Scientific publications of the Saarland University
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Description of Streptomyces explomaris sp. nov., isolated from the coastal soil rhizosphere of Juniperus excelsa and reclassification of Streptomyces libani as a later heterotypic synonym of Streptomyces nigrescens
Full text linkThe strain Je 1-4T
was isolated from the coastal rhizosphere soil of Juniperus excelsa M. Bieb. (Crimean Peninsula). Phylogenetic
studies based on the 16S rRNA gene sequence revealed that Je 1-4T
is phylogenetically close to Streptomyces libani subsp.
libani NBRC 13452T
(JCM 4322) and Streptomyces nigrescens NBRC 12894T
(DSM 40276) with sequence similarities of 99.86 to
99.93%. The genome of strain Je 1-4T
consisted of a linear chromosome with a size of 8.9 Mbp and a G+C content of 70.9mol%.
Digital DNA–DNA hybridization (dDDH) analysis showed that Je 1-4T
is distinct from both S. libani subsp. libani and S. nigrescens
(dDDH values of 66.6% and 66.7%, respectively), while the latter two strains likely represent the same species (dDDH value
of 92.0%). The predominant fatty acids in the strains were iso-C16:0
, anteiso-C17:0
and anteiso-C15:0
, and the major menaqui nones were MK9 H6 and MK9 H8. Based on these genomic and phenotypic data, strain Je 1-4T
represents a novel species of
Streptomyces, for which the name Streptomyces explomaris sp. nov. is proposed. The type strain is Je 1-4T
(=DSM 117375T
=LMG
33490T
). Additionally, we propose that S. libani subsp. libani Baldacci and Grein 1966 (Approved Lists 1980) is a later heterotypic
synonym of S. nigrescens (Sveshnikova 1957) Pridham et al. 1958 (Approved Lists 1980)
Evaluation of Nectin-4 and Trop-2: Implications for Patient Outcomes and Therapy in Penile Cancer
Full text linkMetastatic penile cancer (PC) continues to have a poor prognosis because of inadequate treatment options. Enfortumab vedotin and sacituzumab govitecan are antibody-drug conjugates (ADCs) that have significantly improved the prognosis of several other tumor types and are, therefore, promising candidates for the successful treatment of metastatic PC. We examined the expression of ADC targets Nectin-4 and Trop-2 in an international multicenter cohort of 203 PC patients both in the primary tumor and in metastases. In addition, we evaluated their prognostic values. Either intermediate or high Nectin-4 membrane expression was found in 28.0% of primary tumors and 18.8% of metastases. The expression in primary tumor decreased significantly with increasing T stage (P ≤ .001). It did not correlate with human papillomavirus status (P = .307) and was not associated with metastasis-free survival, cancer-specific survival, or overall survival. Nectin-4 expression levels at the tumor front and in metastases were significantly associated with each other (P = .005). Trop-2 was detected on the membrane of almost all samples with intermediate or high expression (primary tumor, 98.1%; metastasis, 100%). It was not associated with metastasis-free survival, cancer-specific survival, or overall survival. The expression at tumor front was significantly increased in human papillomavirus–positive tumors (P = .005). Neither Nectin-4 nor Trop-2 was found to be of prognostic value in PC. Enfortumab vedotin therapy seems promising for selected patients with high Nectin-4 expression, which should be confirmed in PC metastases before treatment is considered. Trop-2 is highly expressed in almost all PCs, and therefore, it is a very interesting potential target for sacituzumab govitecan therapy. Both ADCs warrant investigation in clinical trials focusing on PC
Test suite optimization for human-in-the-loop testing processes in industry: Addressing slow test feedback and risks from untested changes
Full text linkBackground. Software underpins the digital infrastructure that sustains modern societies. The importance and complexity of software systems necessitate rigorous testing to ensure their quality and, in particular, their correctness. In industrial contexts, humans play a significant role in software testing. They plan and conduct manual tests, make critical decisions about test completion, and monitor for risks, for example, arising from untested code changes. As software systems become more complex, the challenges of testing increase, consuming more time amidst limited resources. At the same time, compiling a test plan and deciding when all risks have been mitigated are particularly demanding tasks for the humans involved. Objective. In this dissertation, we seek to optimize human-in-the-loop testing processes in industrial practice by enhancing their