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In dieser Dissertation werden die Erfahrungen mit verschiedenen Präparationsmethoden für CH-Kavitäten beschrieben, um die Leistung der Kavitäten nach der Herstellung weiter zu steigern. Die Leistung wird anhand von zwei wichtigen HF-Parametern bewertet:
dem elektrischen Feld Ea und der intrinsischen Güte Q0. Im Gegensatz zu normalleitenden (NC) Kavitäten kann die intrinsische Güte von supraleitenden (SC) Kavitäten mit zunehmendem elektrischem Feld erheblich variieren. Das optimale Ergebnis für die Kavitätenpräparation ist die Erhöhung des maximalen elektrischen Feldes unter Beibehaltung eines höheren Q0 über die gesamte Feldspanne. Da Q0 umgekehrt proportional zu den Kavitätsverlusten ist, reduziert eine Erhöhung des Qualitätsfaktors die Kryoverluste für den Betrieb bei gegebenem Feldniveau. Die Entwicklung der Kavitätenperformanz im Verlauf dieser Arbeit dargestellt.
Die meisten SC-Kavitäten sind elliptische Strukturen, welche bei hoher Geschwindigkeit und Tastrate angewendet werden. Die Präparationsmethoden wurden daher überwiegend auf diese Strukturen angewandt und optimiert. Diese Arbeit konzentriert sich auf die Umsetzung der zuverlässigsten und vielversprechendsten Oberflächenbehandlungen mittels des ersten vom IAP entwickelten SC 360MHz CH-Prototyps. Diese Kavität wies nach 11 Jahren Lagerung eine verminderte Leistung auf, welche mit Röntgenstrahlung bei bereits niedrigen elektrischen Feldern einherging. Dies deutet auf eine unbeabsichtigte Belüftung mit normaler Luft hin, durch die Partikel eingeführt wurden, die als verstärkende Quellen von Elektronen fungierten. Außerdem musste der Leistungskoppler aufgrund einer starken Überkopplung neu ausgelegt werden.
Die Kavität wurde für 48 Stunden bei 120◦ C mittels Heizbändern in der Experimentierhalle des IAP’s ausgeheizt, was zu einer Verbesserung des Qualitätsfaktors bei niedrigen Werten und zu einer Verkürzung der für die Konditionierung von Multipacting-Barrieren erforderlichen Zeit führte. Allerdings wurde durch diese Behandlung das maximale erreichbare elektrische Feld weiter verringert. Die Verbesserung der Güte ist auf das Ausgasen der Kohlenwasserstoffe während des Backvorgangs zurückzuführen. Die negative Auswirkung auf das maximale elektrische Feld ist weniger auf das Backen selbst zurückzuführen als auf den Transport der Kavität und die verwendeten Vakuumkomponenten, die in der Versuchshalle gelagert sind.
Die beobachtete Leistungseinschränkung lässt sich hauptsächlich durch Partikel im Inneren des Resonators erklären, da Feldemission bei niedrigen Feldstärken auftrat. Eine Hochdruckspülung mit ultrareinem Wasser (HPR) ist das Standardverfahren, um nach Behandlungen, bei denen das Risiko einer Oberflächenkontamination besteht, eine hohe Reinheit der inneren Oberflächen zu erreichen. Die HPR wurde in Zusammenarbeit mit dem Helmholtz-Institut-Mainz und der Gesellschaft für Schwerionenforschung geplant und durchgeführt. Der Resonator zeigte bereits während der Messung der Q-E-Kurve eine Zunahme der transmittierten Leistung bei konstanter Vorwärtsleistung, was vor der HPR nicht der Fall war. Bei der CW-HF-Konditionierung zeigte die Kavität den höchsten Gradienten bei einem deutlich schwächeren Q-Abfall bei hohen Feldstärken.
Sowohl bei der Messung von 2008 als auch bei der beschriebenen Messung wurde die Kavität mit einer HPR-Behandlung fertiggestellt, aber für die HPR-Behandlung bei HIM in Mainz wurden einige Anpassungen vorbereitet. Der CH Prototyp verfügt über keine zusätzlichen Spülports und wurde daher mit zwei verschiedenen Düsen mit unterschiedlichen Sprühwinkeln gespült, um die erreichbaren inneren Resonatorflächen zu maximieren. Die Verwendung mehrerer Sprühwinkel könnte auch für CH-Kavitäten mit Spülöffnungen von Vorteil sein und sollte für zukünftige HPR-Anwendungen in Betracht gezogen werden.
