A comparative shotgun proteomics study on nasal epithelia from healthy restaurant workers

Funding Information: This work was supported by the Calouste Gulbenkian Foundation, Portuguese Central Administration of Nacional Health System, the Portuguese Rede Nacional de Espectrometria de Massa, the National Institute of Health Doctor Ricardo Jorge, and the Centre for Toxicogenomics and Human Health \u2013 ToxOmics from NOVA Medical School-FCM, UNL. SN and SP were granted with PhD fellowships from Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia. Funding Information: This work was supported by the Calouste Gulbenkian Foundation, Portuguese Central Administration of National Health System, the Portuguese Rede Nacional de Espectrometria de Massa, the National Institute of Health Dr Ricardo Jorge, and the Centre for Toxicogenomics and Human Health \u2013 ToxOmics from NOVA Medical School-FCM, UNL. SN and SP were granted with PhD fellowships from Portuguese Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia. Publisher Copyright: © 2024 The AuthorsNon-smokers exposed to second-hand smoke (SHS) present risk of developing tobacco smoke-associated pathologies. To investigate the airway molecular response to SHS exposure that could be used in health risk assessment, comparative shotgun proteomics was performed on nasal epithelium from a group of healthy restaurant workers, non-smokers (never and former) exposed and not exposed to SHS in the workplace. HIF1α-glycolytic targets (GAPDH, TPI) and proteins related to xenobiotic metabolism, cell proliferation and differentiation leading to cancer (ADH1C, TUBB4B, EEF2) showed significant modulation in non-smokers exposed. In never smokers exposed, enrichment of glutathione metabolism pathway and EEF2-regulating protein synthesis in genotoxic response were increased, while in former smokers exposed, proteins (LYZ, ATP1A1, SERPINB3) associated with tissue damage/regeneration, apoptosis inhibition and inflammation that may lead to asthma, COPD or cancer, were upregulated. The identified proteins are potential response and susceptibility/risk biomarkers for SHS exposure.publishersversionpublishe

Úloha črevných cirkadiánnych hodín v epiteliálnom transporte, proliferácii a tumorigenéze.

AABBSSTTRRAAKKTT Molekulárne cirkadiánne hodiny umožňujú predvídanie zmien v okolitom prostredí. U cicavcov sú molekulárne hodiny prítomné prakticky vo všetkých tkanivách a tvorí ich sústava transkripčno-translačných spätnoväzbových slučiek tzv. hodinových génov. Centrálne hodiny predstavujú vnútorný pacemaker, ktorý sa nachádza v suprachiazmatických jadrách (SCN) hypotalamu a synchronizuje periférne hodiny. Cirkadiánne hodiny v tráviacom trakte a ich podiel na regulácii črevných funkcií sú nedostatočne preskúmané. Preto bolo cieľom tejto práce charakterizovať molekulárne hodiny v jednotlivých častiach čreva potkana a objasniť ich úlohu v regulácii epiteliálneho transportu, bunkového cyklu a nádorovej transformácie v hrubom čreve. Na stanovenie cirkadiánnych profilov expresie hodinových génov v epiteli duodena, jejuna, ilea a hrubého čreva potkana sme použili kvantitatívnu RT-PCR (qPCR). Ďalej sme analyzovali expresiu génov kódujúcich transportéry a kanály umožňujúcich transport NaCl, ako aj regulátorov bunkového cyklu v hrubom čreve. Na detailnejší popis expresie v rámci štruktúr črevného epitelu sme použili laserovú mikrodisekciu. Elektrogénny transepiteliálny transport bol meraný ako skratovací prúd v Ussingových komorách. Na určenie úlohy hodín v procese tumorigenézy sme použili myší model azoxymetánom...AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...Katedra fyziologieDepartment of PhysiologyFaculty of SciencePřírodovědecká fakult

Primary aldosteronism: molecular medicine meets public health.

