Este trabalho descreve o desenvolvimento de dispositivos usando cristais piezelétricos de quartzo com eletrodos separados (ESPC) que, através do monitoramento da freqüência de oscilação e do espectro de impedância, permitem estudar modificações superficiais do cristal. Para o monitoramento de freqüência de oscilação, usou-se um circuito oscilador TTL seguido por um freqüencímetro e por uma interface conectada a um microcomputador. Para o controle desta interface, foi desenvolvida uma biblioteca de ligação dinâmica e um programa em HP-VEE 4.0. Os espectros de impedância foram obtidos via programas que controlam um analisador de impedâncias via protocolo GPIB. O primeiro dispositivo presta-se a estudos de filmes depositados (DFD) e é feito em acrílico com eletrodos em latão posicionados a décimos de milímetros do cristal. Sua parte superior possui uma camisa de água para a termostatização. O segundo, semelhante ao primeiro, permite a modificação de superfície (DMS) de ESPC através da introdução de reagentes na fase gasosa. Os dois dispositivos foram caracterizados e o primeiro mostrou boa linearidade entre as massas depositadas e aquelas estimadas pela equação de Sauerbrey. O DFD foi utilizado para monitoramento em tempo não real da modificação superficial do cristal por N-[3-(trimetoxisilil)propil]-1,2-etanodiamina seguida por reação com ácido iodoacético. Os resultados mostraram evidências de aumento de massa e mudança nas propriedades viscoelásticas do filme. O DMS foi utilizado para monitoramento em tempo real da modificação superficial com trimetilclorosilano (TMCS) e dimetildiclorosilano (DMDCS). Com o TMCS, devido à formação de uma monocamada, não foi possível monitorar variações significativas de freqüência de oscilação. Com o DMDCS foi possível verificar a formação de poli(dimetilsiloxano). / This dissertation describes the development of devices based on separated-electrode piezoelectric quartz crystals (ESPC) to study surface modifications. This is accomplished by monitoring the oscillation frequency and impedance spectrum of the crystal in the cell. A TTL oscillator coupled to a frequency counter and a microcomputer interface is used for monitoring the oscillation frequency. A dynamic link library and HP-VEE 4.0 programs were developed to control the interface. Other programs were also developed to acquire the impedance spectra from a spectrum analyzer by using the GPIB protocol. The first device (DFD) is intended to study deposited films. It is made in Plexiglas, with brass electrodes positioned at a few tenths of millimeters from the crystal surface, and a thermostatic water jacket. The second device (DMS) is similar to the first one, but allows the use of corrosive reagents in the gas phase for surface modification. The performance of both devices was evaluated and the results showed good agreement between the deposited mass and the one predicted by Sauerbrey equation. The DFD was used for offline monitoring of the crystal surface modification by N-[3-(triethoxysilyl)propyl]-1,2-ethanediamine followed by iodoacetic acid. The results show evidences of mass and viscoelastic variations of the film. The DMS was used for on-line monitoring during surface modification with trimethylchlorosilane (TMCS) and dimethyldichlorosilane (DMDCS). Due to the monolayer formed by TMCS, no significant frequency variation could be observed. On the other hand, the polymerization of DMDCS could be monitored.