Die Heliumbehandlung wurde am CH-Prototyp 2,5 Stunden lang durchgeführt und lieferte vielversprechende Ergebnisse in Bezug auf die Güte und die Gradientenoptimierung.
Während dieses Prozesses wurde die emittierte Röntgenstrahlung in Richtung am Arbeitsplatzs gemessen und zeigte starke zeitabhängige Fluktuationen. Dies deutete auf die Beseitigung von Partikeln hin und wurde anschließend durch einen Anstieg des elektrischen Feldes von 8,4 auf 8,7 MV/m bestätigt. Eine unerwartete Auswirkung wurde bei der Q-Steigung im mittleren bis hohen Feld festgestellt, bei der der Qualitätsfaktor im Vergleich zum HF-konditionierten Fall eine Erhöhung von 5% oberhalb von 2MV/m aufwies. Dieser systematische Anstieg wurde für diesen Beschleuniger vor der Behandlung bisher nicht beobachtet. Stickstoffgedopte Kavitäten zeigen ein ähnliches Verhalten, bei dem Wechselwirkungen innerhalb der Oxidschicht mit Änderungen der Qualitätsfaktoren korreliert sind. Da Helium ein nicht reaktives Element ist, sind mögliche Erklärungen für diesen Effekt der Sputterprozess und die Einlagerung von Helium innerhalb der Oberfläche. Eine Serie von Heliumbehandlungen ist geplant, um ein optimiertes und sicheres Rezept für CH-Kavitäten zu finden. Die Q-E-Messung nach der Abkühlung und vor der Behandlung wird auch zeigen, ob der Leistungsgewinn durch ein Aufwärmen auf Raumtemperatur beeinträchtigt wird.
Die in dieser Arbeit skizzierte Behandlungssequenz wird für CH-Kavitäten dringend empfohlen. Das Ausheizen hat sich bei der Verringerung des Multipactings and der Güteabnahme bei hohen Feldern als wirksam erwiesen und bleibt von der anschließenden HPR unbeeinflusst. In dieser Arbeit wurden keine negativen Auswirkungen der HPR auf das Multipactingverhalten festgestellt. Anschließend wird eine CW-HF-Konditionierung durchgeführt, bis keine weitere Leistungszunahme der Kavität mehr zu verzeichnen ist.
Wenn die Kavität immer noch durch Feldemission begrenzt ist, sollte eine Wiederholung der HPR-Behandlung in Betracht gezogen werden, da bei sorgfältiger Durchführung der HPR keine der bisherig gefertigten CH-Kavitäten hierdurch begrenzt war. Es ist auch anzumerken, dass die Heliumbehandlung nur an der 360MHz CH-Kavität durchgeführt wurde, als diese eine geringe Strahlung durch Feldemission aufwies. Das Risiko des Heliumprocessing an CH-Kavitäten unter starker Feldemission ist unbekannt. Es ist zu erwarten, dass die Elektronenströme und damit die Ionenbeschusslawinen zunehmen und ein größeres Risiko für die Beschädigung von der Komponenten darstellen. Nach dem derzeitigen Kenntnisstand sollte die Heliumbehandlung nur für gut vorbereitete Kavitäten mit minimaler Feldemission in Betracht gezogen werden.
Die Arbeit behandelt die Messung von Photonen mit Teilchendetektoren, die auf digitalen Silizium-Pixelsensoren basieren. Diskutiert werden zwei wesentliche Schritte in den Upgrade-Programmen des ALICE-Experiments am CERN-LHC:
1. FOCAL-Detektor-Upgrade (2027): Untersuchung der Detektorantwort des elektromagnetischen Pixel-Kalorimeters EPICAL-2 und der Form elektromagnetischer Schauer durch Teststrahl-Messungen und Monte Carlo Simulationen.
2. ALICE 3-Upgrade (2035): Simulationsstudien zum Untergrund in der Messung von Photonen mit sehr kleinem Transversalimpuls.
Teil 1: Performance des elektromagnetischen Pixel-Kalorimeters EPICAL-2
Detektordesign und Testmessungen: EPICAL-2, ein SiW-Sandwich-Design-Kalorimeter mit ALPIDE Sensoren, besitzt eine Tiefe von ca. 20 Strahlungslängen und etwa 25 Millionen Pixel. Testmessungen wurden an der Universität Utrecht (kosmische Myonen) sowie am DESY und CERN-SPS (Elektronen) durchgeführt.