Primary aldosteronism is the most common single cause of hypertension and is potentially curable when only one adrenal gland is the culprit. The importance of primary aldosteronism to public health derives from its high prevalence but huge under-diagnosis (estimated to be <1% of all affected individuals), despite the consequences of poor blood pressure control by conventional therapy and enhanced cardiovascular risk. This state of affairs is attributable to the fact that the tools used for diagnosis or treatment are still those that originated in the 1970-1990s. Conversely, molecular discoveries have transformed our understanding of adrenal physiology and pathology. Many molecules and processes associated with constant adrenocortical renewal and interzonal metamorphosis also feature in aldosterone-producing adenomas and aldosterone-producing micronodules. The adrenal gland has one of the most significant rates of non-silent somatic mutations, with frequent selection of those driving autonomous aldosterone production, and distinct clinical presentations and outcomes for most genotypes. The disappearance of aldosterone synthesis and cells from most of the adult human zona glomerulosa is the likely driver of the mutational success that causes aldosterone-producing adenomas, but insights into the pathways that lead to constitutive aldosterone production and cell survival may open up opportunities for novel therapies

Úloha črevných cirkadiánnych hodín v epiteliálnom transporte, proliferácii a tumorigenéze.

AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...AABBSSTTRRAAKKTT Molekulárne cirkadiánne hodiny umožňujú predvídanie zmien v okolitom prostredí. U cicavcov sú molekulárne hodiny prítomné prakticky vo všetkých tkanivách a tvorí ich sústava transkripčno-translačných spätnoväzbových slučiek tzv. hodinových génov. Centrálne hodiny predstavujú vnútorný pacemaker, ktorý sa nachádza v suprachiazmatických jadrách (SCN) hypotalamu a synchronizuje periférne hodiny. Cirkadiánne hodiny v tráviacom trakte a ich podiel na regulácii črevných funkcií sú nedostatočne preskúmané. Preto bolo cieľom tejto práce charakterizovať molekulárne hodiny v jednotlivých častiach čreva potkana a objasniť ich úlohu v regulácii epiteliálneho transportu, bunkového cyklu a nádorovej transformácie v hrubom čreve. Na stanovenie cirkadiánnych profilov expresie hodinových génov v epiteli duodena, jejuna, ilea a hrubého čreva potkana sme použili kvantitatívnu RT-PCR (qPCR). Ďalej sme analyzovali expresiu génov kódujúcich transportéry a kanály umožňujúcich transport NaCl, ako aj regulátorov bunkového cyklu v hrubom čreve. Na detailnejší popis expresie v rámci štruktúr črevného epitelu sme použili laserovú mikrodisekciu. Elektrogénny transepiteliálny transport bol meraný ako skratovací prúd v Ussingových komorách. Na určenie úlohy hodín v procese tumorigenézy sme použili myší model azoxymetánom...Department of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Hypothalamic gene expression profiling in mouse strains susceptible or resistant to diet-induced obesity

