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Synthesis and diversification of nitrogen and silicon heterocycles used as organic fluorophores
No full textWith the interest in photocatalytic transformations constantly increasing, the need of new catalysts is raising overproportionally. Transition metal polypyridyl catalysts have been the dominant class of catalysts for many years. However, with an increasing demand and the awareness for sustainability steadily growing, the demand for precious metal free alternatives was never greater. Acridinium salts have shown photophysical properties similar to those of polypyridyl transition metal catalysts and proven to be versatile and adequate alternatives with similar photophysical properties and nearly identical catalytic potential. Unfortunately, instability towards irradiation often limits their applications and reduces their efficiency. Herein, the scalable synthesis of acridinium salts with an enhanced photostability is reported. Tailormade precursors and an optimized catalyst formation through a 1,5-bifunctional lithium reagent allowed for the preparation of eleven 6- and 7-substituted asymmetric acridinium dyes from commercially available starting materials.
Precise optimization of the substrate synthesis led to the preparation of symmetric acridinium catalysts with an enhanced photostability by formation of a 1,5,5’ trifunctional reagent. A highly efficient tree step procedure synthetic route allowed the preparation of four symmetric photostable acridinium salts.
Further, the enhanced photostability could be demonstrated in Ni-catalyzed esterification, where the catalyst loading of both photocatalyst and Ni-catalyst could be significantly lowered from 10 mol% to more sustainable levels, coming closer to industrial requirements. After gaining this insight, we could demonstrate the versatility of photostable acridinium salts in a 96-wellplate screening setup that demonstrated to possibilities of base variations
1,5-Bifunctional reagents give access to a variety of heteroatom bearing fluorophores. Previously, we could demonstrate the formation of a symmetric silicon rhodamines from a 1,5-bifunctional reagent and an ester. Cryogenic mono-metalation allowed for the access of an unsymmetric 1,5 halogenated silane which after exposure to elemental magnesium could be transformed to its 1,5-bifunctional reagent that gave way to a new class of so far unknown unsymmetric silicon rhodamines. Compared to previous rhodamines, unsymmetric Si-rhodamines show a unique hypsochormic shift in absorption of about 200 nm compared to their symmetric analogous while maintaining similar emission properties
Affective Partisan Polarization and Citizens' Attitudes and Behavior in Swiss Democracy
Get PDFThere is a concern that citizens with different political positions and party affiliations increasingly dislike each other. We examine this affective polarization (AP), which is often associated with a weakening of democracy, in the context of Switzerland's multiparty landscape with proportional governmental representation. Evaluating the long-term development of AP in Switzerland with both historical and newly gathered data for 2023, we find hardly any considerable change in AP over the last three decades, except for a substantial jump between 1999 and 2003 and a generally lower level of party sympathy in 2023. Complementary, our analysis of split-ticket voting behavior in national parliamentary elections with continuous data back to 1983 does not support any trend in partisan polarization from a voters' revealed preference perspective. We further find that more affectively polarized individuals report, on average, lower satisfaction with democracy but show a higher willingness to participate in politics across a wide range of different forms of political engagement, even when controlling for individuals' general sympathy towards political parties
Assessing trends and determinants of childhood undernutrition among urban poor and slum children in Bangladesh
Get PDFThis thesis aims to contribute to better understanding about trends and drivers of childhood undernutrition affecting urban poor and slum children in Bangladesh—a rapidly urbanizing country with one of the highest child stunting burdens in the world. The first thesis manuscript used serial population-representative data from Bangladesh Demographic and Health Surveys spanning over nearly two decades (2000-2018), and explored reasons for disparities in childhood linear growth status between urban poor and non-poor children. The manuscript findings highlighted urban poor children—compared to rural or other urban children—as a disproportionately disadvantaged subgroup for child undernutrition, and elicited maternal background factors as among primary predictors of the intra-urban child linear growth gap. The second thesis manuscript used primary data collected in urban slums and explored the role of maternal work as a potential predictor of nutritional outcomes in slum children. It found that children of working mothers were nearly twice more likely to be stunted than children of non-working mothers, but that family structures and availability of childcare support to mothers moderated the association in these settings. The third thesis manuscript then explored the effects of the COVID-19 pandemic on nutritional status of urban slum children, using primary data collected before- and after- the first wave of the pandemic in Bangladesh. It found that the average nutritional status of slum children had generally not deteriorated over the one-year sample period, but variations were apparent by slum area and household migration status
