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The role of Themis2 for beta2 integrin function and neutrophil trafficking in innate immunity
Get PDFLipid metabolism in remyelination: does a hidden dialogue exist between the central nervous system and the periphery?
Get PDFInflammatory demyelinating disorders of the central nervous system (CNS) manifest through the pivotal features of myelin disruption and imbalanced inflammation. Within this category, multiple sclerosis (MS) emerges as a major member, distinguished by the formation of demyelinated lesions induced by inflammation. In response to demyelinating stimuli, the CNS initiates a regenerative process known as remyelination, wherein the active participation of glial cells serves to resolve inflammation and promote repair. However, during the progressive phase of MS, remyelination fails, leading to the exacerbation of disease symptoms. The objective of remyelination is to reinstate the integrity of disrupted myelin sheaths, which predominantly consist of lipids. Lipid metabolism in the CNS is of paramount importance as it fulfills crucial functions in structural support, signaling, and energy storage. Lipids hold a central role in remyelination as the phagocytosis of myelin debris, followed by the intracellular process and extracellular efflux of debris lipid components contribute to a successful lesion repair. Simultaneously, the upregulation of de novo lipid biosynthesis, such as cholesterol, also facilitates the regenerative response. Whereas the significance of lipid metabolism in remyelination has only recently begun to be elucidated, the majority of research in this field focuses on local processes within the CNS. New studies indicate that aberrant systemic metabolism, as observed in conditions like obesity, influences the process of remyelination. Nevertheless, the mechanisms through which peripheral lipid metabolism impacts lesion recovery and the inflammatory capacity remain poorly understood. Thus, in this thesis, I aimed to bridge the existing knowledge gap by exploring, in two distinct projects, how lipid metabolism in both the CNS and the periphery influences the regenerative capacity upon acute demyelination. In Project 1, I investigated the role of lipid storage in microglia/macrophages (phagocytes) and its influence on the process of remyelination. I discovered that the conversion of free cholesterol into cholesterol esters and the subsequent formation of lipid droplets (LD) in phagocytes are essential prerequisites for the process of remyelination. When phagocytes fail to generate LD, the resolution of inflammation is impaired and ultimately remyelination proves unsuccessful. I also found that mice lacking the triggering receptor expressed on myeloid cells 2 (TREM2) fail to produce LD due to their incapability to adapt to surplus cholesterol exposure, consequently leading to the development of endoplasmic reticulum (ER) stress. Mitigating ER stress in TREM2-deficient mice restores LD formation and the inflammation is resolved. Thus, I concluded that the biogenesis of LD in response of acute demyelinating injury constitutes a protective mechanism crucial for the remyelination process. In Project 2, I explored the role of dysregulated peripheral lipid metabolism on the remyelination response. Utilizing a murine model that recapitulates the phenotype of lipodystrophy, I discovered that, after acute CNS demyelinating stimuli, lesion repair is promoted, while the inflammation is resolved. Metabolomic analysis revealed increased levels of branched-chain amino acids (BCAAs) and numerous phosphatidylcholine (PCs) species with polyunsaturated long-chain fatty acids (LCFAs) in the plasma of lipodystrophic mice. Successive proteomic analysis indicated a metabolic activation of brown adipose tissue (BAT) via stimulated thermogenesis. The activation of thermogenesis via the uncoupling protein 1 (UCP1)-dependent pathway, however, did not result in enhanced lesion repair. Therefore, I concluded, that, upon demyelinating injury, lipodystrophic mice exhibit enhanced remyelination and resolution of inflammation, mediated in a UCP1-independent way. In a nutshell, both projects of my thesis aimed to examine the role of lipid metabolism in the context of remyelination, in an effort to gain a deeper understanding of the molecular mechanisms that govern the regenerative process, in order to promote efficient treatment strategies. Undoubtedly, lipids stand at the core of this intricate process, urging further appreciation and investigation into their crucial significance
Formal verification of revocation approaches in identity-based cryptography
Get PDFTraditional public key cryptography relies on certificates to attribute a public key to the owner. This approach produces significant cost in secure communication because certificates need to be downloaded and their validity status needs to be tracked. Identity-based Cryptography (IBC) offers an alternative by deriving public keys directly from their owners’ unique identifiers, e.g. email addresses. This method eliminates the need to manage certificates. A Trusted Third Party (TTP) generates the user’s corresponding secret keys.