efficiency and effectiveness. We target two optimization levers: (1) adopting automated test optimization techniques to improve manual testing; (2) supporting test management and quality assurance in the labor-intensive task of allocating test effort and assessing test completion. To accomplish the former, we explore optimization opportunities in manual testing, in particular, established optimization techniques from automated testing. We strive to understand the prerequisites and limitations for their transferability to existing manual testing processes, and how effective these techniques can be. To realize the latter, we prioritize untested code changes according to estimated risk. Methods and Results. To achieve our objective, we have conducted a series of empirical studies on human-in-the-loop testing, using methods such as field experiments and sample studies with industry partners. Manual test suites offer great optimization opportunities, since they often suffer from long run times—up to five person-months for our industry partners. Based on historical data and stakeholder interviews with our industry partners, we demonstrate the transferability and effectiveness of optimization techniques from automated to manual testing. Our results show that applying test case selection and prioritization to manual testing captures up to 81% of failures while reducing execution time by 43%. The second optimization lever addresses the labor-intensive code and test reviews which our industry partners conduct to mitigate the risks of untested code changes. We explore risk factors for code changes and propose a simple risk-based prioritization approach for untested code changes. In our evaluation using historical quality assurance documents from our industry partners, this approach was able to prioritize risky changes significantly higher than less risky changes. Our studies have demonstrated the suitability and effectiveness of the proposed solutions in practice, and after our studies, many subjects have been convinced to adopt our solutions by embedding them in their testing process. Conclusion. We demonstrate a variety of optimization opportunities and levers for human-in-the-loop software testing. Our empirical studies provide evidence of the feasibility and effectiveness of our optimization techniques in industry contexts. This dissertation constitutes a solid foundation for future research on human-in-the-loop testing processes and facilitates adoption by practitioners through detailed optimization guidelines. Both are crucial contributions to the future of software engineering.Hintergrund. Software bildet das Fundament der digitalen Infrastruktur, auf der moderne Gesellschaften aufbauen. Um die Softwarequalität und in erster Linie die Korrektheit dieser bedeutenden und komplexen Softwaresysteme sicherzustellen, sind gründliche Tests unerlässlich. Im industriellen Kontext spielen Menschen im Softwaretesten eine wichtige Rolle. Sie planen und führen manuelle Tests durch, sie treffen kritische Entscheidungen über den Testabschluss und sie überwachen Risiken, die beispielsweise durch ungetestete Codeänderungen entstehen. Mit der zunehmenden Komplexität von Softwaresystemen wachsen auch die Herausforderungen beim Testen. Das Testen wird dadurch zeitaufwendiger, während die verfügbaren Ressourcen begrenzt bleiben. Besonders anspruchsvolle Aufgaben für die beteiligten Personen sind das Erstellen eines Testplans und die Entscheidung, wann das Testen abgeschlossen ist. Ziele. In dieser Dissertation zielen wir darauf ab, menschengestützte Testprozesse aus der Industrie zu optimieren, indem wir die Effizienz und Effektivität des Testens steigern. Wir konzentrieren uns auf zwei Hebel für die Optimierung: (1) die Übertragung von Testoptimierungstechniken für automatisierte Tests auf das manuelle Testen; (2) die Unterstützung des Testmanagements und der Qualitätssicherung bei der arbeitsintensiven Steuerung des Testaufwands und der Bewertung, ob das Testen abgeschlossen werden kann. Um Ersteres zu erreichen, untersuchen wir Optimierungsmöglichkeiten für das manuelle Testen, insbesondere etablierte Optimierungstechniken aus dem automatisierten Testen. Wir versuchen zu verstehen, unter welchen Voraussetzungen und mit welchen Einschränkungen sie auf bestehende manuelle Testprozesse übertragen werden und wie effektiv diese Techniken dabei sein können. Für Zweiteres setzen wir auf eine risikobasierte Priorisierung ungetesteter Codeänderungen. Methodik und Ergebnisse. Um unser Ziel zu erreichen, haben wir eine Reihe empirischer Studien zu menschengestützten Testprozessen durchgeführt. Dabei kamen