Simulation und Validierung: Das EPICAL-2 wird im Simulationspaket Allpix2 implementiert, um die Testmessungen zu validieren und das Detektorverhalten zu untersuchen. Systematische Variationen bestätigen die Stabilität und Reproduzierbarkeit der Simulation.
Datenaufbereitung und Schauerprofile: Im Rahmen der Datenanalyse werden fehlerhafte Pixel ausgeschlossen, Pixel-Treffer zu Clustern gruppiert, Chips kalibriert und der Strahlwinkel korrigiert. Das longitudinale Profil elektromagnetischer Schauer zeigt, dass das Schauermaximum in der Simulation etwas tiefer liegt als in den Testdaten, was auf zusätzliches Material oder eine unvollständige Beschreibung der Schauerentwicklung in der Simulation zurückzuführen sein könnte. Das laterale Profil zeigt, dass eine Schauertrennung im Millimeter-Bereich möglich ist.
Energieantwort und -auflösung: Die nicht-lineare Energieantwort wird sowohl in Testdaten als auch in Simulationen beobachtet. Die Energieauflösung des EPICAL-2 für Cluster ist besser als für Pixeltreffer und vergleichbar mit dem analogen CALICE-Prototypen. Simulationen ohne Strahlenergie-Fluktuationen zeigen eine bessere Energieauflösung als in den Testdaten.
Teil 2: Untergrund in der Messung von Photonen in ALICE 3
Simulationssetup: Die ALICE 3-Detektorgeometrie wird in GEANT4 implementiert, um den Untergrund in der Messung weicher Photonen zu untersuchen. Simulationen mit PYTHIA und GEANT4 zeigen, dass der Untergrund hauptsächlich aus Zerfallsphotonen und Photonen aus externer Bremsstrahlung besteht.
Ergebnisse der Untergrundstudien: Der Untergrund durch Photonen aus externer Bremsstrahlung dominiert und liegt im Akzeptanzbereich des FCT um einen Faktor von 5 bis 10 über dem theoretischen Signal weicher Photonen. In der Simulation wird das Material zu 8%—14% X0 in ALICE 3 bestimmt, wobei bereits bei 5% X0 der Untergrund genauso stark ist wie das erwartete Signal.
Möglichkeiten zur Untergrundreduzierung: Untersuchungen zeigen, dass ein Elektron-Veto das Signal-zu-Untergrund-Verhältnis um den Faktor 30 verbessern und eine Materialreduktion durch ein optimiertes Strahlrohr um den Faktor 7.
Die Ergebnisse des ersten Teils dieser Arbeit demonstrieren insgesamt die gute Performance des EPICAL-2 in Bezug auf die Energiemessung und die Bestimmung der Schauerform. Darüber hinaus unterstützen sie den Einsatz digitaler Kalorimeter im FOCAL-Upgrade des ALICE-Experiments und zeigen das Potenzial der digitalen Kalorimetertechnologie für zukünftige Hochenergiephysik-Experimente.
Die Ergebnisse des zweiten Teils dieser Arbeit liefern einen wesentliche Beitrag zum geplanten ALICE 3-Upgrade. Weiterhin veranschaulichen sie, wie ein Elektron-Veto und die Reduzierung des Materials zusammen eine vielversprechende Messstrategie bilden können.
In almost all parts of the world the industrialisation grows continuously and thus, the chemical pollution of natural waters has become a major public concern. A major consequence and one of the key environmental problems we are facing today is the increasing contamination of freshwater systems with chemicals. The chemicals are detected in wastewater, surface (river) water, ground water and drinking water ubiquitously in natural waters and not only in industrialised areas. The main point sources for water pollution and the release of these synthetic organic substances of human origin, so called micropollutants (MPs), are wastewater treatment plants (WWTPs). These MPs such as pharmaceuticals, personal care products, disinfectant chemicals, chemicals used in the industry and in households, contraceptives, hormones, food additives, artificial sweeteners, pesticides, biocides, and many emerging contaminants are only incompletely removed by the existing conventional wastewater treatment technologies. The MPs end up in the water cycle and have adverse effects on wildlife aquatic ecosystems and human health even at very low concentrations. Therefore, advanced wastewater treatment (AWWT) technologies, such as ozonation, treatment with activated carbon, biofiltration, membrane bioreactors (MBRs) or exposure to ultraviolet light are investigated as options to upgrade conventional WWTPs. However, several studies show that especially the ozonation of wastewater generates diverse transformation products (TPs) with unknown properties. These TPs could be more toxic than the mother compound. Thus, a post-treatment after the ozonation process is required.