A problem arises when a key is revoked because the owner must be able to continue using their identifier. There are several proposals to handle the revocation problem in IBC. However, there is little abstract analysis of this problem and the security considerations are limited to manual proofs for specific approaches and informal explanations of general properties. This work gives a broader overview: It systematizes revocation mechanisms overall, identifies three classes of revocation mechanisms in IBC and provides a formalization for an automated prover to analyze the security properties expected of each class.
The overall systematization happens along two dimensions: 1. “explicit/implicit” (i.e. validity either needs to be checked or can be ignored by third parties) and 2. “directly/indirectly” (i.e. the mechanism manages revoked keys or it manages valid keys). All identity-based methods are implicit and indirect, obsoleting all secret keys at a certain cue and issuing new material to all non-revoked users. They differ in how users obtain new key material.
This work identifies: 1. The renewal method, where keys are completely replaced, 2. the individual-update method, where each user receives a customized update token for their individual key, and 3. the universal-update method, where all users receive the same update token for their individual keys.
The formalization captures the stages that all classes have in common and the processagnostic security goals they ideally achieve. It is adaptable to each class through the mathematical dependencies it models for the keys and yields a blueprint model for an automated analysis with a trace-based prover tool. The proof-of-concept implementation in Tamarin is the first high-level security analysis of IBC-revocation mechanisms. It confirms both the weaknesses to decryption key exposure that were found in individual update mechanisms if they fail to re-randomize the key in the update process and the collusion attack for the universal update approach that was previously only acknowledged indirectly. It also reconciles the notions of Decryption Key Exposure Resistance and of Forward-/Post-Compromise Security, which were previously never discussed together.
Based on this work, research might consider more detailed mathematical context of a certain IBC scheme and its revocation mechanism if and when the problem of modeling distributive laws is solved. This would allow for a more fine-grained security analysis and increase trust in revocable identity-based mechanisms. More generally, the proposed formalization can be used as blueprint for the formal verification of obsolescence-based revocation mechanisms and applications that use key updates outside of IBC
Unraveling the potential and complex interplay of endolysosomal proteins TRPML1, TPC2, and Rab7a
Get PDFThe endolysosomal network comprises distinct interconnected membrane-bound organelles, namely early endosomes (EEs), recycling endosomes (REs), multivesicular bodies (MVBs), late endosomes (LEs), and lysosomes (LYs). Within eukaryotic cells, LYs serve as primary degradative centers, housing a variety of enzymes that function optimally at their acidic pH and are capable of degrading proteins, lipids, and carbohydrates. Lysosomal function and physiology are regulated by resident proteins and ion channels that facilitate ion movement across the endolysosomal membrane. The transient receptor potential mucolipin channel 1 (TRPML1) and two-pore channel 2 (TPC2) are chief cation channels found in LEs and LYs and share several characteristics. These channels are permeable to calcium (Ca2+) and sodium (Na+) and govern cargo trafficking, vesicle fusion, and membrane dynamics in the endolysosomal system. Additionally, both TRPML1 and TPC2 interact with the mammalian target of rapamycin complex 1 (mTORC1), are involved in lysosomal exocytosis and autophagy, and are activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Dysfunctions or mutations in endolysosomal ion channels have been associated with various channelopathies, encompassing autoimmune disorders, fatty liver disease, neurodegenerative diseases, lung disease, metabolic disorders, and cancer. A prominent group of disorders directly related to lysosomal pathophysiology is known as lysosomal storage diseases (LSDs). Mucolipidosis type IV (MLIV), the autosomal recessive LSD, arises from mutations in the gene encoding TRPML1 (MCOLN1). Typically, MLIV manifests in childhood with neurodegenerative symptoms accompanied by visual and motor impairments. MLIV patients often exhibit an accumulation of lipid products and increased aggregates, such as p62/Sequestosome 1 (SQSTM1), within intracellular organelles.
In Scotto Rosato et al., 2022, we aimed to rescue LSD phenotypes by activating TPC2 using the selective agonist TPC2-A1-P. We utilized different models, including induced pluripotent stem cell (iPSC)-derived neurons, patient fibroblasts, and in vivo MLIV mice. Interestingly, stimulation of MLIV cells with TPC2-A1-P reduced lipid and cholesterol accumulation, reversed the autophagy blockade, and restored cellular ultrastructure. Additionally, MLIV mice treated with TPC2-A1-P ameliorated central nervous system defects and exhibited improved motor performance compared to mice treated with DMSO vehicle control.