Fettleibigkeit hat sich zu einem weltweiten Gesundheitsproblem in der Öffentlichkeit entwickelt. Sie wird durch ein komplexes Ungleichgewicht der Regulation von Appetit und Energiestoffwechsel verursacht, die durch verschiedene Faktoren wie genetische Defekte, Nahrungspräferenzen und Lebensstil kontrolliert werden. Die hochfetthaltige westliche Nahrung ist einer Hauptfaktor, die die Entwicklung von Fettleibigkeit in der menschlichen Bevölkerung fördert. Trotzdem werden nicht alle Konsumenten der Hochfettnahrung fettleibig. In dieser Studie wurden zwei unterschiedliche Mausinzuchtlinien – AKR/J und SWR/J – entweder mit einer hoch fetthaltigen Nahrung oder der Standardnahrung gefüttert. Der AKR/J Stamm repräsentiert ein Mausmodel für diät-induzierte Fettleibigkeit (diet-induced obesity = DIO). Mäuse dieses Stammes wurden fett wenn sie mit der hochfetthaltigen Diät gefüttert wurden, wohingegen sie schlank bei Fütterung mit der Standard-Diät blieben. Im Gegensatz dazu waren die Mäuse des SWR/J Stamm resistent gegenüber der DIO, d.h. es war im Vergleich kein wahrnehmbar Anstieg des Körpergewichts oder von Fettleibigkeit in Mäusen, die mit fetthaltiger Nahrung oder Standard-Diät gefüttert wurden. Genexpressions-Arrays wurden benutzt um differentiell exprimierte Gene im Hypothalamus von AKR/J und SWR/J Mäusen bei fetthaltiger Fütterung zu identifizieren. Um die Kandidatengene, ausgesucht aus der Array Datenanalyse to validieren, wurde Northern Blot Analyse, in situ Hybridisierung und real-time RT-PCR durchgeführt. Hämoglobin alpha, adult chain 1 (Hba-alpha1) ist auf dem Chromosom 11 der Maus (Chromosom 16p13.3 des Menschen) lokalisiert. Die funktionelle Bedeutung der Expression von Hba-alpha1 ist unbekannt. Eventuell erleichtert es den Sauerstofftransport im Gehirn in einer ähnlichen Weise wie das Myoglobin im Skelettmuskel. In dieser Arbeit wurde eine höhere ubiquitäre Expression von Hba-alpha1 im Hirn der SWR/J Maus im Vergleich zur AKR/J Maus beobachtet. Dieser Unterschied könnte mit der höheren Stoffwechselrate der SWR/J Mäuse zusammenhängen. So weit konnte keine direkte Beziehung zwischen Hba-alpha1 Expression und Fettleibigkeit hergestellt werden. Im Gegensatz dazu zeigt die Glyoxalase I (Glo 1) ein spezifisches Expressionsmuster mit stärkster Präsenz im Hippocampus. Im Hypothalamus kann die Glo1 Expression im arquatischen Nukleus (ARC), im ventromedialen hypothalamischen Nukleus (VMH) und im paraventricularen hypothalamischen Nukleus (PVN) detektiert werden. Während die Expression von Glo1 ausserhalb des Hypothalamus ähnlich in beiden Mausstämmen ist, ist die mRNA Expression in der hypothalamischen Region viel stärker in AKR/J im Vergleich zur SWR/J Mäusen. Das Glo1 Gen befindet sich auf Chromosom 17 der Maus (Chr. 6 des Menschen) und an der Entgiftung von Stoffwechselnebenprodukten beteiligt. Außerdem wurde Glo1 auf der Fettleibigkeits-Genkarte vom Menschen verzeichnet und vermutet eine Verbindung zwischen einer abweichenden Expression des Glyoxalase-Systems und Krankheiten wie Krebs und Diabetes. Tumor Nekrose Faktor alpha-induziertes Protein 1 (endothelial) (tumor necrosis factor alpha induced protein 1 (TNFAIP1) ist auf Maus-Chromosom 11(45,10 cM) und Mensch-Chromosom 17q22-q23 lokalisiert. Das Protein ist beim Kalium-Eisen-Transport durch Proteinbindung und bei der Einstellung der spannungsabhängigen Kaliumkanal Aktivitäten involviert. TNFAIP1 lokalisiert sich im ARC, im VMH und PVN. Es wurde durch Hochfett-Diäten in den AKR/J aber nicht SWR/J Mäusen hochreguliert, was an den Filterarrays und den Northern Blots, aber nicht mit der real-time RT-PCR und in situ Hybridisierungen gezeigt werden konnte. Obwohl bei der in situ Hybridisierung eine 1,6fache Steigerung der mRNA Expression im ARC und VMH durch die Hochfettdiät beobachtet werden konnte, war diese Steigerung aufgrund individueller Variationen nicht signifikant. Weitere Experimente mit höherer Stichprobenzahl müssten durchgeführt werden um dieses Ergebnis zu bestätigen. Weil es sich um ein neu annotiertes Gen handelt, ist nicht viel über die pathologische Relevanz bekannt. Bisher hat keine Studie eine Verbindung zwischen TNFAIP1 und Fettleibigkeit beschrieben. Es wird angenommen, dass TNFalpha einen Einfluss auf Körpergewichtsregulation hat und wahrscheinlich durch einen lokalen Prozess im Fettgewebe wirkt. Möglicherweise führt eine erhöhte Sekretion von TNFalpha aus Adipozyten in fettleibigen Versuchstieren/-personen zu einer Induktion von TNFAIP1 im Hypothalamus. Weitere Studien sollten durchgeführt werden um die Funktion von TNFAIP1 im Gehirn aufzuklären

