.2 Asymptomatischer pHPT
2.3.3 Hyperkalzämie
2.4 Therapie des pHPT
2.4.1 Konservative Therapie.
2.4.2 Operative Therapie
2.5 Chirurgie
2.5.1 Chirurgie der Nebenschilddrüse
2.5.2 Minimal-invasives OP-Verfahren
2.5.3 Komplikationen der operativen Therapie
2.5.4 Chirurgie der Schilddrüse
3 Fragestellung/Hypothese
4 Material und Methode
4.1 Patientenselektion und Datenerhebung
4.2 Deskriptive Statistik
4.2.1 Patientenbasisdaten
4.2.2 Diagnostik
4.2.3 Operation
4.2.4 Postoperative Medikation
4.2.5 Histologie.
4.2.6 Postoperativer klinischer Verlauf
4.3 Statistische Analyse
4.4 Methodenbeschreibung PTH-Test
5 Ergebnisse
5.1 Diagnose und Lokalisierung NSD-Adenom
5.2 Diagnose Schilddrüse
5.3 Geschlechterverteilung und Patientenalter
5.4 Begleiterkrankungen
5.4.1 Symptomatischer und asymptomatischer pHPT
5.5 ASA-Klassifikation
5.6 Zielführende Umfelddiagnostik
5.7 Bildgebende Diagnostik
5.8 Anwendung und Dosierung von Mimpara®
5.9 Interpretation Laborwerte
5.9.1 Kalzium
5.9.2 IOPTH
5.9.3 Kreatinin
5.9.4 GFR
5.9.5 Harnstoff
5.10OP-BefundundHistologie
5.10.1 Histologische Schnellschnittdiagnose
5.10.2 Operatives Verfahren
5.11 Postoperative Medikation
5.12 Postoperativer Verlauf
5.12.1 Verweildauer Krankenhaus
5.12.2 Postoperative Verlaufskontrolle
6. Diskussion
7.Schlussfolgerung
8.Danksagun
Racist discourse in a German far-right blog: A corpusdriven approach using word embeddings
No full textNewer forms of racism in the context of right-wing extremism are characterised by an apparent distancing from overt racist devaluations. In addition or even beyond biological features, it is now cultural characteristics attributed to social groups which serve as grounds for practices of othering and social exclusion. This paper analyses racist discourse in the comment sections ofthe influential far-right blog pi-news.com where these practices can be observed in detail. With reference to discourse analytical approaches to racism and using corpus-linguistic, data-driven methods, especially word embeddings and collocations, it is shown how racism is linguistically and discursively expressed. Next to both overt and more implicit racist nominations and predications, the notion of Heimat (‘homeland’) is analysed; it is used to draw racist demarcations without relying on overtly racialising terms
Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
No full textIntroduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.
Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.
Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels
Carbon Capture and Storage on its way to large-scale deployment: Social acceptance and willingness to pay in Germany
No full textCarbon Capture and Storage (CCS) is an emerging technology to mitigate greenhouse gas emissions from fossil fuel-fired power plants. In the wake of a rapidly changing German energy system, CCS can play an important role. By means of an online survey among 130 university students in Dresden, this paper investigates the level and influencing factors of social acceptance of CCS. Furthermore, the individual willingness to pay for CCS and renewable power delivery is measured and compared through a choice model. The survey results reveal that the attitude towards CCS is neutral. Moreover, it is shown that acceptance of CCS is an important factor for the willingness to pay. The level of willingness to pay for CCS technology is much lower than for renewable energy
Nonlinear Optical Microscopy in Thin Film Ferroelectric Materials
No full textThe Nonlinear optical (NLO) microscopy is a very powerful and noninvasive tool to analyze the material properties, such as the local symmetry, as well as to visualize ferroelectric domains and domain walls. As a result, NLO microscopy becomes a very powerful tool in characterization and quality control which are key tasks in material development and device fabrication. One such area where NLO microscopy is widely used, is thin film materials. Thin film and nanosized materials with dimensions ranging from a few micrometers thickness down to atomically thin 2D materials, offer many innovative and intriguing features for applications in electronics, optics, and many other fields. In order to provide physical stability, these thin film and 2D materials are usually supported on substrates and handles, leading to multiple effects, such as thin film resonance and reflections at the thin film-substrate interface, that influence the genuine NLO signal from the sample. These effects are not present in bulk samples; therefore, it is natural to erroneously consider that these effects are also not present in thin film materials. This work tries to identify, quantify, and disentangle the parameters that influence nonlinear microscopy in thin film materials. To achieve this, Second Harmonic Generation (SHG) microscopy and Third Harmonic Generation (THG) microscopy were applied as two archetypal NLO processes. In particular the influence of thin film interference and phase matching on the signal strength is analyzed. Furthermore, key differences between three and four photon processes, such as the role of the Gouy-phase shift and the focal position is studied. This understanding can be extended to other three and four-photon processes, such as Coherent Anti-Stokes Raman Scattering (CARS).