Besides LSDs, a study has revealed an intriguing connection between TRPML1 and triple-negative breast cancer (TNBC). TNBC is known for its aggressive nature, characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. TRPML1 showed an upregulated expression in TNBCs, and its silencing inhibited the growth of these cancer cells by regulating the activity of mTORC1. For this purpose, we extended our work to investigate the proposed role of TRPML1 in TNBC. In Rühl et al., 2021, we successfully developed the first isoform-selective and potent antagonist for TRPML1, namely the steroid 17β-estradiol methyl ether (EDME). To decipher its mechanism of action, we treated the MDA-MB-231 TNBC cell line with EDME and generated a CRISPR/Cas9 TRPML1 knockout (KO) within the same cell line. The results were compelling, showing that both TRPML1 KO and EDME treatment reduced cell migration and invasion compared to the control. The TRPML1 KO line provided further evidence of the on-target effects of EDME. These findings offer valuable insight into the potential of temporarily modulating TRPML1 activity to suppress the growth and migration of aggressive TNBC.
Nevertheless, despite key similarities, TRPML1 and TPC2 also possess distinct properties. Notably, TPC2 can also be activated by the Ca2+-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), rendering the ion selectivity of TPC2, a highly debatable topic. A previous study in our laboratory demonstrated the agonist-dependent activation of TPC2 using small molecule activators, TPC2-A1-P (used in Scotto Rosato et al., 2022) mimicking PI(3,5)P2 activation, and TPC2-A1-N mimicking NAADP activation. Intrigued by the agonist-mediated switch of TPC2 between non-selective selective Ca2+ and selective Na+ states, we further investigated the simultaneous application of these compounds on TPC2 behavior in Yuan et al., 2022. To explore this aim, we tested the co-application of TPC2-A1-P and TPC2-A1-N using Ca2+ imaging via genetically encoded Ca2+ indicator GCaMP6 and electrophysiology patch clamp recordings. Our preliminary investigations aimed at testing various cell lines for the expression of TPC2 (data published in Abrahamian et al., 2021, appendix). The melanoma cell line, SK-MEL-5, showed high expression for TPC2 but not the TPC1 isoform on a transcript level and was selected for further investigation. Indeed, in wild-type (WT) SK-MEL-5 cells, robust Ca2+ responses were evoked that were twice as high as those observed in Hela cells. A TPC2 KO was created in this line to be used as a control for different experiments. As expected, TPC2-deficient SK-MEL-5 cells showed significantly reduced Ca2+ evoked responses. The simultaneous activation of TPC2 by TPC2-A1-P and TPC2-A1-N resulted in increased Ca2+ permeability and flux in WT cells; however, Na+ flux remained unaltered. Our study provides novel insight into the complex interaction of TPC2 with its ligands, demonstrating its preference for potentiating Ca2+ permeability over Na+ in response to signaling cues. This versatile behavior has profound implications on cellular function and physiology, particularly of significance when targeting TPC2 in disease models.
In a pathological context, we expanded our gene expression analysis of endolysosomal cation channels and Rab proteins across various cancer types. We observed the highest expression for MCOLN1, TPCN2, and RAB7A particularly in comparison to other lysosomal genes tested, including MCOLN2, MCOLN3, TPCN1, and RAB7B. Intriguingly, these genes (MCOLN1, TPCN2, and RAB7A) showed the most significant enrichment in melanoma and a hepatocellular carcinoma line, surpassing other cancer types like cervical adenocarcinoma, ovarian cancer, colon adenocarcinoma, lung adenocarcinoma, and pancreatic ductal adenocarcinoma, among others. Consequently, melanoma, a highly aggressive type of skin cancer originating from melanocytes, was our primary focus of investigation. Identified risk factors for melanoma include excessive exposure to ultraviolet (UV) radiation, family history, fair hair, skin, and eye color. In particular, we focused on exploring the role of TPC2 in melanoma, given its substantial expression in this type of cancer, localization to mature melanosomes, as well as its pivotal role in pigmentation. Gain of function (GoF) mutations (G734E and M484L) in human TPC2 have been associated with reduced melanin production and blond hair. In accordance with this finding, our study focused on pigmented in vitro melanoma lines, MNT-1 (human) and B16F10 (mouse). Remarkably, the genetic ablation of TPC2 in MNT-1 demonstrated an increased melanin content and a larger but less acidic