Clinical prediction models in orthopaedics: from data collection to model development and validation
Get PDFRotator cuff disease affects up to 20% of the general population and is a major cause of shoulder pain and disability. Non-operative treatment is usually firstly counseled, but surgery, with the performance of arthroscopically assisted rotator cuff repair (ARCR) is often required. The lack of studies with proper methodology impairs health policy makers in drawing conclusions regarding the safety and the effectiveness of this procedure. In that context, the ARCR_Pred study, a nationwide multicenter cohort funded by the Swiss National Science Foundation and Swiss Orthopaedics, aimed to standardize outcomes, and develop clinical prediction models. This PhD thesis focuses on the implementation of the study, the description of overall and patient characteristics, and the development of prediction models for post-operative stiffness (POSS) and functional outcomes, notably through the patient-reported Oxford Shoulder Score (OSS). In the first thesis manuscript, the successful implementation of the ARCR_Pred study was highlighted: 973 patients were included from 18 Swiss and 1 German orthopedic centers between June 2020 and November 2021, achieving high follow-up rates. After consideration of case-mix variables, the analysis showed no clinically relevant outcome differences between public and private clinics at last follow-up. The second manuscript is a systematic review summarizing the evidence related to prognostic factors for POSS. However, the low quality of the summarized evidence highlighted the need to complement our findings with an expert opinion. A Delphi process was therefore initiated and based on these results, three models were built and compared in the third manuscript. The ARCR_Pred-POSS model had the best discrimination and calibration performance (AUC=0.719 and calibration slope=1.022). Decision-curve analysis showed superior performance of the ARCR_Pred-POSS model in comparison to the surgeon prediction of POSS over the whole range of threshold probabilities. At the risk probability threshold 15%, the model showed a sensitivity of 57% and a specificity of 73%. A user-friendly responsive web-based application was developed. Regarding the prediction of the OSS, the second systematic review identified 22 potential prognostic factors for the improvement of shoulder function after an ARCR, which correspond to the fourth and last manuscript of the thesis. These findings were also completed by an expert opinion involving 52 healthcare professionals (including surgeons and experienced physiotherapists), which led to the identification of 101 factors. The ARCR_Pred-OSS model remains to be developed and validated on the ARCR_Pred data, which would add a patient-relevant outcome to the ARCR_Pred prediction tool. The evaluation of the impact of the use of such prediction tools on surgical and patient-reported outcomes remains also to be assessed. Model optimization should first be sought, by assessing the predictive ability of new factors and modern modeling techniques. Prediction tools in surgery should also incorporate patient and healthcare professionals’ preferences
The role of sleep restriction, light intensity, and chronotype for alertness, mental effort, and cognition
Get PDFShort sleep duration at night and prolonged exposure to artificial light throughout the day are part of today’s lifestyle for many people. Sleep duration as well as light conditions can influence alertness. According to a proposed model (Lasauskaite & Cajochen, 2018) based on Brehm’s motivation intensity theory (Brehm & Self, 1989), alertness would influence perceived task demand, which in turn determines mental effort. Effort is the mobilization of resources to carry out instrumental behavior and expected to manifest in the sympathetic beta-adrenergic impact on the heart, indexed through cardiac pre-ejection period (PEP), but also systolic blood pressure (SBP). In the PhD project presented in this thesis, I have summarized existing
literature on effects of one night of sleep restriction on alertness and cognition and investigated effects of sleep restriction and light intensity on mental effort during a cognitive challenge.