Methoden wie Feldexperimente und Fallstudien mit Industriepartnern zum Einsatz. Manuelle Testsuiten bieten vielfältige Optimierungsmöglichkeiten, denn sie leiden häufig unter langen Laufzeiten – bei unseren Industriepartnern dauert die Ausführung einer manuellen Testsuite bis zu fünf Personenmonate. Auf Grundlage historischer Daten und Stakeholder-Befragungen mit unseren Industriepartnern zeigen wir, dass Optimierungstechniken wirksam vom automatisierten auf das manuelle Testen übertragen werden können. Unsere Ergebnisse demonstrieren, dass die Auswahl und Priorisierung von Testfällen bis zu 81 % der Fehler bei manuellen Tests erfasst und gleichzeitig die Ausführungszeit um 43 % reduziert. Der zweite Hebel für die Optimierung adressiert arbeitsaufwendige Code- und Testreviews, die unsere Industriepartner durchführen, um das Risiko ungetesteter Codeänderungen zu mindern. Hierfür untersuchen wir Risikofaktoren für Codeänderungen und schlagen auf dieser Grundlage einen einfachen risikobasierten Priorisierungsansatz für nicht getestete Codeänderungen vor. Bei der Evaluierung anhand historischer Qualitätssicherungsdokumente unserer Industriepartner war unser Ansatz in der Lage, risikoreiche Änderungen signifikant höher zu priorisieren als weniger risikoreiche. Unsere Studien haben die Praxistauglichkeit und Wirksamkeit der vorgeschlagenen Lösungen bestätigt. Viele der Beteiligten waren nach unseren Studien so überzeugt von unseren Lösungen, dass sie diese in ihren Testprozess integriert haben. Schlussfolgerung. Wir demonstrieren eine Vielzahl von Optimierungsmöglichkeiten und dort ansetzende Hebel für menschengestützte Testprozesse. Unsere empirischen Studien belegen die Anwendbarkeit und Effektivität unserer Optimierungstechniken im Industriekontext. Diese Dissertation bildet eine solide Grundlage für zukünftige Forschung im Bereich menschengestützter Testprozesse und erleichtert Praktikern die Implementierung durch detaillierte Optimierungsrichtlinien. Beide Aspekte liefern einen entscheidenden Beitrag zur Zukunft des Software Engineerings.BMBF "Q-SOFT
Superhedging supermartingales
Full text linkSupermartingales are here defined in a non-probabilistic setting and can be interpreted solely
in terms of superhedging operations. The classical expectation operator is replaced by a pair of
subadditive operators: one defines a class of null sets, and the other acts as an outer integral. These
operators are motivated by a financial theory of no-arbitrage pricing. Such a setting extends the
classical stochastic framework by replacing the path space of the process by a trajectory set, while
also providing a financial/gambling interpretation based on the notion of superhedging. The paper
proves analogues of the following classical results: Doob’s supermartingale decomposition and
Doob’s pointwise convergence theorem for non-negative supermartingales. The approach shows
how linearity of the expectation operator can be circumvented and how integrability properties
in the proposed setting lead to the special case of (hedging) martingales while no integrability
conditions are required for the general supermartingale case
CMC-Based Injectable Hydrogels Crosslinked by Diels–Alder Chemistry for Wound Healing Applications
Full text linkChronic wounds disrupt natural healing and tissue regeneration, posing a major chal lenge in healthcare. Conventional wound care often lacks effective drug delivery, tissue
integration, infection control, and patient comfort. However, injectable hydrogels offer
localized, minimally invasive treatment and conform to irregular wound shapes. This study
presents carboxymethyl cellulose (CMC)-based injectable hydrogels, prepared via Diels–
Alder click chemistry using highly furan functionalized CMC (45%) and a bismaleimide
crosslinker. The hydrogels showed a rapid gelation time (<490 s) under physiological
conditions. The hydrogel exhibited favorable physicochemical and mechanical properties,
as well as sustained curcumin release (∼80% in 5 days). In vitro studies confirmed excellent
biocompatibility with NIH3T3 fibroblasts and notable antibacterial activity against E. coli,
supporting its potential for wound healing applications
Qualitative Enhancement of the Tooth–Filling Interface Using Cold Atmospheric Plasma
Full text linkObjective: To evaluate the effects of cold atmospheric plasma (CAP) on adhesive bonding
in Class II composite restorations in vitro. Methods: Forty-eight standardized Class II
cavities were assigned to six groups (n = 8), varying in phosphoric acid conditioning,
CAP treatment (1.5 W or 3 W), composite filling, and thermo-mechanical loading (TML).