melanosome lumen. Besides, Naringenin, a natural flavonoid, has been found to block TPC2 activity. To expand on this discovery, we performed a screening of novel flavonoids derived from Dalbergia parviflora, which could potentially be more potent than Naringenin. Among the tested compounds, two were prominent hits: MT-8 (O-methylated isoflavone) and UM-9 (tri-O-methylated isoflavan). We validated these compounds using electrophysiology patch clamp experiments, which demonstrated their ability to inhibit TPC2 at a much lower concentration compared to Naringenin. In addition, both MT-8 and UM-9 showed the highest melanin generation in MNT-1 and B16F10 cells, indicating their potential as effective TPC2 antagonists. Accordingly, we sought to elucidate the physiological significance of TPC2 in regulating melanoma phenotypes in these pigmented lines using our hit compounds and the knockout model. Our experiments focused on assessing the impact of TPC2 on melanoma cell behavior, revealing substantial reductions in cell proliferation, migration, and invasion in the TPC2-deficient melanoma cells compared to their WT counterpart. We examined the downstream signaling cascades influenced by the endolysosomal machinery. Exceptionally, the melanoma oncogene, MITF, demonstrated a significant reduction on a protein level in the TPC2 KO MNT-1 cells compared to the WT cells. Further analysis using the protein stability cycloheximide chase experiments revealed that this downregulation was attributed to the proteasomal degradation of MITF. To confirm this observation, we treated the TPC2-deficient cells with the proteasomal inhibitor MG-132, which ameliorated MITF expression to levels comparable to WT cells. Moreover, we explored the signaling pathways known to regulate MITF and melanoma growth, including MAPK, cAMP, canonical Wnt, and Akt pathways. Our result demonstrated an inverse increase in GSK3β levels in TPC2 KO MNT-1 cells compared to WT, solidifying the role of GSK3β in melanoma as the negative regulator of MITF, promoting its proteasomal degradation. Intriguingly, a proteomic analysis of the TPC2 interactome unveiled Rab7 as an interaction partner of TPC2. Rab7a, a small guanosine triphosphatase (GTPase), serves as a lysosomal marker and plays critical roles in the trafficking and degradation of molecules, fusion of late endosomes and autophagosomes, and lysosomal biogenesis. Nevertheless, the functional impact of Rab7a on TPC2 channel activity and the consequent pathophysiological relevance of this interaction remains unclear. In our study (Abrahamian et al., 2023, appendix), we first reproduced the co-immunoprecipitation (Co-IP) data from Lin-Moshier et al., 2014 and further performed fluorescence resonance energy transfer (FRET) experiments, confirming the physical interaction between Rab7a and TPC2. Moreover, utilizing endolysosomal patch-clamp and Ca2+ imaging techniques, we demonstrated that Rab7a strongly enhances the activity of TPC2, establishing the functional interaction between these two lysosomal proteins. To explore the potential implications in melanoma, we generated different knockout models using CRISPR/Cas9, employed selective small molecule antagonists and agonists, and performed siRNA knockdown (KD) and overexpression (OE) studies in a range of melanoma lines with different mutational backgrounds. Interestingly, we observed significantly diminished melanoma cell proliferation, migration, and invasion in most MITF-dependent melanoma lines upon KO or KD of Rab7a or TPC2. However, most MITF-independent lines exhibited no alternations in melanoma phenotypes upon Rab7a or TPC2 depletion. Furthermore, we identified a positive correlation between the transcript levels of Rab7a and TPC2 in these melanoma lines, as well as a positive correlation between the protein expression of Rab7a with MITF and GSK3β. Consistent with the data obtained from the MITF-dependent pigmented MNT-1 line, the loss or pharmacological inhibition of Rab7a or TPC2 decreased the protein expression of MITF and β-Catenin, while GSK3β protein levels were increased. These findings corroborate the proposed model of the connection between the Wnt/β-Catenin pathway, MITF, and the endolysosomal machinery in melanoma20. In addition, we performed different rescue experiments in the Rab7a and TPC2 knockout lines. Our results demonstrated that the OE of Rab7a only partially alleviated the phenotype of TPC2 KO, whereas TPC2 OE effectively rescued the Rab7a KO phenotype. Based on these findings, we identified Rab7a as a melanoma oncogene and an effector of TPC2, highlighting their potential as targets for therapeutic interventions in melanoma.
