Assessment of circadian dysregulation as a pathological factor in non-communicable diseases and proposal of chronobiological interventions for their treatment

We propose involvement in projects on:

1. Timing in patients

We can determine an accurate circadian phase of a patient (limited n due to cost and labor-intensive approach, requires repeated sampling in in dim light) from saliva or plasma and analyzing DLMO melatonin (ELISA/RIA), or by isolating mRNA from buccal swabs and analyzing clock gene expression by RT qPCR. We can isolate live fibroblasts from skin biopsy, transduce with circadian luminescence marker (Bmal1-Luc delivered by lentivirus) and analyze period of peripheral clock (limited n due to cost and labor-intensive approach). We can provide MCTQ questionnaires (e.g. online form), analyze chronotype and social jetlag of patients (unlimited n) and correlate with other variables (e.g. diagnosis).

2. Dosing time effects

We can test drugs in circadian reporter cell lines, tissue explants (e.g. SCN) from Per2Luc mice, or fibroblasts from skin biopsies and look for circadian effects.

We can test dosing time effects in vitro or in vivo (mice, rats) by analyzing gene expression (RNAseq, RT qPCR), metabolome, or other parameters (in collaboration by sending samples).

3. Circadian biomarkers

We can correlate biomarker data with chronotype, sleep or other circadian parameters of patients (e.g. data from online questionnaire).

We offer expertise and skills

(A) time-resoved omics experiments – planning sampling from patients/animals/cells for a successful experiment, isolating RNA for RNAseq, managing transcriptomic (Biocev, Prague), proteomic (IPHYS, Prague) or metabolomic (IPHYS, Prague) analysis of samples within our service facilities, general bioinformatics and specialized time-resolved analyses to generate final data, including advanced plotting and statistics.

Recent publication:

Sládek M., Houdek P., Čajka T., Sumová A. PLoS Biol. 2025 Sep 26;23(9):e3003404. doi: 10.1371/journal.pbio.3003404.

(B) population studies – power analysis, preparation of specialized online questionnaires (either Google forms or programmed interactive version that provides feedback to the subject such as calculation of chronotype and social jetlag using iMCTQ), analysis of medium or large datasets, including multivariable regression models, visualization of final data.

Recent publications:

Sládek M., Klusáček J., Hamplová D., Sumová A. Sleep. 2023 Jun 13;46(6):zsad037. doi: 10.1093/sleep/zsad037.

Sládek M., Kudrnáčová Röschová M., Adámková V., Hamplová D., Sumová A. Sci. Rep. 2020, 10:1419, DOI: :10.1038/s41598-020-58413-9

(C) real time circadian luminescence monitoring of clock gene expression – preparation of reporter cell lines, including primary human cells transfected by lentiviral circadian reporters, PER2::LUC mice and preparation of explants to monitor circadian clock in specific tissue (SCN, choroid plexus, peripheral tissues), circadian luminescence microscopy on Luminoview LV200 (Olympus) including expertise in time-resolved image analysis and corresponding statistics.

Publications:

Nováková M., Sládek M., Sumová A. Chronobiol. Int. 30(4): 607-617, 2013, doi: 10.3109/07420528.2012.754455

Nováková M., Praško J., Látalová K., Sládek M., Sumová A. Bipolar Disorders, 17: 303-314, 2015, doi: 10.1111/bdi.12270

Sládek M., Houdek P., Myung J., Semenovykh K., Dočkal T., Sumová A. Fluids Barriers CNS. 2024 May 27;21(1):46. doi: 10.1186/s12987-024-00547-3

Expertise overview:

Experimental methods: molecular biology – RT qPCR, transfection or lentiviral/AAV transduction of cells/tissue explants with fluorescence or bioluminescence markers, plasmid cloning, access to various omics services; biochemistry – detection of proteins and hormones by RIA/ELISA/LUMINEX or IHC/Western blot; sample preparation – laser-capture microdissection, cryocut (frozen tissues), vibratome (live tissues), RNA isolation, stereotaxic surgery; fluorescence (confocal) and bioluminescence (Luminoview LV200) microscopy; long-term circadian luminescence detection by photomultiplier tubes (Lumicycle) and 384-well plate reader

Statistics and data analysis: time-series analysis of image, luminescence or omics data, including rhythm detection; analysis of large datasets (e.g. questionnaires) including multidimensional regression; Python-based data processing and visualization.