Wedge-shaped samples were used for the experiments here, whose thickness was varied from bulk thickness down to approximately 50 nm. In both cases, it was found that the signal in the back reflection is the phase-matched co-propagating signal and not the counter-propagating signal which may naively be expected. It was also found that the signal from the surrounding material, and support does not affect the SH signal from the sample because the second-order nonlinear tensor is only available in non-centrosymmetric material. However, the signals from the surrounding do affect the TH signal from the sample because a third-order nonlinear tensor is available in every material. Furthermore, the THG signal from the thin film starts to vanish as the thickness increases, opposite to what happens in SHG. To back up the experimental findings, two numerical models were used. The first model is the numerical simulation, while the second is a semi-analytical paraxial model.
This thesis lays the groundwork for performing quantitative NLO -spectroscopy on thin films and 2D materials, as it identifies and quantifies the impact of the corresponding sample and setup parameters on the NLO signal, in order to distinguish them from genuine material properties.:1. Introduction 1
2. Theoretical background 7
2.1. Non-linear optical polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2. The non-linear susceptibility tensor . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3. Phase-matching and emission efficiency . . . . . . . . . . . . . . . . . . . . . . 12
2.4. Nonlinear effects in focused Gaussian beams . . . . . . . . . . . . . . . . . . . 17
3. Methodology and principle 25
3.1. Lithium niobate (LN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2. Wedge preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3. Data generation & processing . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4. Simulation models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4. SHG in thin films 37
4.1. Coherence interaction length . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.1. Experimental data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.1.2. Comparison of numerical and experimental data . . . . . . . . . . . . . 44
4.2. Thin film interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.3. Influence of NA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.4. Reproducibility of experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.5. Detection depth of coherent interaction length oscillations . . . . . . . . . . . 54
4.6. Sensitivity to focus positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5. THG in thin films 57
5.1. Coherence interaction length . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.1.1. Quantifying the coherence interaction length . . . . . . . . . . . . . . . 61
5.2. Comparison of simulated and experimental data . . . . . . . . . . . . . . . . . 63
5.3. Thin film interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6. Conclusion and outlook 67
Appendices 69
A. Additional reflective layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