Overall, our findings advance our understanding of the therapeutic potential and interplay of lysosomal proteins in the context of neurodegenerative disorders and cancer, with a particular focus on breast cancer and melanoma. Through different works, we demonstrated that mutations, alterations in expression, or dysregulated activity of TRPML1 or TPC2 could result in detrimental effects on cellular functions and associated signaling cascades. Consequently, the investigation of endolysosomal cation channels and proteins and the generation of novel selective small molecule agonists and antagonists targeting these channels can offer valuable insights into the molecular mechanism underlying disease heterogeneity, shed light on the variations in disease manifestation, and uncovers novel opportunities for drug repurposing
Rapid electric-field molecular fingerprinting
Get PDFMenschliches Blutplasma besteht aus einer komplexen Mischung von Biomolekülen. Im Vergleich zu invasiven klinischen Tests, bei denen die molekulare Abdeckung und die Analysetiefe oft begrenzt sind, stellt die Infrarotspektroskopie komplexer Bioflüssigkeiten einen wertvollen, minimal-invasiven diagnostischen Ansatz dar. In mehreren Studien wurde die Wirksamkeit des blutbasierten molekularen Infrarot-Fingerabdrucks zur Bestimmung des Gesundheitszustands einer Person untersucht. Hierbei wurden hauptsächlich Fourier-Transform-Infrarotspektrometer eingesetzt. Aufgrund der limitierten Sensitivität der aktuell verfügbaren Geräte gestaltet sich die Detektion schwacher Infrarotabsorption schwierig. Daher wurde in dieser Dissertation die Femtosekundenlasertechnologie zur Entwicklung eines schnell scannenden, feldauflösenden Infrarot-Spektrometers mit Vorteilen in Bezug auf Sensitivität, Zeitgenauigkeit und Durchsatz genutzt, um die diagnostischen Möglichkeiten zu verbessern.
Ein zuvor entwickelter Yb:YAG-Scheibenlaseroszillator, welcher 16-fs-Pulse mit einer Repetitionsrate von 28 MHz emittiert, wurde genutzt, um 60-fs-Pulse im mittleren Infrarotbereich zwischen 6,5 µm und 11 µm (-20 dB) zu erzeugen. Es wurden zwei neue Methoden zur schnellen elektro-optischen Abtastung mit kHz-Raten demonstriert. Bei der ersten Methode variierte die optische Verzögerung mithilfe einer Sonotrode. Ein Teil der optischen Leistung des 16-fs-Pulses wurde als Gate für die elektro-optische Abtastung genutzt. Die Gate-Pulse wurden an der Sonotrode reflektiert, um schnelle Verzögerungsscans zu ermöglichen, bei der 38.000 Spuren der mittleren Infrarot-Wellenform pro Sekunde erfasst wurden. In einem einzigen Scan, der in 26 µs durchgeführt wurde, wurde ein Dynamikumfang des elektrischen Feldes von 150 mit einer zeitlichen Präzision von 34 Attosekunden an den Nulldurchgangspunkten erreicht.
Bei der zweiten Methode wurden die Gate-Pulse für die elektro-optische Abtastung von einem Er:Fiber-Laseroszillator geliefert. Eine elektronisch gesteuerte Modulation der Repetitionsrate wurde verwendet, um die relative Verzögerung zwischen den Pulsen im mittleren Infrarotbereich und den Gate-Pulsen zu scannen. Die neue elektro-optische Verzögerungsverfolgungstechnik ermöglichte die genaue Abbildung der relativen Verzögerung, indem eine schmalbandige Mittlere-IR-Wellenform mit einer bekannten optischen Frequenz als Kalibrationssignal erzeugt und durch elektro-optisches Scannen erfasst wurde. Die Er:Fiber-Laserpulse wurden in zwei geteilt, wobei ein Teil als Gate für das Kalibrationssignal und der andere Teil als Gate für die breitbandige mittlere IR-Wellenform diente, die aus der Probenküvette austrat. Es werden 2.800 Wellenformspuren pro Sekunde erfasst.
In früheren Arbeiten über hochpräzise Dual-Oszillator-Spektrometer wurde ein konstanter Offset zwischen den Repetitionsraten verwendet. Dies entspricht bei Repetitionsraten im MHz-Bereich Verzögerungsbereichen im Nanosekundenbereich, die für die Gasphasenspektroskopie geeignet sind. Für die Kondensationsphasenspektroskopie hingegen sind deutlich kürzere Verzögerungsbereiche in der Größenordnung von Pikosekunden erforderlich. Das Dual-Oszillator-Schnellabtastungs-Spektrometer mit elektro-optischer Verzögerungsnachführung ermöglicht eine präzise Abtastung von Pikosekunden-Verzögerungen mit einem höheren Tastverhältnis als bisherige Methoden. In einem einzigen Scan, der in 357 µs durchgeführt wurde, wurde eine Zeitpräzision von 50 Attosekunden für die Nulldurchgänge der Wellenform im Verzögerungsfenster mit der höchsten Signalstärke erreicht. Die schnelle Messung mit kHz-Raten "friert" das technische Rauschen im sub-kHz-Bereich ein und ermöglicht die Erfassung dynamischer Prozesse, z. B. in der Durchflusszytometrie. Bei statischen Proben wird die Sensitivität durch die Mittelung wiederholter Messungen der erfassten Wellenformen erhöht.
