Projets de recherche

Code: 241A02MT

In sports drug testing, isotope ratio mass spectrometry is employed to enable the differentiation between endogenous and exogenous sources of urinary steroids, i.e. steroids produced inside the body or administered. Testosterone or testosterone-prohormones may be used by athlete for performance enhancement. As these steroids are endogenous, their presence alone in human urine is not suspicious. Only if urinary concentrations may exceed individual thresholds, a confirmation of the exogenous source becomes necessary and carbon isotope ratios proved to be the method of choice here.

Sample preparation protocols for isotope ratio mass spectrometry-based method are complicated and time-consuming as the purity of analytes is crucial and a prerequisite for valid isotope ratio determinations. Currently applied sample preparations encompass solid and liquid-liquid extraction steps to pre-purify and concentrate urine samples prior to the final clean-up step employing high performance liquid chromatography and fraction collection. This powerful tool for sample clean-up comes along with relatively long run-times per sample of more than 45 min and it may be necessary to use even a two-fold separation here.

This research project aims for improving this crucial step in sample preparation and shorten the run-times per sample by employing supercritical fluid chromatography coupled to a novel fraction collection tool. In supercritical fluid chromatography, pressurized carbon dioxide is used as solvent offering unique features for chromatographic separation not directly comparable to the commonly applied mixtures of water and organic solvent. Therefore, a novel sample preparation method suitable for isotope ratio mass spectrometry will be developed and validated within this project investigating the benefits of supercritical fluid chromatography in sports drug testing. To evaluate the new method and facilitate its implementation into routine doping controls, reference population-based values will be determined and compared to existing routine applications.

The aim of the project is to develop an anti-doping education framework for the World ParaVolley Association (WPV) For a complete list of National Federations involved, refer to World ParaVolley’s membership.

The proposed objectives will be achieved through a mixedmethods approach delineating action-oriented research. The mixed methods approach will be crucial to understanding the sports person's experience with NADA’s anti-doping educational awareness interventions and, thereby, the measures to improve them.

This project investigates the beliefs, attitudes, and awareness of doping among student-athletes in Singapore. A mixed methodologies of quantitative online surveys and qualitative interviews, focusing on student-athletes attending the National University of Singapore (NUS). In hopes to offer insight into Singapore student-athletes’ doping vulnerability, experience of the anti-doping system, and the factors shaping these aspects. Additionally, shedding light on nonsport influences on substance use among young people in larger society and how these influences might indirectly affect student-athletes’ doping attitudes in sport.

The proposed project will identify and characterize sportspecific stressors contributing to doping vulnerability among athletes, to include exploring their impact, frequency, severity, timing, and duration. Additionally, it seeks to investigate how psychological flexibility moderates the relationship between these stressors and doping vulnerability, thereby protecting athletes from doping.

A qualitative approach and informed by a co-design process with national anti-doping and sporting organizations. This project seeks to interview women in three countries to 1) investigate the role of support personnel (e.g., coaches, trainers, partners) in facilitating or preventing PIED use, and 2) to understand the broader psycho-socio-cultural factors that contribute to facilitating or preventing PIED use among female athletes. Investigating when, why, and how women choose to use or avoid PIEDs this project will gain important insights on women’s lived experiences with PIEDs.

A cross-sectional survey design and recruit a large sample of elite athletes (1500) from the said countries. The survey will include a number of validated screening tools in Spanish about self-compassion, mindfulness, sport anxiety, perfectionism, and doping susceptibility. Participants will also be invited to take part in a podcast series and to share their insights and experiences on doping in sport. In order to contribute towards more informed decision-making playing a crucial role in the prevention of doping behavior in the PANRADO Region, while focusing on a positive psychology approach emphasizing the role of mindfulness and selfcompassion.

