1,740 research outputs found

    Advanced glycation end products and age-related diseases in the general population

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    In this thesis, epidemiological, nutritional, and gut microbiome related studies are presented to illustrate the relation of advanced glycation end products (AGEs) with age-related diseases. The studies are embedded in the Rotterdam Study, a cohort of the Dutch general population of middle-aged and elderly adults. The amount of skin AGEs measured as SAF was used as a representative of the long-term AGE burden. Chapter 1 gives an overview of the whole thesis (Section 1.1) and gives a brief introduction to AGEs and their implications in disease pathophysiology. Chapter 2 focuses on the interplay of AGEs in the skin and clinical and lifestyle factors, and Chapter 3 concerns the link of skin and dietary AGEs with age-related diseases. Chapter 4 discusses the interpretations and implications of the findings, major methodological considerations, and pressing questions for future research

    Advanced glycation end products and age-related diseases in the general population

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    In this thesis, epidemiological, nutritional, and gut microbiome related studies are presented to illustrate the relation of advanced glycation end products (AGEs) with age-related diseases. The studies are embedded in the Rotterdam Study, a cohort of the Dutch general population of middle-aged and elderly adults. The amount of skin AGEs measured as SAF was used as a representative of the long-term AGE burden. Chapter 1 gives an overview of the whole thesis (Section 1.1) and gives a brief introduction to AGEs and their implications in disease pathophysiology. Chapter 2 focuses on the interplay of AGEs in the skin and clinical and lifestyle factors, and Chapter 3 concerns the link of skin and dietary AGEs with age-related diseases. Chapter 4 discusses the interpretations and implications of the findings, major methodological considerations, and pressing questions for future research

    Exploiting molecular vulnerabilities in genetically defined lung cancer models

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    Lung cancer is the leading cause of cancer-related death worldwide, with approximately 1.8 million deaths in 2020. Based on histology, lung cancer is divided into non-small cell lung cancer (NSCLC) (85 %) and small cell lung cancer (SCLC) (15 %). The most common types of NSCLC are lung squamous cell carcinoma (LUSC), large-cell carcinoma (LCC), and lung adenocarcinoma (LUAD). LUAD, the largest subgroup of NSCLC, is characterized by genomic alterations in oncogenic driver genes such as KRAS or EGFR. Mutations in the kinase domain of EGFR result in aberrant signaling activation and subsequent cancer development. Tyrosine kinase inhibitors (TKIs) selectively target and inhibit mutant kinases, thereby killing oncogene-addicted cancer cells. The introduction of TKIs into clinical practice shifted NSCLC treatment from cytotoxic chemotherapy towards precision medicine, improving both survival and the quality of life during therapy. Patients with canonical EGFR mutations like the point-mutation L858R or exon 19 deletions mutations, which account for the majority of EGFR mutations, respond well to EGFR targeted TKIs. However, rare mutations like insertions in exon 20 insertions still represent challenging drug targets. C-helix‚Äď4-loop insertion mutations in exon 20 push the C-helix into the active, inward position without altering the binding site for TKIs. This leaves the binding site for TKIs in kinases with exon 20ins mutations highly similar to wild type (WT) EGFR. Thus, the challenge in the development of exon 20 inhibitors is the design of wild type sparing small molecules. Here, we analyzed a novel small molecule EGFR inhibitor (LDC0496) targeting an emerging cleft in exon 20-mutated EGFR to achieve selectivity over the wild type. In contrast to classical EGFR TKIs, LDC0496 reduces the cellular viability of EGFR exon 20 mutated cells but spares wild type EGFR. Targeted therapy inevitably results in the development of on- or off-target resistance. Drug induced resistance mutations require the constant development of novel drugs targeting the diverse landscape of resistance mechanisms. We detected BRAF mutations in EGFR-driven lung cancer patients as a resistance mechanism to EGFR inhibitors. Notably, we also detected co-occurrence of EGFR and BRAF mutations before treatment start. Combination treatment of EGFR and mitogen-activated protein kinase kinase (MEK) inhibition displayed activity in BRAF- and EGFR-mutated xenograft studies, therefore providing a treatment strategy to overcome BRAF mutation as a resistance mechanism. Compared to NSCLC, SCLC lacks druggable targets and the initial chemosensitive state rapidly turns into a chemoresistance state. SCLC is genetically defined by a biallelic loss of tumor suppressors RB1 and TP53 and alterations of MYC family members. The transcription factor MYC is a challenging target that cannot be directly targeted. Therefore, alternative strategies are needed, for example targeting its co-factors, such as the MYC-interacting zinc finger protein 1 (MIZ1). To study the complex interplay of Myc‚ÄďMiz1 in SCLC, we developed a novel mouse model with a truncated Miz1, which is unable to stably bind chromatin (RPMM: Rb1fl/flTrp53fl/flMycLSL/LSLMIZ1‚ąÜPOZfl/fl). Compared to Miz1 wild type the characterization of the novel mouse model revealed tumor-onset, localization, size and immune infiltration to be unaffected by the ablation of the Miz1-POZ domain, but mice with Miz1-‚ąÜPOZ live longer, exhibit an increased number of apoptotic cells and are more sensitive towards chemotherapy. We found that truncated Miz1 alter SCLC tumorigenesis towards a less aggressive phenotype and prolongs the chemosensitive state. Our study highlights alternative strategies to define novel vulnerabilities and options to overcome chemoresistance