Das Spektrometer wurde in der multizentrischen klinischen Studie "Lasers4Life" getestet, um zu untersuchen, wie gut Personen mit Lungen-, Prostata-, Brust- und Blasenkrebs im therapienaiven Zustand anhand von molekularen Infrarot-Fingerabdrücken des Blutplasmas identifiziert werden können. Die Studie wurde in Zusammenarbeit mit Spezialisten des Universitätsklinikums der LMU durchgeführt. Die Blutplasmaproben von mehr als 5.300 Probanden wurden mit dem beschriebenen feldauflösenden Spektrometer analysiert. Das Spektrometer erwies sich als robust genug, um reproduzierbare Messungen in großem Maßstab an klinischen Proben durchzuführen und Personen mit Lungenkrebs von nicht symptomatischen Kontrollpersonen mit einer Genauigkeit von 80% zu unterscheiden. Die Ergebnisse für die anderen Krebsarten waren im Vergleich dazu weniger vielversprechend. Der erfolgreiche Abschluss der ersten groß angelegten klinischen Studie mit einem feldauflösenden Infrarotspektrometer ist ein ermutigendes Zeichen für die Entwicklung eines neuen schnellen, minimal-invasiven Ansatzes zur Überwachung der menschlichen Gesundheit.Human blood plasma comprises a complex mixture of biomolecules. Compared to invasive clinical testing, which is often limited in molecular coverage and depth of analysis, infrared spectroscopy of complex biofluids presents a valuable, minimally invasive, single-measurement diagnostic approach. Several recent studies have explored the efficacy of blood-based infrared molecular fingerprinting to determine the state of an individual's health. Commercially available Fourier transform infrared spectrometers were predominantly applied. However, the limited sensitivity of currently available devices makes it difficult to detect weak infrared absorption. In this thesis, the power of femtosecond laser technology has been harnessed to develop a rapid-scanning field-resolving infrared spectrometer with advantages in sensitivity, timing precision, and throughput, in the hope of improving diagnostic capabilities.
To achieve this, a previously developed Yb:YAG thin-disk laser oscillator emitting 16-fs pulses at a repetition frequency of 28 MHz was utilized to generate few-cycle mid-infrared radiation spanning from 6.5 µm to 11 µm (-20 dB). This thesis demonstrates two new techniques for rapid electro-optic sampling at kHz rates. The first technique used an ultrasonic sonication device called a sonotrode, which vibrates at 19 kHz, to vary optical delay. A fraction of the optical power of the 16-fs pulse is picked off to act as the gate for electro-optic sampling. The gate beam was reflected off the sonotrode to facilitate ultra-rapid delay scanning, with 38,000 traces of the mid-IR waveform acquired per second. In a single scan acquired in 26 µs, an electric-field dynamic range of 150 was achieved, with a timing precision of 34 attoseconds at the zero-crossing points.
The second method involved a dual-oscillator approach, in which the gate pulses for electro-optic sampling were sourced from a second Er:fiber laser oscillator. The relative delay between the mid-infrared and gate pulses was scanned by an electronically controlled modulation of the pulse repetition frequency of the second oscillator with respect to the first. The novel electro-optic delay tracking technique facilitated precise mapping of the relative delay, wherein a narrowband mid-infrared waveform with a known optical frequency was produced as a delay calibration signal and captured using electro-optic sampling. The Er:Fiber laser pulses were split into two, with one part serving as a gate for the delay calibration signal to track the relative delay with high precision and the other gating the broadband mid-IR waveform that came out through the cuvette containing the sample. By modulating the repetition frequency of the Er:Fiber laser at a frequency of 1.4 kHz, traces of the mid-IR waveform are acquired at a rate of 2,800 per second.