B. Focus fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
List of Figures 78
List of Tables 79
Acronyms 82
Own Publications 93Die nichtlineare optische Mikroskopie (NLO) ist ein sehr leistungsfähiges und nichtinvasives Instrument zur Analyse der Materialeigenschaften, z. B. der lokalen Symmetrie, sowie zur Visualisierung ferroelektrischer Domänen und Domänenwände. Dadurch wird die NLO-Mikroskopie zu den wichtigsten Aufgaben bei der Materialentwicklung und der Herstellung von Bauelementen gehören. Ein solcher Bereich, in dem die NLO-Mikroskopie weit verbreitet ist, sind Dünnschichtmaterialien. Dünnschicht- und Nanomaterialien mit Abmessungen von wenigen Mikrometern Dicke bis hin zu atomar dünnen 2D-Materialien bieten viele innovative und faszinierende Eigenschaften für Anwendungen in der Elektronik, Optik und vielen anderen Bereichen. Um die physikalische Stabilität zu gewährleisten, werden diese Dünnschicht- und 2D-Materialien in der Regel auf Substraten und Handlegriffen getragen, was zu zahlreichen Effekten führt, wie z. B. Dünnschichtresonanz und Reflexionen an der Grenzfläche zwischen Dünnschicht und Substrat, die das echte Signal der Probe beeinflussen. Diese Effekte sind bei Bulk-Proben nicht vorhanden; daher ist es naheliegend, fälschlicherweise anzunehmen, dass diese Effekte auch bei Dünnschichtmaterialien nicht vorhanden sind. In dieser Arbeit wird versucht, die Parameter, die die nichtlineare Mikroskopie in Dünnschichtmaterialien beeinflussen, zu identifizieren, zu quantifizieren und zu entflechten. Zu diesem Zweck wurden die Mikroskopie der zweiten Harmonischen (SHG) und die Mikroskopie der dritten Harmonischen (THG) als zwei archetypische NLO-Prozesse untersucht. Insbesondere wird der Einfluss von Dünnschichtinterferenzen und Phasenanpassung auf die Signalstärke analysiert. Darüber hinaus werden die wichtigsten Unterschiede zwischen Drei- und Vier-Photonen-Prozessen, wie die Rolle der Gouy-Phasenverschiebung und der Fokusposition, untersucht. Dieses Verständnis kann auf andere Drei- und Vier-Photonen-Prozesse, wie z. B. die kohärente Anti-Stokes-Raman-Streuung (CARS), ausgeweitet werden.
Für die Experimente wurden keilförmige Proben verwendet, deren Dicke von der Bulk-Dicke bis hinunter zu etwa 50 nm variiert werden. In beiden Fällen wurde festgestellt, dass es sich bei dem Signal in der Rückreflexion um das phasenangepasste Mitausbreitungssignal handelt und nicht um das Gegenausbreitungssignal, das man naiverweise erwarten könnte. Es wurde auch festgestellt, dass das Signal aus der Umgebung das SH-Signal der Probe nicht beeinflusst, da der nichtlineare Tensor zweiter Ordnung nur in nicht-zentrosymmetrischem Material vorhanden ist. Die Signale aus der Umgebung beeinflussen jedoch das TH-Signal der Probe, da ein nichtlinearer Tensor dritter Ordnung in jedem Material vorhanden ist. Außerdem verschwindet das THG-Signal des dünnen Films mit zunehmender Dicke, anstatt wie bei SHG zuzunehmen. Um die experimentellen Ergebnisse zu untermauern, wurden zwei numerische Modelle verwendet. Bei dem ersten Modell handelt es sich um eine numerische Simulation, bei dem zweiten um ein halbanalytisches paraxiales Modell.