    Molecular profiling of sex-specific podocyte stress response in response to mTOR inhibition

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    Strong sexual dimorphism exists in many kidney diseases, progression to End-Stage Kidney Disease and renal graft functions. However, molecular mechanisms remain poorly understood. Podocytes are the most important cell-type of the glomerulus regulating integrity and function of the glomerular filtration barrier. Sex differences in their molecular components and functional stress responses might be crucial for these clinically relevant sex differences. Therefore, these cells were chosen as targets for the following study investigating intrinsic sexual dimorphism on genomic and proteomic levels and sex-specific differences in response to mTOR inhibition by rapamycin in an in vivo stress model in mice. Metabolomic studies of renal cortex tissues from the same experimental groups were included to investigate the functional consequences of sexual dimorphic molecular signatures. Male and female ROSAmT/mG-NHPS2(podocin)Cre mice with GFP-expression restricted to podocytes were treated with rapamycin or vehicle for three weeks. Renal functional parameters were monitored using metabolic cages prior to sacrifice. The genetic mouse model allowed isolation of highly pure podocytes by sequential perfusion of kidneys and fluorescence activated cell sorting after tissue digestion. Kidneys were perfused for subsequent podocyte isolations for sequencing and proteomics or directly snap frozen for histology and metabolomics. qRT-PCR, western blots and immunohistological stainings were performed for validation of omics studies and further molecular characterizations. Although kidney function and morphology remained normal in all experimental groups, RNA sequencing revealed strong intrinsic sex-differences with more than 1700 mRNA transcripts significantly sex-differently expressed. Furthermore, stress responses due to mTOR inhibition displayed sexual dimorphism in transcriptomes and metabolomes and signaling differences as assessed by western blots. Gene set enrichment analyses of sequencing data pointed to sex-differences in enriched GOs related to transcription, cytoskeleton and focal adhesion in male and mitochondria and translation in female podocytes. Proteomics displayed in addition to significant enrichment in mitochondrial proteins increased abundance of basal cell proteins in females. Rapamycin treatment abolished significant sex differences, yet rather due to increased changes in male podocytes whereas the female transcriptome remained more stable towards treatment-induced changes. Metabolomics further supported this finding that females appeared to be less sensitive towards mTOR inhibition by showing less accumulation of amino acid pool and glycolysis metabolites after rapamycin. In conclusion, for the first-time podocyte-specific sexual dimorphism in gene regulation and protein expression patterns were identified together with sex-different metabolic changes in stress responses, which might contribute significantly to sex differences in renal disease susceptibilities and progression.Viele Nierenerkrankungen, die zeitliche Entwicklung hin zum terminalen