Previously reported dual-oscillator spectrometers with sub-femtosecond timing precision have applied a constant offset between the repetition frequencies of the two oscillators. For MHz repetition rates, this corresponds to nanosecond delay ranges well suited to gas phase spectroscopy. Condensed phase spectroscopy, on the other hand, requires much smaller delay ranges on the order of picoseconds due to the shorter dephasing times of the excited molecular vibration states. The dual-oscillator rapid scanning spectrometer with electro-optic delay tracking fills this space by enabling precise scanning of picosecond delays with a much higher duty cycle. For a single scan acquired in 357 µs, a timing precision of 50 attoseconds was achieved at the zero-crossings of the waveform in the delay window with the strongest signal strength. Rapid measurement at kHz rates 'freezes' technical noise in the sub-kHz range and enables the capture of dynamic processes, such as in flow cytometry. For static samples, averaging repeated measurements also enhances the sensitivity of the captured waveforms. Nonlinear spectroscopy techniques, such as pump-probe, photon echo, and coherent anti-stokes Raman scattering, can benefit from the versatile method of electro-optic delay tracking.
The developed instrument was put to the test in the Lasers4Life multi-centric clinical study to evaluate how accurately individuals with lung, prostate, breast, and bladder cancer could be identified at therapy naïve states from infrared molecular fingerprints of blood plasma. The study, which was conducted in collaboration with specialists at the LMU University Hospital, involved more than 5300 individuals. The blood plasma samples were analyzed over several months using the field-resolving spectrometer described in this thesis. The newly developed device proved robust enough to perform reproducible large-scale measurements on clinical samples, classifying individuals with lung cancer from non-symptomatic control individuals with an accuracy of 80 %. The results for the other cancer types were less promising in comparison. The successful completion of the first large-scale clinical study with a field-resolving infrared spectrometer is an encouraging sign for developing a rapid, minimally-invasive, single-measurement approach to monitoring human health
Assoziationsstudien zu Aggression und suizidalem Verhalten
Get PDFEs wird angenommen, dass suizidales Verhalten ein multifaktorielles Geschehen ist, welches durch Interaktionen verschiedener Faktoren entsteht. Eine Heritabilität von bis zu 50 %, auch weitgehend unabhängig von psychiatrischen Erkrankungen, wurde detektiert. Persönlichkeitsmerkmale wie z. B. aggressives Verhalten gelten als intermediärer Phänotyp mit einer Heritabilität von bis zu 47 %. Daraus ergab sich die Hypothese, dass Aggression als Persönlichkeitsmerkmal auch unabhängig von einer psychiatrischen Diagnose einen Risikofaktor für Suizidalität darstellt und, dass es gemeinsame genetische Varianten suizidalen und aggressiven Verhaltens gibt.
Es wurde eine Genomweite Assoziationsstudie (GWAS) in einer neuropsychiatrisch gesunden Probandengruppe durchgeführt, in der single nucleotide polymorphismen (SNPs) auf eine Assoziation mit dem Endophänotypen „Aggression“ untersucht wurden. Die dabei am signifikantesten mit Aggression assoziierten SNPs wurden im Anschluss auf eine Assoziation mit suizidalem Verhalten in zwei Gruppen mit an Schizophrenie erkrankten Patienten untersucht.
Es fanden sich 27 mit Aggression assoziierte SNPs mit p ≤ 0,0001, wobei einer der SNPs eine nahezu genomweit signifikante Assoziation nach Bonferroni-Korrektur zeigte. Bei weiterer Analyse dieser vielversprechendsten SNPs in der Gruppe der schizophrenen Patienten zeigte sich eine signifikante Assoziation nach multiplem Testen des Weiteren eine nominal signifikante Assoziation und zwei Trends zu einer Assoziation mit dem Phänotyp „Suizidversuch“. In einer Replikationsstichprobe zeigte einer der 4 SNPs eine unterschwellige Tendenz. Die Varianz suizidalen Verhaltens, die durch SNPs der GWAS „Aggression“ erklärt werden kann, lag bei 1,4 %. Die in der vorliegenden Arbeit dargestellten Ergebnisse lassen die Annahme zu, dass es einen gemeinsamen genetischen Hintergrund aggressiven und suizidalen Verhaltens geben und Aggression einen Anteil an der genetischen Varianz suizidalen Verhaltens haben könnte. Es wurden neue Genbereiche detektiert, die signifikant mit Aggression assoziiert sind und möglicherweise einen Einfluss auf suizidales Verhalten zu haben scheinen. Einige der SNPs liegen in, bzw. in der Nähe von Genen, bei denen ein Zusammenhang mit der neuronalen Entwicklung, u. A. im Bereich der axonalen Wegfindung, des Axonwachstums und der GTPase-Aktivierung angenommen werden kann. Inwiefern die neurobiologischen Mechanismen Einfluss auf die Genese von Aggression und von Suizidalität nehmen, gilt es noch herauszufinden. Die Ergebnisse könnten somit für das Verständnis der Genetik der Suizidalität und von Persönlichkeitsmerkmalen wichtig sein, auch wenn weitere Forschung mit größeren Stichproben auf dem Gebiet notwendig ist.It is assumed that suicidal behaviour is a multifactorial event that arises from the interaction of various factors. A heritability of up to 50 % was detected, also largely independent of psychiatric diseases. Personality traits such as aggressive behaviour are considered as intermediate phenotypes. Aggression has a heritability of up to 47 %.