Diese Arbeit legt den Grundstein für die Durchführung quantitativer NLO -Spektroskopie an dünnen Schichten und 2D-Materialien, da sie die Auswirkungen der entsprechenden Proben und Einrichtungsparameter auf das NLO-Signal identifiziert und quantifiziert, um sie von den echten Materialeigenschaften zu unterscheiden.:1. Introduction 1
2. Theoretical background 7
2.1. Non-linear optical polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2. The non-linear susceptibility tensor . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3. Phase-matching and emission efficiency . . . . . . . . . . . . . . . . . . . . . . 12
2.4. Nonlinear effects in focused Gaussian beams . . . . . . . . . . . . . . . . . . . 17
3. Methodology and principle 25
3.1. Lithium niobate (LN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2. Wedge preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3. Data generation & processing . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4. Simulation models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4. SHG in thin films 37
4.1. Coherence interaction length . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1.1. Experimental data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.1.2. Comparison of numerical and experimental data . . . . . . . . . . . . . 44
4.2. Thin film interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.3. Influence of NA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.4. Reproducibility of experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.5. Detection depth of coherent interaction length oscillations . . . . . . . . . . . 54
4.6. Sensitivity to focus positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5. THG in thin films 57
5.1. Coherence interaction length . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.1.1. Quantifying the coherence interaction length . . . . . . . . . . . . . . . 61
5.2. Comparison of simulated and experimental data . . . . . . . . . . . . . . . . . 63
5.3. Thin film interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6. Conclusion and outlook 67
Appendices 69
A. Additional reflective layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
B. Focus fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
List of Figures 78
List of Tables 79
Acronyms 82
Own Publications 9
Development of novel transient Foamy Virus (TraFo) vectors - Combining ancient viruses with bacterial CRISPR nucleases for efficient genome editing
No full textKnowledge on the human genome and specific sequences associated with human diseases is continuously growing. The ability to connect human genetics to cellular mechanisms and physiology raises the need for medicine to get to gene specific therapeutics. In order to achieve gene-specific modification, tools are required to enable sequence-specific DNA cleavage. Not long ago, the RNA-guided endonuclease Cas9 was shown to effectively facilitate gene editing in humans. Cas9 endonuclease, which is naturally part of an adaptable bacterial immune system, can be easily adjusted to recognize and cleave specific DNA sequences in a 20 nt RNA-DNA complementary manner. The easy adjustability and high efficiency of Cas9 gave rise to hopes that this genome engineering tool could pave the way to ‘gene surgery’ in humans.
However, to achieve DNA cleavage, the endonuclease and its guiding RNA need to be sufficiently accessible in the nucleus of target cells. Viruses, which evolution has made well adapted to transfer their own genetic information into cells can be exploited for transfer of foreign genetic material. Replication deficient retroviruses therefore represent interesting vehicles for gene delivery. Retroviruses preferentially incorporate their own genetic information in the form of RNA into viral particles. Typically, viral RNA of retroviruses is reverse transcribed into DNA during viral infection and integrated into host cell chromosomes. In this respect, integration-competent or integration-deficient lentiviral (HIV-derived) vectors (ICLV/IDLV) were reported to be efficient ‘gene shuttles’ for Cas9 delivery.
In contrast, up to now Foamy viruses (FV), which represent a distinct subfamily in the family of retroviruses have not previously been tested for their efficiency to transduce CRISPR/Cas9 components. FV show several unique characteristics some of which make them interesting candidates for gene therapy, such as high transduction efficiency on a wide variety of human cell lines or a special capability to efficiently transfer and provide non-viral RNA in target cells.
In this thesis the unique characteristic of FVs, which allow for the efficient transduction of non-viral RNAs, was exploited for transient FV mediated (TraFo) Cas9 expression. It is shown in this thesis that gene knock-out (KO) achieved with TraFo Cas9 particles appears to have several advantages over ICLV or IDLV mediated Cas9 transduction. In this work, it could be demonstrated that a single application of TraFo Cas9 supernatant results in high efficiency of GFP KO in osteosarcoma cells (U2OS). The efficiency of gene KO with TraFo Cas9 particles exceeded gene KO frequencies achieved with similar volumes of ICLV or IDLV supernatant for Cas9 transduction. In addition, transient Cas9 delivery achieved with TraFo particle supernatant resulted in remarkably reduced Cas9 off-target cleavage compared to corresponding infections with ICLV or IDLV particles. The results show, that TraFo Cas9 represents an interesting addition to the currently utilized methods for transient Cas9 delivery. One particular feature of TraFo particle transduction is especially noteworthy – TraFo mediated transduction does not depend on any particular adjustment on the encapsidated non-viral RNA sequence (such RNA only needs to be present in sufficient amounts during virus assembly) nor does it depend on any modification of viral proteins. The easy adaptability of TraFo mediated non-viral RNA transfer is an especially remarkable feature, since science continues to both developing new variants of Cas9 and continues to find new and interesting members of the pool of CRISPR enzymes. In this regard TraFo particles represent interesting vehicles to transiently provide mRNA transcripts of such new protein candidates in cells.