Evaluations included dye penetration, adhesive layer morphology, resin tag length, and
hybrid layer thickness. Results: CAP combined with phosphoric acid (H3PO4) significantly
increased hybrid layer thickness and resin tag length (p < 0.006). The lowest dye penetration
was observed in Groups 1 and 4. Conclusions: CAP in combination with phosphoric acid
improved the adhesive interface in Class II cavities. CAP alone showed limited benefits,
and higher power levels may negatively affect bonding
Investigations on the Particle Fouling and Backwash Efficiency During Microplastic Microfiltration–Particle Size Aspects
Full text linkThe characteristics of polystyrene (PS) microplastic (MP) microfiltration by a cellulose
acetate (CA) membrane were investigated within this study. Particle sizes and pore sizes
were selected in a comparable range in order to challenge the dead-end microfiltration.
Backwashing experiments round up the investigations. Microfiltration characteristics and
particle size measurements, as well as a particle fouling analysis by different methods, were
applied in the study in order to provide a comprehensive picture of particle deposition and
particle fouling structuring. The particle removal efficiency was particle-size-dependent,
and especially small particles were further reduced during the proceeding filtration, while
the larger particles were already removed within the first minutes of filtration. This
observation was attributed to the pore blocking (internal and/or complete) and build up of the filter cake. The difference in the particle-fouling structure at low and elevated
filtration pressure significantly influences the backwashing efficiency. The particle fouling
resulting from low-pressure filtration was completely removed due to the backwashing
procedure applied, while an increased filtration pressure resulted in a different particle fouling structure, which negatively influenced the backwashing efficiency. This knowledge
of the formation and structure of the MP particle fouling and its removal by backwashing
is a prerequisite for further process development
Microtubule polymerization generates microtentacles important in circulating tumor cell invasion
Full text linkCirculating tumor cells (CTCs) have crucial roles in the spread of tumors during metastasis. A decisive step is the extravasation of CTCs from the blood stream or lymph system, which depends on the ability of cells to attach to vessel walls. Recent work suggests that such adhesion is facilitated by microtubule (MT)-based membrane protrusions called microtentacles (McTNs). However, how McTNs facilitate such adhesion and how MTs can generate protrusions in CTCs remain unclear. By combining fluorescence recovery after photobleaching experiments and simulations we show that polymerization of MTs provides the main driving force for McTN formation, whereas the contribution of MTs sliding with respect to each other is minimal. Further, the forces exerted on the McTN tip result in curvature, as the MTs are anchored at the other end in the MT organizing center. When approaching vessel walls, McTN curvature is additionally influenced by the adhesion strength between the McTN and wall. Moreover, increasing McTN length, reducing its bending rigidity, or strengthening adhesion enhances the cell-wall contact area and, thus, promotes cell attachment to vessel walls. Our results demonstrate a link between the formation and function of McTNs, which may provide new insight into metastatic cancer diagnosis and therapy
Determinants of reliability of self-reported height and weight and their impact on medication dosing: a cross-sectional study
Full text linkObjective Patient-reported anthropometric measures,
such as height and weight, are frequently used in clinical
practice but are susceptible to reporting biases. This
study aims to investigate the determinants of reliability
of patient-reported anthropometric measures in patients
in cardiology and general practice and their impact on
potential medication dosing.