The present thesis was part of the BMBF-funded TransRisk project dealing with “the characterisation, communication, and minimisation of risks of emerging pollutants and pathogens in the water cycle”. One main objective was the investigation of conventional treated wastewater after a full-scale ozonation with four post-treatments (each non-aerated and aerated granular activated carbon (GAC) filtration and biofiltration) in comparison to a MBR treatment of raw (untreated) wastewater separately and in combination with an additional ozonation on a pilot WWTP. For this purpose, the wastewater samples were characterised with a comprehensive battery of in vitro and in vivo bioassays. The in vitro bioassays were performed to detect endocrine activities (such as (anti)estrogenic and (anti)androgenic activities), genotoxicity, and mutagenicity. The results showed a decreased estrogenic activity due to the conventional wastewater treatment as well as the ozonation, but a distinct increase of the anti-estrogenic activity and the mutagenicity in the ozonated wastewater, possibly caused by new formed TPs, that were reduced after the post-treatments whereas the GAC filtration performed better than the biofiltration. The in vivo bioassays included for example the impact of the wastewater on mortality, reproduction, development, and energy reserves of the test organisms. The in vivo on-site tests with the mudsnail Potamopyrgus antipodarum and with the amphipod Gammarus fossarum indicated a major impact of conventional treated wastewater, ozonated wastewater, and MBR treated wastewater. The flow channel experiments in the laboratory with Gammarus pulex pointed to a serious impact of an estrogenic effluent on life-history traits of the amphipod. Finally, an ozonation of the wastewater with subsequent GAC filtration represented the most promising option. In addition, chemical analyses of 40 selected MPs, so called tracer substances, performed in parallel to the in vitro and in vivo bioassays underlined this assumption.
A second main objective was the optimisation of the preparation of water and wastewater samples for ecotoxicological in vitro bioassays because common sample preparation techniques are predominantly adapted for chemical analyses. Therefore, the impact of sample filtration, long-term acidification with following neutralisation as well as the enrichment with solid phase extraction (SPE) in combination with short-term acidification were investigated using amongst others raw (untreated) wastewater, hospital wastewater, conventional treated and ozonated wastewater, surface water, and ground water. Overall, eleven in vitro bioassays were performed for the detection of endocrine activities, genotoxicity, and mutagenicity. The results show that sample filtration and acidification/neutralisation significantly affected the outcome of the bioassays especially the anti-estrogenic activity and the mutagenicity whereas the sample filtration had a minor impact than the acidification. Thus, the testing of untreated (waste)water samples is advisable because the sample is minimally processed. Furthermore, the SPE extracts showed in parts high cytotoxic effects whereby no conclusions on the results of the bioassays were possible. However, the enrichment of endocrine activity and mutagenicity was predominantly effective but depended on the used SPE cartridge and the pH value of the (waste)water samples. Based on the results the use of a Telos C18/ENV cartridge and an acidified sample is recommendable. In the end, there is a need to optimise the sample preparation for in vitro bioassays to reach their maximum outcome for the best possible assessment of the water quality.
Autophagy is an important degradation pathway mediating the engulfment of cellular material (cargo) into autophagosomes followed by degradation in autophagosomes.
Different stress stimuli, e.g. nutrient deprivation, oxidative stress or organelle damage, engage autophagy to maintain cellular homeostasis, recycle nutrients or remove damaged cell organelles. Autophagy not only degrades bulk cytoplasmic material but also selective autophagic cargo, for example lysosomes (lysophagy), mitochondria (mitophagy), ER (ER-phagy), lipid droplets (lipophagy), protein aggregates (aggrephagy) or pathogens (xenophagy). Selective autophagy pathways are regulated by selective autophagy receptors which bind to ubiquitinated cargo proteins and link them to LC3 on the autophagosomal membrane.
Ubiquitination is an essential post-translational modification controlling different cellular processes such as proteasomal and lysosomal degradation or innate immune signaling.
M1-linked (linear) poly-Ubiquitin (poly-Ub) chains are exclusively assembled by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the M1 poly-Ub-specific OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN). In addition to key functions in innate immune signaling and nuclear factor-κB (NF-κB) activation, M1 ubiquitination is also implicated in the regulation of autophagy.