Therefore, the hypothesis arose that aggression as a personality trait represents a risk factor for suicidal tendencies (also independent of psychiatric diseases) and that there are common genetic variants of suicidal behaviour and aggression.
A genome-wide association study (GWAS) was performed in a healthy group of subjects, in which single nucleotide polymorphisms (SNPs) were examined for an association with the endophenotype "aggression". The SNPs most significantly associated with aggression were then examined for an association with suicidal behaviour in two groups of subjects with patients suffering from schizophrenia.
There were 27 SNPs associated with aggression with p ≤ 0.0001, one of which showed an almost genome-wide significant association after Bonferroni correction. Further analysis of these top SNPs in the schizophrenic group revealed one significant association after multiple testing, one nominally significant association, and two SNPs with a trend towards an association with the suicide attempt phenotype. In a replication sample, one of the 4 SNPs showed a subliminal trend. The variance of suicidal behaviour that can be explained by SNPs of the GWAS "aggression" was 1.4 %. The results presented in this study allow the assumption that there is a common genetic background of aggressive behaviour and suicidality, and that aggression could contribute to the genetic variance of suicidal behaviour. New gene regions were detected that are significantly associated with aggression and may appear to have an impact on suicidal behaviour. Some of the SNPs are located in or near genes thought to be involved in neuronal development, including axonal pathfinding, axon outgrowth and GTPase activation. The extent to which the neurobiological mechanisms influence the genesis of aggression and suicidal tendencies remains to be determined. The results could be important for understanding the genetics of suicidality and personality traits, although further research in the area is needed. Additional studies in larger samples would be needed to increase the level of significance
Wirksamkeit der monoklonalen Antikörperkombination Tixagevimab/Cilgavimab als COVID-19-spezifische Präexpositionsprophylaxe bei Patienten mit hämatologischen Erkrankungen
Get PDFExcited state dynamics in novel energy conversion materials
Get PDFThe photophysical properties of light-absorbing semiconducting materials are of significant interest, as these materials are essential for light-induced energy conversion processes such as those in solar cells. This work investigates photo-induced excited state dynamics in semiconductors using confocal microscopic measurement techniques, focusing specifically on the radiative photoluminescent decay of both static and diffusing excited states. The thesis begins with an introduction to the physical description of excited states in materials, comparing their characterization in semiconductors to that in molecules. Additionally, the excited state can represent a mobile state, driven by a chemical potential. A subsequent chapter explains the dynamics of diffusion combined with photophysical decay, linking measured data with diffusion functionality through various numerical and analytical approaches. Chapter three is introducing the confocal microscope and the implemented advanced time-resolved and spectroscopy techniques. Furthermore, a detailed description of the signal generation is given determining the measured photo luminescence. The final section explains the numerical analysis of the measured data. These foundational chapters serve to introduce the main projects discussed in this work. In methyl ammonium lead iodide (MAPbI 3 ) thin films, the diffusive transport of excited ambipolar charge carriers is hindered by optical phonons and lattice fluctuations. Additionally, the disorder induced by phase transitions in thin films was found to halt this transport. In ceasium formamidium lead triiodide (CsFAPbI 3 ) quantum dot films, restructuring of the clustering improved photophysical properties and consequently enhanced power conversion efficiency. For lead-free perovskite systems, the inferior solar cell performance was attributed to strong localization of excited states due to coupling to lattice phonons and immobile defect states. The novel approach using wurster-benzodithiophene-dialdehyde covalent-organic-framework (WBDT COF) highly crystaline thin films, a tunable material composed solely of organic molecules, exhibited semiconductor-like behavior, with disorder identified as a critical factor influencing excited state diffusive transport. Finally, measurements on rare-earth-ion metallic-organic-frameworks (REI MOFs) revealed a non-parametric downconversion process in ytterbium 2,5-dihydroxy-1,4-benzoquinone metallic-organic-frameworks (DHBQ MOFs), demonstrated through second-order time correlation function analysis of emitted photons