Nierenversagen, sowie die Funktion von Nierentransplantaten weisen einen starken sexuellen Dimorphismus auf. Die zugrundeliegenden molekularen Mechanismen sind jedoch weiterhin nur unzureichend erforscht. Podozyten sind die wichtigsten glomerul√§ren Zellen, die die Integrit√§t und Funktion des glomerul√§ren Filters regulieren. Geschlechterunterschiede in ihren molekularen Komponenten und funktionellen Stressantworten k√∂nnten daf√ľr eine entscheidende Rolle spielen. Daher wurden diese Zellen in der folgenden Arbeit ausgew√§hlt, um intrinsische Geschlechterunterschiede auf Genom- und Proteomebene, sowie sex-spezifische Unterschiede in der Stressantwort durch mTOR-Signalweghemmung mittels Rapamyzin in einem in vivo Mausmodell zu untersuchen. Um Auswirkungen auf den Metabolismus zu adressieren, wurden die Studien durch Metabolomics von Nierenkortexgewebe der gleichen experimentellen Gruppen erg√§nzt. M√§nnliche und weibliche ROSAmT/mG-NHPS2(podocin)Cre-M√§use mit spezifischer GFP-Expression auf Podozyten wurden 3 Wochen mit Rapamyzin oder Vehikel behandelt. Metabolische K√§fige erm√∂glichten eine genaue Bestimmung der Nierenfunktionsparameter. Aufgrund des genetischen Mausmodells konnten spezifisch reine Podozytenfraktionen nach Nierenperfusion, Verdauungsschritten und fluoreszenzaktiviertem Zellsortieren isoliert werden. Diese wurden f√ľr Sequenzierungen und Proteomics verwendet. Weitere Nieren wurden f√ľr histologische Analysen und Metabolomics direkt schockgefroren.qRT-PCR, Westernblots and immunohistologische F√§rbungen wurden zur Validierung der Omicsstudien und f√ľr weitere molekulare Charakterisierungen eingesetzt. Trotz normaler Nierenfunktion und Nierenmorphologie bei allen experimentellen Gruppen fanden sich bei der RNA-Sequenzierung hochsignifikante Geschlechterunterschiede bei mehr als 1700 mRNA-Transkripten. Ver√§nderungen induziert durch die Stressantwort auf mTOR-Hemmung wiesen ebenso sexuellen Dimorphismus im Transkriptom und Metabolom auf, sowie Signalwegsunterschiede, wie mithilfe von Westernblots gezeigt werden konnte. Gengruppen-Enrichment Analysen deuteten auf Geschlechterunterschiede in angereicherten Genontologien f√ľr Transkription, Zytoskelett und Fokale Adh√§sionen in m√§nnlichen und mitochondrialer Proteine und Translation in weiblichen Podozyten hin. Proteomics wies zus√§tzlich zu mitochondrialen Proteinen eine Anreicherung von basalen Zellproteinen bei weiblichen Podozyten auf. Rapamyzin beseitigte die signifikanten Geschlechterunterschiede, allerdings eher aufgrund erh√∂hter Ver√§nderungen bei m√§nnlichen Podozyten bei insgesamt stabilerem weiblichen Transkriptom. Metabolomics best√§tigte dieses sex-differente Verhalten in Bezug auf Rapamyzinbehandlung. Weibliche Podozyten erschienen stressresistenter und zeigten eine geringere Akkumulation im Aminos√§urenpool und von Metaboliten der Glykolyse als m√§nnliche Podozyten. Zusammenfassend konnte in dieser Arbeit erstmals gezeigt werden, dass Podozyten signifikante Unterschiede in der Genregulation und Proteinexpression aufweisen zusammen mit geschlechtsspezifischen metabolischen Stressanpassungen, was entscheidend zu den Geschlechterunterschieden der Entwicklung von Nierenerkrankungen und deren Progress beitragen k√∂nnte