The ability of TraFo particles to provide the RNA sequence needed to guide Cas9 (termed sgRNA) to its target DNA sequence in cells was additionally investigated. It was assumed that typically engaged RNA polymerase (RNAP) III transcription of sgRNAs hampers transduction with TraFo particles, since RNAP III-derived transcripts are not actively exported into the cytoplasm and show low stability. An additional CRISPR enzyme Csy4 was used, which is able to specifically cleave RNA. This enabled TraFo mediated transfer of RNAP II transcripts (with active nuclear export and higher stability than RNAP III transcripts) with embedded sgRNA sequences. It was demonstrated that a simultaneous infection of cells with TraFo particles providing bicistronic transcripts of Cas9 and Csy4 on the one side and RNAP II-derived transcripts with embedded sgRNA sequences on the other, enabled reasonable GFP gene inactivation in U2OS cells. Gene KO with RNAP II transcripts as a result significantly exceeds TraFo transduction of RNAP III-derived sgRNA.
Interestingly, with regard to gene KO, it was found that de novo transcription of sgRNAs from viral DNA (by integration-competent or integration-deficient retroviral vector [ICRV/IDRV] transduction) when combined with TraFo Cas9 transduction was superior to a TraFo transduction of sgRNA transcripts. IDRV mediated transduction was optimized in order to minimize the risk of unfavorable genome modification of cells by viral DNA integration. By adding the coding sequence of a fluorescent marker to the viral vector, it was demonstrated that a smaller number of viral particles helps to significantly lower the frequency of viral DNA integration. In addition, the expression of a fluorescent marker opened up the opportunity to further reduce the cell fraction with continuous marker gene expression by flow cytometric cell sorting.
The IDRV/ICRV sgRNA and TraFo Cas9 delivery system was then challenged for use on immortalized and primary T cells. Primary T cells represent interesting targets for genetic engineering since modified T cells can be utilized as ‘living drugs’ (by expression of chimeric antigen receptors – CARs) against cancer cells. Efficient gene inactivation was observed on the immortalized T cell line – Jurkat. Transduction of primary T cells pointed to certain restrictions of the split two-virus delivery system for sgRNA and Cas9 transduction. However, despite certain limitations, it was possible to demonstrate that this FV-derived Cas9 delivery system is also feasible on primary tissue, and further optimization could make it an interesting alternative delivery method for CAR therapy.
The ability of IDRV vector genomes to provide repair template donor DNA to induce homologous recombination (HR) was additionally investigated. DNA double-strand breaks in eukaryotic cells are typically repaired by the error prone non-homologous end joining pathway (often leading to frame-shift mutations by small insertions or deletions) or HR. Delivery of a homologous DNA sequence during DNA cleavage enables site-specific integration of exogenous DNA sequences. The work of this thesis showed that IDRV vector genomes providing repair template donor DNA allow for HR in a homology length dependent manner. Besides the length of homology, it was also observed, that the length of sequence which should be integrated (KI) remarkably influences the frequency of HR. HR is therefore engaged significantly more frequently if single nucleotides, rather than a whole gene, are provided as sequences within a repair template. In addition, viral vectors were augmented with additional fluorescent marker sequences. It could subsequently be demonstrated that the majority of cells showed accurate sequence-specific DNA integration. Furthermore, several indications were found, which lead to the assumption that the ratio of KI to homologous sequence markedly influences the accuracy of HR.
Using the previously obtained knowledge it was further possible to tag an essential human protein by FV vector mediated transient Cas9 and repair template transduction. It was found that the large packaging capacity of FV vectors can be exploited to enable selection and flow cytometric sorting of cells with correct site-specific DNA integration.