Este trabalho descreve o desenvolvimento de dispositivos usando cristais piezelétricos de quartzo com eletrodos separados (ESPC) que, através do monitoramento da freqüência de oscilação e do espectro de impedância, permitem estudar modificações superficiais do cristal. Para o monitoramento de freqüência de oscilação, usou-se um circuito oscilador TTL seguido por um freqüencímetro e por uma interface conectada a um microcomputador. Para o controle desta interface, foi desenvolvida uma biblioteca de ligação dinâmica e um programa em HP-VEE 4.0. Os espectros de impedância foram obtidos via programas que controlam um analisador de impedâncias via protocolo GPIB. O primeiro dispositivo presta-se a estudos de filmes depositados (DFD) e é feito em acrílico com eletrodos em latão posicionados a décimos de milímetros do cristal. Sua parte superior possui uma camisa de água para a termostatização. O segundo, semelhante ao primeiro, permite a modificação de superfície (DMS) de ESPC através da introdução de reagentes na fase gasosa. Os dois dispositivos foram caracterizados e o primeiro mostrou boa linearidade entre as massas depositadas e aquelas estimadas pela equação de Sauerbrey. O DFD foi utilizado para monitoramento em tempo não real da modificação superficial do cristal por N-[3-(trimetoxisilil)propil]-1,2-etanodiamina seguida por reação com ácido iodoacético. Os resultados mostraram evidências de aumento de massa e mudança nas propriedades viscoelásticas do filme. O DMS foi utilizado para monitoramento em tempo real da modificação superficial com trimetilclorosilano (TMCS) e dimetildiclorosilano (DMDCS). Com o TMCS, devido à formação de uma monocamada, não foi possível monitorar variações significativas de freqüência de oscilação. Com o DMDCS foi possível verificar a formação de poli(dimetilsiloxano). / This dissertation describes the development of devices based on separated-electrode piezoelectric quartz crystals (ESPC) to study surface modifications. This is accomplished by monitoring the oscillation frequency and impedance spectrum of the crystal in the cell. A TTL oscillator coupled to a frequency counter and a microcomputer interface is used for monitoring the oscillation frequency. A dynamic link library and HP-VEE 4.0 programs were developed to control the interface. Other programs were also developed to acquire the impedance spectra from a spectrum analyzer by using the GPIB protocol. The first device (DFD) is intended to study deposited films. It is made in Plexiglas, with brass electrodes positioned at a few tenths of millimeters from the crystal surface, and a thermostatic water jacket. The second device (DMS) is similar to the first one, but allows the use of corrosive reagents in the gas phase for surface modification. The performance of both devices was evaluated and the results showed good agreement between the deposited mass and the one predicted by Sauerbrey equation. The DFD was used for offline monitoring of the crystal surface modification by N-[3-(triethoxysilyl)propyl]-1,2-ethanediamine followed by iodoacetic acid. The results show evidences of mass and viscoelastic variations of the film. The DMS was used for on-line monitoring during surface modification with trimethylchlorosilane (TMCS) and dimethyldichlorosilane (DMDCS). Due to the monolayer formed by TMCS, no significant frequency variation could be observed. On the other hand, the polymerization of DMDCS could be monitored.

Kvantdatorer är en unik form av datorer som har fördelar över traditionella datorer i speciellaanvändningsområden. Ett av de områdena är den teoretiska möjligheten att knäcka deasymmetriska krypteringsmetoder som dagens kommunikation förlitar sig på. Arbetet inriktarsig på just RSA som idag är den vanligaste krypteringsmetoden. Säkerheten hos RSA ärbaserad på att faktoriseringsproblemet och svårigheten av att faktorisera stora tal vilket ärnågot traditionella datorer behöver hundratals år för att klara av. Kvantdatorer med sinenorma beräkningskapacitet kan potentiellt klara av samma primtalsfaktorisering under enbråkdel av tiden. Dagens kvantdatorer är inte tillräckligt utvecklade och saknarberäkningsförmågan för att vara ett hot, uträkningsförmågan beror på mängden kvantbitarsom är sammankopplade. Det största hindret kvantdatorer står inför är framsteg inomfeltolerans som ger möjligheten att bygga kvantdatorer med ett större antal kvantbitar som ärihopkopplade. När kvantdatorer når en punkt med tillräckligt många kvantbitar för att hotakryptering är en svår fråga att besvara men enligt studien bör det komma att ta många årinnan det kvantdatorer blir relevanta för det syftet. För att kunna göra en kvalificerad gissningpå när detta kommer inträffa kombineras flera experters syn av ämnet och en framtidsprognosbaserad på en regressionsanalys. Att nå möjligheten att knäcka RSA 2048 mellan år 2064 och2066 med 50 tusen kvantbitar anses vara någorlunda rimligt enligt den data och uträkningarsom utförts i arbetet.