LUBAC and OTULIN control autophagy initiation and maturation and the autophagic clearance of invading bacteria via xenophagy. However, additional functions of LUBAC- and OTULIN-regulated M1 ubiquitination in autophagy are largely unknown and it also remains unexplored if LUBAC and OTULIN control other selective autophagy pathways in addition to xenophagy. This study aimed to unravel the role of LUBAC- and OTULIN-controlled M1 ubiquitination in bulk and selective autophagy in more detail.
In this study, characterization of OTULIN-depleted MZ-54 glioblastoma (GBM) cells revealed that OTULIN deficiency results in enhanced LC3 lipidation in response to autophagy induction and upon blockade of late stage autophagy with Bafilomycin A1 (BafA1). Furthermore, electron microscopy analysis showed that OTULIN-deficient cells have an increased number of degradative compartments (DGCs), confirming enhanced autophagy activity upon loss of OTULIN. APEX2-based autophagosome content profiling identified various OTULIN-dependent autophagy cargo proteins. Among these were the autophagy receptor TAX1BP1 which regulates different forms of selective autophagy (e.g. lysophagy, aggrephagy) and the glycan-binding protein galectin-3 which serves key functions in lysophagy, suggesting a role of OTULIN and M1 poly-Ub in the regulation of aggrephagy and lysophagy.
Abstract 2
To study aggrephagy, protein aggregation was induced with puromycin which causes premature termination of translation and accumulation of defective ribosomal products (DRiPs). Loss of OTULIN increased the number of M1 poly-Ub-positive foci and insoluble proteins and reduced the levels of soluble TAX1BP1 and p62 in response to puromycin-induced proteotoxic stress.
Intriguingly, upon induction of lysosomal membrane permeabilization (LMP) with the lysosomotropic drug L-Leucyl-L-Leucine methyl ester (LLOMe), M1 poly-Ub strongly accumulated at damaged lysosomes and colocalized with TAX1BP1- and galectin-3-positive puncta. M1 poly-Ub-modified lysosomes formed a platform for NF-κB essential modulator (NEMO) and inhibitor of κB (IκB) kinase (IKK) complex recruitment and local NF-κB activation in a K63 poly-Ub- and OTULIN-dependent manner. Furthermore, inhibition of lysosomal degradation enhanced LLOMe-induced cell death, suggesting pro-survival functions of lysophagy following LMP. Enrichment of M1 poly-Ub at damaged lysosomes was also observed in human dopaminergic neurons and in primary mouse embryonic cortical neurons, confirming the importance of M1 poly-Ub in the response to lysosomal damage.
Together, these results identify OTULIN as a negative regulator of autophagy induction and the autophagic flux and reveal OTULIN-dependent autophagy cargo proteins.
Furthermore, this study uncovers novel and important roles of M1 poly-Ub in the response to lysosomal damage and local NF-κB activation at damaged lysosomes.
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with an onset in early development. ASD has varying degrees of severity and thus affects people differently throughout their lives. Early diagnosis of ASD is essential to provide children with individually-tailored support.8 Eye-tracking may contribute to an earlier diagnosis: Several studies showed differences in eye movements between people with autism spectrum disorder (ASD) and typically developing controls (TD). Different eye movements may contribute to different visual perception that perpetuates to problems in attention, communication and social interaction.
Eye movements are divided into: (1) Fixations (2) Saccades (fast and short eye movements) and (3) Smooth Pursuit Eye Movements (SPEM). SPEM follow the target in a continuous manner. The latter are the subject of the present thesis. SPEM consist of two phases: the open loop phase (= phase of initiation, first 50- 100ms) and the closed loop phase (= phase of maintenance, after about 100ms). SPEM are usually measured by a gain index. It is defined as the ratio of smooth pursuit velocity and visual target velocity and ideally equals to 1.2
In young children, corneal-reflection (CR) eye-tracking is usually applied to quantify eye movement. It allows precise measurements without the use of potentially intrusive devices.
Studies in ASD reported deficits in open loop and closed loop pursuit in children and adults with a mean age of 19.32 (TD) and 20.04 (ASD) years. However, SPEM in preschoolers with ASD remain understudied, although this developmental phase is crucial to the development of non-social and social attentional abilities.
In the present study 66 toddlers and preschoolers (18 to 72 months; ASD: n = 33, TD: n = 33) with matched cognitive abilities and sex were assessed. The main objective was to compare the gain index (Smooth Pursuit Gain = SPG). SPEM were compared between groups with gain index as a dependent measure. We hypothesized that participants with ASD show lower average gain compared to the control group.