In summary, the results of this thesis demonstrate for the first time that FV mediated non-viral mRNA Cas9 transduction in combination with retroviral delivery of sgRNA (and repair template sequence) are a promising basis for several different interesting applications with relevance for not only basic research, but also for gene therapy.:1. Introduction 1
1.1 Gene therapy 1
1.2 Viral vectors for gene therapy 2
1.3 History of retroviral research 2
1.4 Taxonomy of Retroviruses 3
1.5 Foamy Viruses 4
1.5.1 Morphology of Foamy Virus 6
1.5.2 Foamy Virus replication 7
1.5.3 Foamy virus proteins, as part of a viral vector system 10
1.6 Genetic engineering 14
1.6.1 ‘DNA scissors’ – Zinc-finger and Transcription-activator like effector nucleases 15
1.6.2 History of CRISPR/Cas9 as a tool for genetic engineering 16
1.7 CRISPR/Cas immunity in prokaryotes 18
1.8 CRISPR/Cas9 functioning 21
1.9 Double-strand break repair in eukaryotic cells 21
1.9.1 Classical NHEJ 23
1.9.2 Homologous recombination 24
1.9.3 DSB repair in vertebrates 26
1.10 DSBs in context of CRISPR/Cas9 cleavage 27
1.11 Thesis Aim: CRISPR/Cas9 transduction with FV particles 28
2. Materials and Methods 30
2.1 Materials 30
2.1.1 Chemicals 30
2.1.2 Buffers and Solutions 30
2.1.3 Bacterial Growth Media 33
2.1.4 Cell Culture Media 34
2.1.5 Antibodies 34
2.1.6 Enzymes 35
2.1.7 Commercial Kits and additional reagents 36
2.1.8 Size Markers 36
2.1.9 Antibiotics 36
2.1.10 Bacterial strains 37
2.1.11 Cell lines 37
2.1.12 Devices and Software 37
2.1.13 Oligonucleotides 38
2.1.14 Plasmids 46
2.1.15 sgRNA sequences 56
2.1.16 Consumable material 57
2.2 Molecular Biology Methods 58
2.2.1 Restriction of DNA 58
2.2.2 Polymerase chain reaction 59
2.2.3 Gibson assembly 60
2.2.4 Agarose gel electrophoresis 60
2.2.5 Ligation 61
2.2.6 Cultivation of bacteria 62
2.2.7 Transformation 62
2.2.8 Plasmid Preparation 63
2.2.9 Sequencing 65
2.3 Cell culture methods 66
2.3.1 Passaging of cells 66
2.3.2 Cell counting 66
2.3.3 Freezing and thawing of cells 66
2.3.4 Seeding and fixation of cells for microscopy 67
2.4 Virological Methods 67
2.4.1 Polyethyleneimine transfection 67
2.4.2 Integration-competent, integration-deficient and ‘transient’ retroviral vectors 68
2.4.3 Infection of adherent cells 70
2.4.4 Infection of suspension cells 71
2.4.5 Flow cytometry 72
2.4.6 Multiplicity of infection (MOI) 72
2.4.7 Particle preparation 73
2.5 Nucleic acid composition in viral particles and culture cells 73
2.5.1 Isolation of total RNA from viral particles 73
2.5.2 RNA isolation from culture cells 73
2.5.3 Reverse transcription of viral or cellular RNA 73
2.5.4 DNA isolation from culture cells 74
2.5.5 Quantitative PCR (qPCR) analysis 74
2.5.6 T7 endonuclease assay 75
2.6 Protein biochemistry methods 76
2.6.1 Cell lysates 76
2.6.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 76
2.6.3 Semi-dry Western Blot 77
2.6.4 Immunodetection 78
2.6.5 Stripping of Western blot membranes 78
2.6.6 Immunostaining of cells for FACS analysis 78
2.7 Microscopy methods 79
2.7.1 Fluorescence microscopy 79
2.7.2 Confocal Laser scanning Microscopy (CLSM) 79
2.7.3 Live-cell imaging 79
3. Results 80
3.1 Transient foamy virus transduction of non-viral mRNA transcripts 80
3.2 Transient foamy virus transduction of Cas9-encoding mRNA transcripts 81
3.3 Cas9-encoding nucleic acids and their ‘effects’ in cells after retroviral transduction 84
3.4 Off-target analysis after TraFo Cas9 delivery 87