Partial Post Quantum Cryptography migration of GitLab Community Edition source code with 3 main contributions 1. Devloped RubyCrypt - a simple scanner to assist the Cryptographic Inventory Compilation of Ruby apps 2. Configured git to use PQC signature (CRYSTALS-Dilithium) for commit signing 3. Included CRYSTALS-Dilithium to ssh_data, a common cryptographic Ruby gem used by GitLab (& GitHub):1. Introduction 2. Theoretical Background - Post Quantum Cryptography 2.1. Code-based Cryptography 2.1.1. McEliece Cryptosystem 2.2. Lattice-based Cryptography 2.2.1. CRYSTALS-Dilithium 3. Post Quantum Cryptography Migration of GitLab - a Case Study 3.1. Problem Statement 3.2. Related Work 3.2.1. Software Tools for Static Program Analysis 3.3. Chosen Approach 4. Implementation 4.1. Cryptographic Inventory Compilation 4.1.1. Results 4.2. Migration Planning 4.3. Migration Execution 4.3.1. PQC Commit Signatures in git 4.3.2. Including Dilithium to ssh_data 5. Conclusion and Outlook 6. References List of Tables List of Figures List of Source Code Acronyms Notation / Partielle Migration des GitLab Community Edition Source Codes auf Verfahren der Post-Quanten-Kryptographie mit 3 Hauptergebnissen 1. Entwicklung von RubyCrypt - einem simplen Scanner zur Unterstützung der Inventarisierung verwendeter Kryptographie in Ruby-Anwendungen 2. Konfiguration von git zur Verwendung des quantensicheren Signaturalgorithmus CRYSTALS-Dilithium zur Signatur von Commits 3. Integration von CRYSTALS-Dilithium in ssh_data, ein populäres kryptographisches Ruby gem welches in GitLab (und GitHub) verwendet wird:1. Introduction 2. Theoretical Background - Post Quantum Cryptography 2.1. Code-based Cryptography 2.1.1. McEliece Cryptosystem 2.2. Lattice-based Cryptography 2.2.1. CRYSTALS-Dilithium 3. Post Quantum Cryptography Migration of GitLab - a Case Study 3.1. Problem Statement 3.2. Related Work 3.2.1. Software Tools for Static Program Analysis 3.3. Chosen Approach 4. Implementation 4.1. Cryptographic Inventory Compilation 4.1.1. Results 4.2. Migration Planning 4.3. Migration Execution 4.3.1. PQC Commit Signatures in git 4.3.2. Including Dilithium to ssh_data 5. Conclusion and Outlook 6. References List of Tables List of Figures List of Source Code Acronyms Notation

In Saccharomyces cerevisiae nimmt die membrangebundene RING-Ub-Ligase Doa10 eine bedeutende Rolle in der Proteinqualitätskontrolle (PQC) des Endoplasmatischen Retikulums (ER) und des Nukleus ein. Doa10 katalysiert dabei die Verknüpfung K48- verbundener Ub-Ketten auf Proteine, die entweder in der ER-Membran oder löslich im Cytosol oder dem Nukleoplasma vorliegen. Diese Markierung leitet die Degradation dieser Proteine ein. Interessanterweise kooperiert Doa10, im Gegensatz zu anderen RING-Ub-Ligasen, mit zwei Ub-konjugierenden Enzymen (E2), um ihre Substrate zu prozessieren. In dieser Arbeit wird veranschaulicht, wie die beiden hochspezialisierten E2 Enzyme Ubc6 und Ubc7 sequentiell agieren, um Doa10 Substrate zu modifizieren. Zuerst wird ein einzelnes Ub-Molekül Ubc6-abhängig an ein Substrat konjugiert (Initiation). Von diesem Rest ausgehen katalysiert Ubc7 die Ausbildung einer K48-verbundenen Ub-Kette (Elongation). Die Fähigkeit von Ubc6 nicht nur Lysine, sondern auch hydroxylierten Aminosäuren wie Serin und Threonin mit Ub-Molekülen zu verknüpfen, erweitert das Substratspektrum von Doa10 und ermöglicht die Prozessieren von Proteinen, die keine zugänglichen Lysinreste exponieren. Weiterhin wird gezeigt, dass ein Überangebot von Ubc6 den Doa10-abhängigen Substratabbau beeinträchtigt. Dies weist darauf hin, dass die Generierung eines effizienten Poly-Ub-Signals einer streng kontrollierten Koordination beider E2 Enzyme am Doa10-Ligase-Komplex unterliegt. / In Saccharomyces cerevisiae, the membrane-bound RING-type Ub ligase Doa10 is a key player of Protein Quality Control (PQC) in the endoplasmic reticulum (ER) and the nucleus. Doa10 promotes lysine 48-linked poly-ubiquitylation of proteins that either reside in the ER membrane or are soluble in the cytosol or the nucleus and thereby labels them for degradation. Strikingly, in contrast to other RING Ub ligases, which typically employ a single Ub conjugating enzyme (E2) for substrate ubiquitylation, the Doa10 ligase requires two of such enzymes for client processing. This study demonstrates that the highly specialized E2 enzymes Ubc6 and Ubc7 act in a sequential manner on Doa10 client proteins. In a first step Ubc6 attaches a single Ub molecule to a substrate (priming), which is followed by the elongation of this moiety with K48-linked Ub chains by Ubc7 (elongation). The ability of Ubc6 to conjugate Ub not only to lysine but also to hydroxylated amino acids like serine and threonine broadens the substrate range of Doa10 and allows processing of proteins, which do not expose accessible lysine residues. Overproduction of Ubc6 was shown to impair Doa10 dependent substrate degradation. Apparently, the generation of a productive K48-linked poly-Ub signal requires a tightly coordinated activity of the individual E2 enzymes at the Doa10 ligase complex.

Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>