    The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

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    Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

    Deciphering the vascular disease mechanisms underlying hypertensive disorders of pregnancy

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    Summary. As the prevalence of hypertensive orders increases, particularly pre-eclampsia superimposed on a background of chronic hypertension (SPE), so too does the global disease burden they represent. Not only are these disorders detrimental to both mother and child during the course of pregnancy, they also have lasting longterm effects to future cardiovascular health for both. Despite this, relatively little is known about the generation and development of these multifactorial disorders. In combination with the effects of potential treatments to offspring health, the resultant scope for therapeutic interventions is severely limited. The stroke-prone spontaneously hypertensive (SHRSP) rat is an established model of chronic hypertension during pregnancy that can be further stressed by infusion of angiotensin II (ANGII) in mid-gestation to create a pre-eclamptic phenotype that closely mimics the clinical manifestation of SPE in humans. This thesis aimed to optimise and characterise a novel rodent model of SPE to provide a useful tool in understanding the underlying pathophysiology of the condition and in testing potential therapeutic strategies. The objectives were to assess the maternal, fetoplacental and neonatal response to SPE development in the SHRSP; evaluate the use of magnesium sulphate (MgSO4) as a preventative therapeutic in the context of SPE; investigate the underlying genetic mechanisms that may influence abnormal uterine artery remodelling due to maternal hypertension; and, finally, to validate these genetic mechanisms in vitro. A variety of in vivo and ex vivo techniques were employed in the generation and assessment of the optimised SPE rodent model. Pregnant SHRSP dams infused with 750ng/kg/min ANGII were found to exhibit signs of impaired maternal cardiovascular, renal and placental function alongside the abnormal uterine artery remodelling already characteristic of the SHRSP. Further, the offspring of these dams were more likely to be growth restricted and preliminary evidence suggested neonatal gene expression may be altered. When MgSO4 was administered in a preventative capacity in daily drinking water, it was shown to improve maternal blood pressure, proteinuria and weight. However, MgSO4 was ineffective at improving maternal cardiac function or uteroplacental flow and was observed to worsen fetal growth restriction. To better understand the maternally-derived factors in this impaired uterine artery remodelling associated with hypertensive pregnancy, RNA-sequencing was used to assess the genetic profiles of early pregnancy uterine arteries in SHRSP and normotensive WKY. Though the two strains shared a conserved response to pregnancy, there were striking differences in pathways related to vascular function, notably reactive oxygen species (ROS) production and calcium (Ca2+) signalling. Finally, using a combination of whole uterine arteries and vascular smooth muscle cells (UAVSMCs) derived from them, the gene expression patterns relating to ROS and Ca2+ were investigated to validate them. Though studies were preliminary and sample sizes small, there was evidence of altered ROS production, NOX subunit expression and UAVSMC Ca2+ release between WKY and SHRSP. This work has provided information on an optimised, novel rodent model of superimposed pre-eclampsia that may be used as a potential tool in investigating the pathophysiology of the condition or in assessing the long-term consequences of an adverse in utero environment. It has also deepened our understanding of the effects of prolonged MgSO4 exposure during pregnancy and highlighted the need for an optimised, standardised dosing regime in humans. Furthermore, this work has generated novel insights into the genetic factors that influence uterine artery remodelling and their functional consequences in early hypertensive pregnancy

    Strategies to Improve Antineoplastic Activity of Drugs in Cancer Progression

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    The aim of this Special Issue is to collect reports regarding all the recent strategies, directed at the improvement of antineoplastic activity of drugs in cancer progression, engaging all the expertise needed for the development of new anticancer drugs: medicinal chemistry, pharmacology, molecular biology, and computational and drug delivery studies
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