Investigating the Role of Mineralocorticoid Receptor Signaling in Cancer Biology in the Genomic Era

In the last decades, advances that take place in the next-generation sequencing and bioinformatics research have helped reveal tissue- and cancer-specific gene expression patterns and mutation landscapes. Indeed, such data are now easily accessible via online genome browsers and different types and levels of public data compendia. Appropriate use of these tools eventually can lead to better patient stratification for diagnosis, prognosis, and therapy of cancers. Mineralocorticoid receptor (MR), encoded by NR3C2 gene, has long been implicated in the development and progression of multiple cancers. Nevertheless, MR has remained relatively understudied at the genomic and transcriptomic levels. In this review, we present the current, literature-based state of knowledge on the role of MR primarily in epithelial cancers. At the same time, we summarize the gene expression, mutation, and copy number variation data on MR obtained from The Cancer Genome Atlas (TCGA). We also show that MR expression could be a promising prognostic marker in different cancers using online tools for survival data analysis. Accordingly, this review strongly demonstrates the emerging potential of studying MR using available tools from the genomics/transcriptomics field for improving cancer diagnosis and prognostication

Understanding the functional involvement of ERɑ and hypoxia in the pathophysiology of breast cancers with different molecular subtypes

Breast cancer is the leading cause of cancer-related mortality for women. Dysregulated RNA polymerase III (Pol III) transcription of tRNA is significantly implicated in cancer progression. Oestrogen receptor alpha (ERɑ) potentiates ~75% of breast tumours, and resistance to endocrine therapies that target ERɑ activity is a major clinical problem urgently requiring significant advances in research to improve survival outcomes for women with endocrine-resistant disease. The ERɑ is a prolific transcription factor that upregulates many pro-tumorigenic genes, including tRNAs in response to hormone activation. This thesis sought to investigate the mechanism driving ERɑ-dependent tRNA expression. Analysis of public ChIP-seq data showed ERɑ was physically associated with ~50% of tRNA loci in breast cancer cells. ERɑ recruitment to tRNA genes was mediated by protein-protein interactions of ERɑ with Pol III-specific TFIIIC, determined by qPLEX-RIME and coimmunoprecipitation. FOXA1, a modulator of ERɑ activity, was enriched at tRNA promoters, suggesting FOXA1 may facilitate ERɑ recruitment to Pol III-transcribed genes and hormone-dependent activation of transcription. Further exploration of this ERɑ-FOXA1-Pol III axis could lead to novel and necessary developments in the treatment of advanced ERɑ-driven breast cancer. Altered Na+ homeostasis is a critical determinant of breast cancer progression. Intratumoral hypoxia is linked to disruption of many cellular processes, including ion transport, which has significant implications in therapy resistance and advanced disease. This thesis aimed to delineate hypoxia-dependent changes in Na+ transport. Optimisation of reference genes (RGs) for studying alterations in gene expression in hypoxic breast cancer cell lines by RT-qPCR identified RPLP1 and RPL27 as suitable RGs for such investigations. RNA-seq and RT-qPCR found hypoxia enhanced Na+ transporter gene expression in ERɑ+ breast cancer cell lines, particularly by upregulating Na+/K+- ATPase (NKA) and epithelial Na+ channel (ENaC), highlighting a new mechanism by which hypoxia may contribute to breast cancer progression and therapy resistance. Conversely, voltage gated Na+ channels (VGSCs) were not affected by low O2 tension, but ERɑ was shown to mediate expression of some VGSC isoforms. Understanding changes in Na+ handling in advanced breast cancer is imperative and may result in the development or repurposing of targeted therapies aimed at modulating Na+ transport to improve breast cancer outcomes