Code: 23GD04KL

The abuse of gene transfer methods in sports is a real and growing concern, the more successes one realizes in gene therapy treatment of hereditary diseases, the more likely the abuse in healthy individuals becomes to improve sports performance. Although the administration manner remains unclear, one can speculate that the most likely method would be a local injection of the transgene(s). Published PCR based methods target cDNA sequences of these transgenes, more specifically the exon-exon junctions. In collaboration with the group of Haisma et al. (University of Groningen, Netherlands), we successfully transferred their published targeted NGS approach to detect exon-exon junctions of 5 potential doping genes to the UZ-Ghent NGS core. However, these proof-of-concept (POC) studies were performed for only a limited number of potential doping genes and with high quality DNA extracted from cell lines. The quality of the input material is similar as the quality from DNA extracted from a fresh blood sample. We would like to optimize this protocol on cfDNA extracted from urine samples so noninvasive samples can be obtained from an athlete. In the POC study, probes were designed to capture the exon-exon boundaries of all transcripts of EPO, GH1, GH2 and IGF. We will now expand the target region with additional potential doping genes, including the coding regions of these genes to evaluate other relevant gene doping marks. A genomic fingerprint will be added so samples can be assigned to correct individual. This method ensures the accurate identification and tracking of biological samples in various fields, including genetics, forensics, and clinical research. Single nucleotide Polymorphisms (SNPs) are genetic variations that involve a single nucleotide change at a specific position in the DNA sequence. They are commonly used as genetic markers due to their abundance and stability. These SNPs should exhibit high variability among individuals or populations to differentiate samples accurately. The unique genetic profiles generated by SNP genotyping allow for accurate sample identification, preventing errors and ensuring the integrity of data and results. In a second work package we will reinvent the data analysis part. Manual counting of the reads (as published by De Boer et al. 2019) is not feasible, especially not if the number of samples to be tested increase. We will further optimize the data analysis to an (semi) automated pipeline for read counting and program an additional module to take non-human traces and gDNA variants into account. Overall, these adjustments to the initial proof of concept study will prepare this analysis to be implemented as a gene doping test.

Code: 23A10XT

Isotope ratio mass spectrometry's (IRMS) efficiency for detecting pseudo-endogenous steroids depends on the difference between the isotopic labeling of the formulations available in the market and the natural isotopic labeling of the endogenously produced steroids by the athletes. The more frequent presence of pharmaceutical formulations with values close to the endogenous ones is becoming challenging and in some populations makes the current detection approach useless. This phenomenon already observed for testosterone preparations lead WADA to establish some countermeasures as the ABP endocrine module (for their indirect detection) or the analysis of the actual testosterone esters (the most frequent way of administration) in blood samples or dried blood spots (DBS). We are here proposing a study directed to verify the confirmation capacity of nandrolone esters in DBS or blood and the effect of the administration over the parameters of the endocrine module. This will be compared with the current approach by GC-MS/MS (for the Initial testing procedure- ITP) and GC-C-IRMS (for the confirmation procedure- CP). Formulations not detectable with the current approach will be included in the study.

Code: 23GD03HS

Gene doping is defined as “the non-therapeutic use of genes, genetic elements and/or modified cells that have the capacity to enhance athletic performance” and is categorized as a threat to the integrity of sport and the health of athletes by the World Anti-Doping Agency (WADA). The only direct approaches for detecting gene doping are PCR assays, so introducing a novel alternative approach for rapid and accurate identification of gene doping is vital. In this project, a novel ultrasensitive detection platform will be designed based on the combination of 2 different technologies i.e. CRISPR and Microfluidics. CRISPR/CAS systems can survey the whole genome and detect transgenic DNA (tDNA) using a single-stranded guide RNA (gRNA). In the presence of a target gene, a ternary complex (Cas12/gRNA/tDNA) will be formed and the quenched fluorescence probe will be degraded by collateral cleavage activity of the ternary complex. Microfluidics fluorescence detection can provide exquisite sensitivity. The combination of CRISPR/CAS as a biological gene doping detection system and a microfluidics system can provide multiplex gene doping detection (up to 96 samples with 96 detection assays per chip). Pre-amplification technologies such as recombinase polymerase amplification will be applied in order to reach single molecule (1 aM) sensitivity. Here, we will investigate more than 20 genes including CSH1, CSH1,CSH2, CSHL1, EPO, GDFB, GH1, GH2, IGF1, IGF2, MYOG, PPARd, VEGF, etc. as a proof of concept. However, the number of assay combinations could be 96 samples with 96 detection assays (96 different Cas/gRNAs). The gRNAs will be designed to target exon-exon junctions, specific sequences of viral vectors, such as CMVp (cytomegalovirus promoter), TkpA (thymidine kinase polyA from herpes simplex virus), hexon (major virus capsid protein from adenovirus), and CMVp (cytomegalovirus promoter) etc. The use of combinations of different gRNAs for the same gene (several gRNAs for different exon-exon junctions) with virus targeted-gRNAs will allow us to detect doping genes and different vectors simultaneously and dramatically increase the successful detection of tDNAs. Moreover, specific CRISPR-reference materials (C-RM) containing modified transgenic sequences will be designed to avoid false positive results due to cross-contamination. Based on the WADA guidelines for gene doping, appropriate no template controls (NTC) and positive template controls (PTC) will be used to determine the specificity, efficiency, sensitivity and limit of detection of the assay. The performance efficiency of the detection platforms will be tested in spiked blood samples. These multiplexed gene doping detection CRISPR-equipped microfluidic platforms could be an ideal and rapidly deployable detection approach, which is accurate, inexpensive and rapid, allowing for anti-gene doping assays in WADA-accredited laboratories.