We could show a significant group influence on the gain when considering interactions between target velocity and group (p = 0.041). The TD group showed a greater dependence on the increasing object speed than the ASD group with a trend of -0.30 ± 0.11 in the TD group and a trend of -0.13 ± 0.12 in the ASD group. Across groups, the gain decreased with increasing target velocity and dropped faster in vertical than in horizontal trials. Additionally, participants showed a lower SPG in vertical sequences than in horizontal sequences. This supports the general validity of the measure.
Toddlers and preschoolers represent a group that has been subject of little research to date. In addition, there has been only a limited number of studies analyzing SPEM in ASD. To check for a possible group difference without interactions a study with a larger sample size at fixed target velocity and target direction should follow.
Inorganic phosphate is one of the most abundant and essential nutrients in living organisms. It plays an indispensable role in energy metabolism and serves as a building block for major cellular components such as the backbones of DNA and RNA, headgroups of phospholipids and in posttranslational modifcations of many proteins. Disturbances in cellular phosphate homeostasis have a detrimental effect on the viability of cells. There- fore, both the import and export of phosphate is strictly regulated in eukaryotic cells. In the eukaryotic model organism Saccharomyces cerevisiae, the uptake of phosphate is carried out either by transporters with high affinity or by transporters with low affinity, depending on the cytosolic phosphate concentration. While structures are available for homologues of the high-affinity transporters, no structures of low-affinity transporters have been solved so far. Interestingly, only the low-affinity transporters have a regulatory SPX domain, which is found in various proteins involved in phosphate homeostasis.
In this work, structures of Pho90 from Saccharomyces cerevisiae, a low-affinity phosphate transporter, were solved by cryo-EM, providing insights into its transport mechanism. The dimeric structure resembles the structures of proteins of the divalent anion symporter superfamily (DASS) and of mammalian transporters of the solute carrier 13 (SLC13) family. The transmembrane domain of each protomer consists of 13 helical elements and can be subdivided into scaffold and transport domains. The structure of ScPho90 in the presence of phosphate shows the phosphate binding site within the transporter domain in an outward-open conformation with a bound phosphate ion and two sodium ions. In the absence of phosphate, an asymmetric dimer structure was determined, with one protomer adopting an inward-open conformation. While the dimer contact and the scaffold domain are identical in both conformations, the transport domain is rotated by about 30° and shifted by 11 Å towards the cytoplasmic side, leading to the accessibility of the binding pocket from the cytoplasm. Based on these findings and by comparison with known structures, a phosphate transport mechanism is proposed in the present work that involves substrate binding on the extracellular side, conformational change by a rigid-body motion of the transport domain, in an "elevator-like" motion, and substrate release into the cytoplasm. The regulatory SPX domain is not well resolved in the ScPho90 structures, so that no direct conclusions were drawn about its regulatory mechanism. The findings provide new insights into the function and mechanism of eukaryotic low-affinity phosphate transporters.
While eukaryotic cells express various phosphate import proteins, most eukaryotes have only a single highly conserved and essential phosphate exporter. These exporters show no sequence homology to other transporters of known structure, but also possess a regulatory SPX domain. In this work, the structural basis for eukaryotic phosphate export is investigated by elucidating the structures of the homologous phosphate exporters Syg1 from Saccharomyces cerevisiae and Xpr1 from Homo sapiens, using cryo-EM. The structures of ScSyg1 and HsXpr1 show a conserved homodimeric structure and the transmembrane part of each protomer consists of 10 TM helices. Helix TM1 establishes the dimer contact by means of a glycine zipper motif, which is a known oligomerization motif. Helices TM2-5 form a hydrophobic pocket that has density for a lipid molecule. Whether the lipid binding into the hydrophobic pocket has an allosteric effect on the phosphate export activity or only serves protein stabilization is not known. Helices TM5-10 form a six-helix bundle, which constitutes a putative phosphate translocation pathway in its center. This bundle is formed by the protein sequence annotated as EXS domain.
The respective phosphate translocation pathways of ScSyg1 and HsXpr1 show structural differences. While the translocation pathway in HsXpr1 is accessible from the cytoplasm, in ScSyg1 it is closed by a large loop of the SPX domain. Interestingly, this loop is not conserved in higher eukaryotes and is therefore not present in HsXpr1. Another difference are distinct conformations of helix TM9. In ScSyg1, TM9 adopts a kinked conformation, which results in the translocation pathway being open to the extracellular side. In contrast, TM9 adopts a straight conformation in HsXpr1, resulting in the placement of a highly conserved tryptophane residue in the middle of the translocation pathway. As a result, the translocation pathway in HsXpr1 is closed to the extracellular side.