Design Cross-sectional study.
Setting and methods 730 patients were recruited at the
Clinic of Cardiology, Angiology and Intensive Care Medicine
of Saarland University Hospital and a general medicine
practice from November 2015 to December 2018. We
assessed self-reported height and weight and compared
them to calibrated measures immediately afterwards.
Weight and height (optional with medical history) were
self-reported via questionnaire. Interviews were conducted
by female or male nursing staff or physicians.
Outcome measures The main outcomes were the
deviation between patients’ self-reported height and
weight from objective calibrated measures, as well as the
amount of misdosing of exemplary drugs based on this
deviation.
Results The mean height (SD) of the participants (36%
were patients) was 170.92 (9.34) cm. Patients significantly
overestimated their height by 1.82 cm (range: −8.00 to
11.00 cm). Misreporting was best predicted by age, with
older patients providing more height overestimations. The
mean weight was 84.25 (17.41) kg and was significantly
underestimated by 1.49 kg (range: −36.00 to 26.00 kg).
Misreporting was best predicted by higher body mass
index, cognitive impairment and a longer duration since
the last weighing, and self-reporting by questionnaires was
associated with a higher under-reporting of weight. Unlike
females, male patients exhibited a more pronounced
tendency to under-report their weight when responding
to questionnaires compared with face-to-face interviews.
Comparison of doses for low-molecular-weight heparin
according to self-reported versus calibrated weight
revealed potential underdosing and overdosing in 17% and
77% of all patients, respectively. For the cytostatic agent
doxorubicin, for instance, underdosing and overdosing
would have been applied in 40% and 43% of all patients,
respectively.
Conclusions and relevance Self-reported height and
weight are often invalid, especially in patients who are older and overweight. Misreporting can lead to
inappropriate drug dosing. Calibrated measurement of
height and weight remains part of good clinical practice,
and if self-reporting is unavoidable, personal interviews
should be preferred over questionnaires. Trial registration number https://clinicaltrials.gov/study/
NCT0432105
Ca2+-entry into Red Blood Cells: Channel Mediated Influx and its Detection
Full text linkBlood is an essential connective tissue that performs several critical functions in the
human body. Red blood cells (RBCs), constituting around 45% of the blood volume, are
responsible for gas exchange and nutrient transport. Mature RBCs have a unique biconcave
shape and are filled with hemoglobin (Hb), but they lack a nucleus and organelles. Because
of their structure, RBCs rely on ion regulation as one of the key factors influencing cell
behavior. Ion transport across RBC membranes occurs through a combination of passive
channels, transporters and active pumps.
Ca2+, as a universal signalling molecule, plays an essential role in RBCs to regulate cellular
activities. Due to the large gradient across the membrane, a small number of channel
openings can lead to acute and severe changes in free Ca2+ levels. The plasma membrane
Ca2+-ATPase (PMCA) is one of the key factors in maintaining the Ca2+ gradient in
RBCs. In addition, several channels have been identified as sources of Ca2+ entry in
erythrocytes, including Piezo 1, transient receptor potential vanilloid type 2 (TRPV2) and
Cav2.1 channels. Piezo 1 channels, which are sensitive to mechanical stimuli, help regulate
RBC volume and contribute to RBC deformability. TRPV2 and Cav2.1 channels in RBCs
support RBC adaptability under physiological and pathological conditions. When the
intracellular free Ca2+ concentration changes, it can trigger several downstream pathways
to regulate cell behavior. For example, the Gárdos channel regulates K+ efflux in response
to Ca2+ levels, affecting cell volume and shape.