In a series of meta-analyses, I summarized 44 studies that investigated alertness or cognitive performance after one night of sleep restriction (chapter 4). Separate analyses were run for subjective sleepiness, sustained attention, choice reaction time tasks, cognitive throughput, working memory, and inhibitory control, and, if available, for reaction time and accuracy. Significant increases in subjective sleepiness were observed (p .073).
In two experimental studies, I investigated the influence of sleep duration and light intensity on mental effort (chapter 5). Therefore, 40 participants (24 women) were recruited for Study 1 and 24 participants (16 women) for Study 2. The experimental protocol for both studies included a sleep-duration manipulation (5 h vs. 8 h, within-subjects) during the night before an experimental session in the morning. In Study 1, experimental sessions consisted of an 8-min cardiovascular baseline, 15-min exposure to the experimental light condition (1 lux vs. 65 lux at eye level, between-subjects), and a 5-min auditory 2-back task. For Study 2, a 5-min Karolinska Drowsiness Test was added after the baseline period and each experimental session then had two blocks of 15-min light exposure (100 lux vs. 500 lux, within-subjects) and 5-min modified auditory Sternberg task each. Cardiovascular measurements were recorded throughout the entire experimental sessions. In Study 1, mental effort, reflected through cardiovascular reactivity, was not significantly influenced by sleep duration nor light intensity. Mental effort during the first minute of task performance was higher in the participants who reported lower alertness (p = .041), suggesting disengagement from task performance in participants reporting high alertness. Study 2 showed stronger effort after sleep restriction,
indicated through SBP and DBP reactivity (ps < .001). PEP response was stronger in the higher light intensity condition (p = .032). These studies, for the first time investigated mental effort during a cognitive challenge after experimentally manipulated sleep duration and in different light intensities.
Chronotypes indicate a preference for a certain time of day, going along with sleep timing and the circadian clock, which is indicated by the time of evening melatonin onset. Early types exert less effort in a morning cognitive challenge, which corresponds to their optimal time, compared to an afternoon challenge (Carbajal et al., 2019). In an additional explorative analysis on Study 1, I investigated effects of chronotypes, determined from self-reported sleep time and from timing of melatonin onset, on mental effort during a cognitive challenge in the morning (chapter 6). Applying a Bayesian statistical approach, results indicated inconclusive results for PEP (BF10 = 0.631) and task performance (BF10 = 0.814), and no differences in SBP (BF10 = 0.268) between sleep onset-derived chronotypes. Chronotypes derived from timing of melatonin onset did not differ in alertness, task performance, or mental effort (BFs10 < 0.263), when the timing of the cognitive challenge was adapted to habitual sleep times, thus minimizing differences in circadian timing.
Taken together, results of the empirical studies suggest that sleep restriction would increase mental effort, but excessive sleepiness might result in disengagement from task performance. Effects appeared smaller than expected, therefore future studies should employ larger samples to prevent a lack of statistical power
The dynamic transcriptional landscape of intussusceptive angiogenesis
No full textVascular Endothelial Factor-A (VEGF) is the master regulator of vascular growth
in development and tissue repair and is the key target for therapeutic angiogenesis.