The main focus of this thesis is the application of the nonperturbative Functional Renormalization Group (FRG) to the study of low-energies effective models for Quantum Chromodynamics (QCD). The study of effective field theories and models is crucial for our understanding of physics, especially when we deal with fundamental interaction theories like QCD. In particular, the ultimate goal is the understanding of the critical properties of these models in such a way that we can have an insight on the actual critical phenomena of QCD, with a special focus on its chiral phase transition. The choice of the FRG method derives from the fact that it belongs to the class of functional non-perturbative methods and has also the advantage of linking physics at different energy scales. These features make FRG perfectly compatible with the task of studying non-perturbative phenomena and in particular phase transitions, like the ones expected for strongly interacting matter. However, the functional nature of the FRG approach and of the Wetterich equation has a consequence that its exact resolution is hardly possible, and an ansatz for the effective action is generally needed. In this work we choose to adopt the local-potential approximation (LPA), which prescribes to stop at zeroth order in the expansion in derivative operators of the quantum effective action, including only the quantum effective potential. In this work we exploited the key observation that the FRG flow equation can be cast, for specific models and truncation schemes, in the form of an advection-diffusion, possibly with a source term. This type of equation belongs to the class of problems faced in the context of viscous hydrodynamics. Therefore, an innovative approach to the solution of the FRG flow equation consists in the choice of a method developed specifically for the resolution of this class of hydrodynamic equations. In particular, the Kurganov-Tadmor finite-volume scheme is adopted. Throughout this work we apply this scheme to the study of different physical systems, showing the reliability and the flexibility of this approach.
In the first part of the thesis, we discuss the well-known O(N) model, using the hydrodynamic formulation to solve the FRG flow equation in the LPA truncation. We focus on the study of the critical behaviour of the system and calculate the corresponding critical exponents. Particular attention is given to the error estimation in the extraction of critical exponents, which is a needed and not widely explored aspect. The results are well compatible with others in the literature, obtained with different perturbative and nonperturbative methods, which validates the procedure. In the second part of the thesis, we introduce the quark-meson model as a low-energy effective model for QCD, with a specific focus on its chiral symmetry-breaking pattern and the subsequent dynamical quark-mass generation. The LPA flow equation is of the advection-diffusion type, with an extra source contribution which is due to the inclusion of fermionic degrees of freedom. We thus adopt the developed numerical techniques to derive the phase diagram of the model, which is in agreement with the one obtained with other techniques in the literature.
We also follow another possible way for the study of the critical properties of the quark-meson model: the so-called thermodynamic geometry. This approach is based on the interpretation of the parameter space of the system as a differential manifold. One can then obtain relevant information about the phase transitions from the Ricci scalar. We studied the chiral crossover investigating the behavior of the Ricci scalar up to the critical point, featuring a peaking behavior in the presence of the crossover. We then repeated this analysis in the chiral limit, where the phase transition is expected to be of second order. Via this geometric technique it is possible to have a different view on the chiral phase transition of QCD. This is the case since this approach is based on the calculation of quantities which are influenced by higher-order momenta of the thermodynamic potential, thus allowing for a more comprehensive analysis of the phase transition.
Finally, we exploit the numerical advancement to face the issue of the regulator choice in the FRG calculations. This is one of the most delicate issues which arise when using approximations to solve the FRG flow equation and deserves extensive investigation. In particular, we performed a vacuum parameter study and used the RG consistency requirement to determine the impact of the choice of the regulator on the physical observables and on the phase diagram of the model. Via this study we develop a systematic method to comparison the results obtained via different regulators. We show the importance of the choice of an appropriate UV cutoff in the determination of UV-independent IR observables and, consequently, the impact on the latter that the truncation of the effective average action and the choice of the regulator have.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Mitochondrial RNA granules (MRGs) are membraneless, highly specialized compartments that play an essential role in the post-transcriptional regulation of mitochondrial gene expression. This regulation is crucial for maintaining energy production, controlling metabolic functions and ensuring homeostasis in cells. Dysregulation of mitochondrial genes has been linked to various human diseases, including neurodegenerative and metabolic disorders as well as certain types of cancer.