3.5 Transient fomy virus transduction of Cas9 and sgRNAs 89
3.6 Retroviral vectors providing sgRNAs and a fluorescent protein 92
3.6.1 Gene knock-out with retroviral vectors under saturated conditions 92
3.6.2 MOI adjusted ID sgRNA vector supernatants for comparison of residual vector integration 94
3.6.3 Gene knock-out in murine embryonic fibroblasts 95
3.7 Influence of Cas9 expression on IDRV vector genome integration 96
3.8 TraFo Cas9 mediated T cell receptor knock-out in immortalized and primary human T cells 97
3.9 Homology-directed repair after FV CRISPR/Cas9 mediated double-strand breaks 99
3.9.1 Length of homologous donor DNA and its influence on HDR 100
3.9.2 Effect of freezing viral supernatants on the frequency of HDR 102
3.9.3 Effect of donor DNA mismatches on the frequency of HDR 104
3.10 Investigation on donor DNA integration with additional fluorescent markers 105
3.11 Lentiviral and foamyviral transduction of HDR donor DNA 107
3.12 HDR mediated single nucleotide substitutions after TraFo CRISPR/Cas9 delivery 109
3.13 Tagging of an endogenous protein after TraFo CRISPR/Cas9 delivery 111
3.13.1 Specific CRISPR/Cas9 mediated cleavage of endogenous hPLK1 gene 111
3.13.2 Homology-directed repair of the hPLK1 gene for endogenous gene tagging 113
3.13.3 Confocal fluorescence microscopy analysis of GFP-Plk1 HeLa cell populations 118
4. Discussion 120
4.1 Genetic engineering – potential and risks 120
Chapter I Transient FV vectors – mRNA delivery vehicles for CRISPR/Cas9 mediated gene editing 122
4.2 Non-viral Cas9-encoding mRNA transfer in foamy virus particles 122
4.2.1 Fate of Cas9-encoding nucleic acids in cells after TraFo Cas9 transduction 124
4.2.2 Potential adjustments to further improve TraFo Cas9 transduction 125
4.2.3 Lentiviral in contrast to TraFo transduction of Cas9-encoding nucleic acids 126
4.3 Efficiency of Cas9-mediated gene knock-out with different retroviral vectors 127
4.4 Type of retroviral Cas transduction and its influence on the specificity of Cas9 cleavage 127
4.5 Alternative approaches to deliver Cas9-encoding mRNA in human cells 129
4.6 Transient sgRNA transduction with TraFo particles 131
Chapter II Delivery of foreign DNA with FV-derived vectors – enabling gene knock-out and homology-directed repair 133
4.7 Gene inactivation by TraFo Cas9 transduction and sgRNA expression from retroviral vector genomes 133
4.7.1 Gene editing in immortalized and primary T cells 135
4.8 Homology-directed repair with IDRV genomes 137
4.8.1 The influence of the length of sequence homology on HR 138
4.8.2 The influence of freezing viral supernatants on HR 139
4.8.3 Widening the applicability of TraFo vector particles for improved HR 140
4.8.4 The influence of mismatching nucleotides on HR 140
4.8.5 Visualization of inaccurate HR or additional dsDNA integration 141
4.8.6 The influence of the ratio of knock-in and homologous sequence on the accuracy of HR 142
4.8.7 Alternatives to double-stranded donor DNA 143
4.9 Endogenous gene tagging with IDPV donor DNA transduction 145
4.9.1 Alternative approaches for endogenous protein tagging 146
5. Conclusion 148
6. Summary 150
6.1 Summary 150
6.2 Zusammenfassung 153
7. Supplementary 157
8. References 159
9. Appendices 182
9.1 Indices 182
9.1.1 Abbreviations 182
9.1.2 Index of Figures 185
9.1.3 Index of Tables 187
9.2 Curriculum Vitae 188
9.3 Publication Record 189
9.4 Congress Contributions 189
9.5 Patent Applications 189
10. Statement of Authorship 19