However, its therapeutic modes of action are still elusive. In fact, at therapeutic doses,
VEGF stimulates vascular growth in skeletal muscle through intussusception, also known
as splitting angiogenesis, rather than through the commonly studied process of
sprouting (1). A key feature of intussusception is the formation of endothelial cellular
processes projecting into the vascular lumen, termed intraluminal sprouts. These
projections from opposite sides of the vascular wall fuse to form transluminal pillars,
which then grow in circumference, merge laterally, and eventually divide the affected
vessel into two new ones (2). However, the specific functional cell states that endothelial
cells may adopt in response to VEGF in order to perform intussusceptive remodeling
remain unknown. Therefore, here we set out, for the first time, to capture such dynamic
and transient endothelial cell phenotypes by analyzing the transcriptomic changes
induced by VEGF delivery to skeletal muscle, at single-cell resolution and over time. In
particular we sought to:
1. Identify which endothelial communities are induced by VEGF delivery
2. Investigate the defining biological processes and markers of such communities, and
validate them in vivo
3. Seek intussusceptive-defining processes by comparing the identified functional
states of VEGF-induced intussusception against those of sprouting angiogenesis
C–H functionalization reactions catalyzed by artificial metalloenzymes
No full textCsingle bondH functionalization, a promising frontier in modern organic chemistry, facilitates the direct conversion of inert Csingle bondH bonds into many valuable functional groups. Despite its merits, traditional homogeneous catalysis, often faces challenges in efficiency, selectivity, and sustainability towards this transformation. In this context, artificial metalloenzymes (ArMs), resulting from the incorporation of a catalytically-competent metal cofactor within an evolvable protein scaffold, bridges the gap between the efficiency of enzymatic transformations and the versatility of transition metal catalysis. Accordingly, ArMs have emerged as attractive tools for various challenging catalytic transformations. Additionally, the coming of age of directed evolution has unlocked unprecedented avenues for optimizing enzymatic catalysis. Taking advantage of their genetically-encoded protein scaffold, ArMs have been evolved to catalyze various Csingle bondH functionalization reactions. This review delves into the recent developments of ArM-catalyzed Csingle bondH functionalization reactions, highlighting the benefits of engineering the second coordination sphere around a metal cofactor within a host protein
The molecular basis of the impact of GPCR phosphorylation on arrestin interactions and receptor endocytosis
No full textThis thesis describes the influence of G protein-coupled receptor (GPCR) phosphorylation on its interaction with arrestin2, and subsequent role in the formation of arrestin2 signaling complexes and receptor internalization.
Chapter 1 provides a general introduction to GPCRs and their intracellular partner proteins: G proteins, G protein-coupled receptor kinases (GRKs) and arrestins. It describes the role of phosphorylation motifs in the arrestin interaction, introduces biased signaling and receptor internalization. Furthermore, recent advancements in solving GPCR-GRK and GPCR-arrestin complex structures are presented.
Chapter 2 focuses on the functional dynamics of arrestin2. It provides a protocol for the stable expression and purification of arrestin2 and an advanced NMR analysis comprising amide backbone assignments and relaxation-dependent functional dynamics. This revealed pronounced micro- to millisecond motions of apo arrestin2, which may lie on the pathway to activation.
Chapter 3 describes the interaction of arrestin2 with various CC chemokine receptor 5 (CCR5) C-terminal tail phosphopeptides. The presented structural and functional analysis revealed a key phosphorylation motif, pXpp, responsible for robust arrestin2 recruitment. This motif is found in many other receptors and appears as one of the determinants of specific arrestin2 vs. arrestin3 recognition.
Chapter 4 presents insights into clathrin-mediated endocytosis (CME), which is the dominant mechanism of GPCR internalization. The interactions of arrestin2 with the main constituents of CME, the clathrin protein and the adaptor protein 2 (AP2), were investigated, the role of receptor phosphorylation in this process analyzed, and the in vitro results further validated through cellular functional assays.
Chapter 5 describes new findings on the molecular mechanism of arrestin-biased signaling in the 1-adrenergic receptor (1AR) by carvedilol. A structural and biochemical analysis of the 1AR-arrestin2 complex revealed that arrestin-biased signaling occurs by conformational exclusion of G protein binding, rather than by selection of arrestin2 binding.
Chapter 6 is a mini-review focusing on recent advances in solving GPCR-arrestin complex structures. The review also provides future directions of possible research to obtain a deeper understanding of arrestin signaling.
Chapter 7 describes attempts to form stable complexes of full-length CCR5 with the G protein receptor kinase GRK2 as well as arrestin2, and subsequent analyses of the complexes by negative-stain EM and cryo-EM.