To understand the Ca2+-related pathways in RBCs, it is important to quantify the Ca2+
concentration in RBCs under different conditions. However, current methods based on
fluorescence intensity can only provide semi-quantification, because of a strong influentially
by the hemoglobin. Fluorescence lifetime is one of the intrinsic properties of fluorophores
that is independent on fluorophore concentration, initial disturbance conditions and
fluorescence intensity. The characteristics of fluorescence lifetime provide a potential
method for the quantification of Ca2+.
This study outlines the methods for studying RBCs under different experimental conditions.
Blood samples from different sources, including healthy individuals, marijuana smokers
and patients with specific diseases, were processed to isolate RBCs. Isolated RBCs
were subjected to further steps for Ca2+ measurement based on fluorescence intensity.
Immunofluorescence and western blot techniques were used to detect and quantify specific
proteins within the RBC samples. In addition, the Ca2+ indicator-loaded RBCs were
imaged using fluorescence lifetime imaging microscopy (FLIM).
The data presented in the results part shows that an activation of the Piezo 1 channel
caused by the application of Yoda 1, one of Piezo 1 activators, leads to an increase of
intracellular Ca2+ in two phases, which can be regulated by multiple factors. Measurements
of RBCs from people with spectrin mutation or thalassemia β mutation provide evidence that membrane and cytoskeletal structure may influence Piezo 1 channel function, causing
a different Yoda 1-induced intracellular Ca2+ increase. PMCA show strong inhibitory
effects on the first phase of Yoda 1-induced Ca2+ increase. Cl− conductance has a
positive influence on Yoda 1-induced Ca2+ entry and maintenance of high free Ca2+ levels.
However, the pathway involved is still unknown. The Gárdos channel not only induces K+
efflux and hyperpolarization in response to a Yoda 1-induced Ca2+ increase, its opening
probability can also influence the Cav2.1 channel activity involved in Yoda 1-induced
Ca2+-entry. TRPV2 is a non-selective cation channel recently discovered in RBCs. RBCs
show a heterogeneous response to activation of TRPV2 with an increase of free Ca2+
concentration. In marijuana consumers and sickle cell patients, after application of Δ9-
Tetrahydrocannabinol (Δ9-THC), a TRPV2 activator, RBCs have a higher induced Ca2+
concentration, a higher number of responding RBCs, and even hyperpolarization. It is
indicating that these RBCs are more sensitive to Δ9-THC. Sickle RBCs have an enhanced
response to Δ9-THC in the deoxygenated condition. This suggests that oxidative stress
may alter the activity of the channels. In addition, the studies of patient RBCs also
suggest that the cell age may influence channel activity and sensitivity.
The analysis of FLIM images of X-Rhod-1-loaded RBCs with low or high intracellular
Ca2+ shows different lifetimes with the lowest fluorescence signal originating in the
RBC itself. The number of fitted lifetime components is different between 1-photon
excitation and 2-photon excitation. The environment and the concentration of Hb in
RBCs can influence the lifetime components and the amplitude-averaged lifetime of XRhod-
1. Other factors such as excitation wavelength, acquisition time, use of CellTak and
Ca2+-ionophore Bromo-A23187 show no effect on the lifetime parameters of X-Rhod-1.
With the chosen measurement parameters, the normalized amplitude-average lifetime is a
promising parameter to be used to establish a calibration curve for Ca2+quantification.Blut ist ein wichtiges flüssiges Bindegewebe, das im menschlichen Körper mehrere wichtige
Funktionen erfüllt. Die roten Blutzellen (Erythrozyten), die etwa 45% des Blutvolumens
ausmachen, sind für den Gasaustausch und den Nährstofftransport verantwortlich.
Vollständig ausgebildete Erythrozyten haben eine einzigartige bikonkave Form und sind
mit Hämoglobin (Hb) gefüllt, besitzen jedoch keinen Zellkern und keine Organellen.