MRGs are composed of different RNA species, including mitochondrial precursor RNA (pre-RNA), mature tRNAs, rRNAs and mRNAs complexed with multiple proteins involved in RNA processing and mitoribosome assembly. However, despite the significance of MRGs, their protein composition, structural organization, stability and dynamics during stress conditions remain elusive. In the study reported here, I adopted a three-step approach to address the aforementioned fundamental issues.
First and foremost, I identified the protein composition of MRGs and unveiled their architectural complexity. To characterize the MRG proteome, I applied the cutting-edge TurboID-based proximity labeling approach combined with quantitative mass spectrometry. Proximity labeling was conducted on 20 distinct MRG-associated human proteins, resulting in the identification of more than 1,700 protein-protein interactions. This expansive dataset enabled me to create a comprehensive network, providing valuable insights into both the (sub)architecture as well as the core structure of MRGs in-depth.
Secondly, I investigated the spatio-temporal dynamics of MRGs under various mitochondrial stress conditions. To monitor the morphological alterations and compositional changes of MRGs, I utilized time-resolved confocal fluorescence microscopy and proteomics, respectively. In this analysis, I applied IMT1, the first specific inhibitor that selectively targets mitochondrial transcription. Using this methodology, I pinpointed precise conditions that triggered MRGs’ disassembly during stress, followed by their reassembly when nascent RNA production was restored. The results of this examination elucidate that MRGs are highly dynamic and stress adaptive structures, capable of rapid dissolution and reassembly, a process closely connected to mitochondrial transcription.
Thirdly, I aimed to explore the impact of RNA turnover on MRGs’ integrity during stress, employing confocal fluorescence microscopy and quantitative real-time PCR. I observed that depletion of MRG proteins associated with RNA degradation counteracts MRGs’ disassembly under stress conditions, a phenomenon attributed to the accumulation of double-stranded RNA (dsRNA). These results emphasize the critical role of pre-RNA turnover in maintaining MRG integrity and reveal that MRGs can be stabilized by dsRNA.
Taken together, the comprehensive investigation reported in this thesis has substantially broadened and deepened our understanding of MRGs’ complexity. By identifying their molecular structure and dynamics, I have gained significant insights into the fundamental characteristics and biological functions of MRGs in cellular processes. This knowledge contributes to the identification of disease-related pathways linked to mitochondrial gene expression and may inspire future studies to develop novel therapeutic approaches.
Inflammation is a crucial host defense mechanism activated in response to injury or infection. Its primary goal is to eliminate the source of the disturbance, repair the damaged tissue, and restore homeostasis. Inflammatory processes can be recognized through increased blood flow, higher vascular permeability, and the recruitment of leukocytes and plasma proteins to the tissue. A pathogen-induced inflammation triggers various pro- and anti-inflammatory processes. Local tissue cells and Toll-like receptors call upon innate immune cells like neutrophils, dendritic cells (DCs), and monocytes to respond to the intruder. They move across the endothelium and respond to local signals by releasing mediators or cytotoxic compounds, phagocytosing, or polarizing. To study local pathogen-induced inflammation, a zymosan-induced inflammation model was used in the hind paws of mice, which caused a Toll-like receptor 2 mediated inflammation. Multi-Epitope-Ligand-Cartography (MELC) was used for multiple sequential immunohistochemistry with 40 different antibodies on the same tissue. Bioinformatic analysis and graphical representation revealed a specific inflammatory architecture consisting of three major areas based on macrophage polarization and their cellular neighborhoods: a core region containing the pathogen, a pro-inflammatory region containing M1-like macrophages, and a region containing anti-inflammatory cells. This discovery highlights the coexistence of pro- and antiinflammatory processes during an ongoing inflammation and challenges the concept of a gradual temporal transition from pro- to anti-inflammation. Flow cytometry of the whole paw was performed to support and refine the MELC results. Eosinophils were used as a specific immune cell population to investigate their role in the inflammatory structure. They were found to be present in all three inflammatory regions, adapting their cytokine profile according to their localization. Depleting eosinophils reduced Interleukin 4 (IL-4)- levels, increased edema formation, and mechanical and thermal hypersensitivities during inflammation resolution. In the absence of eosinophils, pro- and anti-inflammatory region could not be determined in the inflammatory architecture, neutrophil numbers increased, and efferocytosis and M2-macrophage polarization were reduced. IL-4 administration restored these regions, normalized neutrophil numbers, efferocytosis, M2-macrophage polarization, and resolution of zymosan-induced hypersensitivity. The results show that eosinophils expressing IL-4 support the resolution of inflammation by enabling the development of an anti-inflammatory framework that encloses pro-inflammatory regions.