Chapter 8 is a protocol for the formation of a stable complex of full-length CCR5 with the super-agonist chemokine [6P4]CCL5 and the Gi protein for cryo-EM analysis. Additionally, the chapter describes the backbone NMR assignment of [6P4]CCL5
When evolution meets drug discovery in "plasmodium falciparum"
No full textMalaria has plagued mankind for thousands of years, shaping the evolution of the human genome. Despite the availability of vaccines, the disease causes approximately 600`000 deaths annually and remains a global health burden. Malaria is caused by the Plasmodium spp., an apicomplexan parasite transmitted through the bite of Anopheles spp. mosquitos. The widespread use of artemisinin-based combination therapies have reduced malaria-related deaths. However, the emergence of drug resistance to current and new antimalarials underscores the urgent need for novel small-molecule chemotherapy with known mechanisms
of action.
The design of antimalarial chemotherapy relies on drugs that can selectively kill the parasite without harming human cells. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway represents a good antimalarial drug target because this pathway is present in the Plasmodium spp. but is absent in humans. Located in the relic plastid, the apicoplast, the MEP pathway contains seven enzymes prone to inhibition by small molecule drugs. This inspired the development of the MepAnti library, a library which contains 432 compounds derived from 12 structurally diverse scaffolds with potent activity against the enzymes of the MEP pathway. In this PhD project, I aimed to identify a novel chemical scaffold from the MepAnti library that can be used as a good chemical starting point for a next-generation antimalarial class.
I first screened the MepAnti library against the asexual blood stage Plasmodium falciparum through a combination of phenotypic and target-based screening. Four scaffolds were discovered to have potent activity against P. falciparum in vitro. Using a combination of isopentenyl pyrophosphate (IPP) chemical rescue and assessment of possible delayed death phenomenon, only the β-aza fosmidomycin analogues exhibited MEP pathway selectivity in P. falciparum . Moreover, I demonstrated that the three other scaffolds likely have targets outside the apicoplast. Taking into account antiplasmodial activity, cytotoxicity and physicochemical properties, I selected the hydroxy benzamides to progress for further target deconvolution studies.
We then established the integral solvent-induced protein precipitation (iSPP), a quantitative mass spectrometry-based proteomics technique that can be used for target-engagement studies in P. falciparum . The iSPP technique was validated in P. falciparum lysates by the observed target-engagement of four out of the six antimalarials tested. In addition, potential secondary targets were identified for two antimalarials. The iSPP is adaptable across lysates from different organisms and can be used as a complementary technique for target deconvolution studies.
Finally, I performed target deconvolution on the most potent derivative of the hydroxy benzamides, HIPS5367. In a first step, HIPS5367 was evaluated in a panel of strains that
exhibit resistance to standard and new antimalarials. HIPS5367 exhibited no cross-resistance in these strains. Three resistant lines were generated de novo from three independent flasks following resistance selection studies with HIPS5367. Whole genome sequencing of the parent and resistant lines, coupled with the analysis of variants identified in the mapped reads, led to the identification of point mutations in the P. falciparum multidrug resistance protein 1 (pfmdr1) gene. These mutations are unique in the resistant lines and were not found in the
parent line. With CRISPR/Cas9-based gene editing, one polymorphism was confirmed to be the primary mediator of HIPS5367 resistance. Even though this finding needs further
experimental validation, we have strong indications that these mutations in PfMDR1 likely result in the sequestration of HIPS5367 in the digestive vacuole, away from its primary site of action in the cytosol. In agreement to this, I demonstrated that HIPS5367 binds to cytosolic ribosomal subunits using the iSPP profiling. In line with this, the surface sensing of translation (SUnSET) assay demonstrated that this compound inhibits protein translation in P. falciparum . Hydroxy benzamides are a novel chemical class with a good safety window and represent a good chemical starting point for a next-generation antimalarial class.
The presented PhD thesis here underscores the value of using complementary techniques in drug discovery. These strategies will be instrumental in uncovering the mechanisms of action and resistance of new chemotypes, contributing to the development of next-generation antimalarials. Furthermore, this study emphasizes that compounds frequently target multiple proteins or pathways in the parasite. This multi-target strategy is beneficial in combating a parasite with a strong tendency for evolutionary adaptation