Aufgrund dieser Struktur sind die Erythrozyten auf die Ionenregulierung als einen der
Schlüsselfaktoren zur Steuerung des Zellverhaltens angewiesen. Der Ionentransport durch
die Erythrozytenmembranen erfolgt durch eine Kombination aus passiven Transporteren,
Kanälen und aktiven Pumpen.
Ca2+ als universelles Signalmolekül spielt in Erythrozyten eine wesentliche Rolle bei
der Regulierung der zellulären Aktivitäten. Aufgrund des großen Gradienten über die
Membran kann bereits eine kleine Anzahl von Kanalöffnungen zu akuten und signifikanten
Veränderungen der freien Ca2+-Konzentration führen. Die Plasmamembran-Kalzium-
ATPase (engl. PMCA) ist einer der wichtigsten Faktoren für die Aufrechterhaltung
des Ca2+-Gradienten in Erythrozyten. Darüber hinaus wurden in der Vergangenheit
mehrere Kanäle als Quellen für den Ca2+-Eintritt in Erythrozyten identifiziert, darunter
Piezo 1, der Transient-Receptor-Potential-Vanilloid-Kanal Typ 2 (TRPV2) und Cav2.1-
Kanäle. Piezo 1-Kanäle, die empfindlich auf mechanische Reize reagieren, helfen bei
der Regulierung des Volumens der Erythrozyten und tragen zur Verformbarkeit der
Erythrozyten bei. TRPV2 und Cav2.1 in Erythrozyten vermitteln zelluläre Reaktionen auf
mechanische und thermische Belastungen und unterstützen so ihre Anpassungsfähigkeit
unter physiologischen und pathologischen Bedingungen. Wenn sich die intrazelluläre
freie Ca2+-Konzentration ändert, kann dies verschiedene nachgeschaltete Signalwege zur
Regulierung des Zellverhaltens auslösen. So reguliert der Gárdos-Kanal beispielsweise den
K+-Efflux als Reaktion auf den Ca2+-Spiegel und beeinflusst folglich auch das Zellvolumen
und die Zellform.
Um die Ca2+-abhängigen Signalwege in Erythrozyten zu verstehen, ist es wichtig, die
Ca2+-Konzentration im Zellinnern unter verschiedenen Bedingungen zu quantifizieren.
Die derzeitigen Methoden, die auf der Fluoreszenzintensitätsmessungen basieren, können
jedoch nur eine Semiquantifizierung liefern, da die Messungen durch das Hämoglobin stark
beeinflusst werden. Die Fluoreszenzlebensdauer ist eine der intrinsischen Eigenschaften von
Fluorophoren, die unabhängig von deren Konzentration, anfänglichen Störungsbedingungen
und der Fluoreszenzintensität ist. Die Eigenschaften der Fluoreszenzlebensdauer bieten
ein potentielles Maß zur Quantifizierung der Ca2+-Konzentration.
In dieser Studie werden die Methoden zur Untersuchung von Erythrozyten unter verschiedenen
Versuchsbedingungen beschrieben. Blutproben unterschiedlichen Ursprungs, darunter
gesunde Personen, Marihuana-Raucher und Patienten mit bestimmten Krankheiten, wurden
zur Isolierung der Erythrozyten aufbereitet. Die isolierten Erythrozyten wurden weiteren Schritten zur Kalziummessung anhand der Fluoreszenzintensität unterzogen.
Immunfluoreszenz- und Western-Blot-Techniken wurden eingesetzt, um spezifische Proteine
in den Erythrozytenproben nachzuweisen und zu quantifizieren. Darüber hinaus
wurden die mit Ca2+-Indikatoren beladenen Erythrozyten mittels Fluoreszenzlebensdauer-
Mikroskopie (engl. FLIM) abgebildet.
Die im folgenden Kapitel vorgestellten Daten zeigen, dass die Aktivierung des Piezo 1-
Kanals durch den Piezo 1-Aktivator Yoda 1 zu einem zweiphasigen intrazellulären Ca2+-
Anstieg führen kann, der durch mehrere Faktoren gesteuert werden kann. Untersuchungen
an Erythrozyten von Menschen mit einer Spektrin- oder einer Beta-Thalassämie-Mutation
liefern Beweise dafür, dass die Membran- und Zytoskelettstruktur die Funktion des Kanals
Piezo 1 beeinflussen kann. Dies hat unterschiedliche Yoda 1-induzierte intrazelluläre Ca2+-
Anstiege zur Folge. Die PMCA zeigt starke hemmende Effekte auf die erste Phase des
Yoda 1-induzierten Ca2+-Anstiegs. Die Cl−-Leitfähigkeit hat einen positiven Einfluss auf
den Yoda 1-induzierten Ca2+-Anstieg und die Aufrechterhaltung eines hohen freien Ca2+-
Spiegels. Der beteiligte Signalweg ist jedoch noch unbekannt. Der Gárdos-Kanal induziert
nicht nur den K+-Efflux und die Hyperpolarisation als Reaktion auf den Yoda 1-induzierten
Ca2+-Anstieg, sondern seine Öffnungswahrscheinlichkeit kann auch die Aktivierung des
Cav2.1-Kanals beeinflussen, der am Yoda 1-induzierten Ca2+-Anstieg beteiligt ist. TRPV2
ist ein kürzlich in roten Blutzellen entdeckter nicht-selektiver Kationenkanal. Erythrozyten
zeigen eine heterogene Reaktion auf die Aktivierung von TRPV2 durch eine erhöhte
freie Ca2+-Konzentration. Nach Zugabe von Δ9-Tetrahydrocannabinol (Δ9-THC), einem
TRPV2-Aktivator, wiesen die Erythrozyten von Marihuana-Konsumenten und Sichelzellpatienten
eine höhere induzierte Ca2+-Konzentration, eine höhere Anzahl reagierender
Erythrozyten und sogar eine Hyperpolarisation auf. Dies deutet darauf hin, dass diese
Erythrozyten empfindlicher auf Δ9-THC reagieren. Außerdem reagieren sichelförmigen
Erythrozyten im desoxygenierten Zustand verstärkt auf Δ9-THC. Somit gibt es Hinweise,
dass oxidativer Stress die Aktivität der Kanäle verändern kann. Darüber hinaus legen
die Studien mit Erythrozyten von Patienten nahe, dass das Alter der Erythrozyten die
Aktivität und Empfindlichkeit der Kanäle beeinflussen kann.
Die Analyse der FLIM-Bilder von mit X-Rhod-1 gefärbten Erythrozyten mit niedrigem
oder hohem intrazellulärem Ca2+ zeigt unterschiedliche Lebensdauern, wobei das Fluoreszenzsignal
geringster Intensität von den Erythrozyten selbst ausgeht. Die Anzahl der
per Regression angepasster Lebensdauerkomponenten ist zwischen 1-Photonen-Anregung
und 2-Photonen-Anregung unterschiedlich. Eine unzureichende Fokussierung während
der Ablichtung kann die Auflösung der Anzahl der angepassten Lebenszeitkomponenten
beeinflussen. Auch die Umgebung und die Hb-Konzentration in den Erythrozyten können
die Lebenszeitkomponenten und die amplitudengemittelte Lebensdauer von X-Rhod-1
beeinflussen. Andere Faktoren wie Anregungswellenlänge, Aufnahmezeit und die Verwendung
verschiedener Farbstoffe wie CellTak und Ca2+-Ionophor Bromo-A23187 haben
keinen Einfluss auf die Lebenszeitparameter von X-Rhod-1. Mit den im Zuge dieser Analyse
bestimmten Messparametern ist die normalisierte amplitudengemittelte Lebensdauer
ein vielversprechender Parameter, der zur Erstellung einer Kalibrierungskurve für die
Ca2+-Quantifizierung verwendet werden kann