5 May 2024
Job Information
- Organisation/Company
AMU Institut de chimie radicalaire- Research Field
Chemistry
Chemistry » Biochemistry
Technology » Materials technology- Researcher Profile
Recognised Researcher (R2)
Leading Researcher (R4)
First Stage Researcher (R1)
Established Researcher (R3)- Country
France- Application Deadline
30 Jun 2024 - 22:00 (UTC)- Type of Contract
Temporary- Job Status
Full-time- Offer Starting Date
1 Oct 2024- Is the job funded through the EU Research Framework Programme?
Not funded by an EU programme- Is the Job related to staff position within a Research Infrastructure?
No
Offer Description
Introduction-Context
The generation of free radicals in biological systems was discovered about 60 years ago. Radicals are chemical species with an unpaired electron resulting in a high reactivity and thus, a short lifetime. Cellular radicals are involved in physiological processes regulating redox signalling or immune defense. Increased production of free radicals may, however, result in the structural damage to biomolecules, leading to lipid peroxidation, posttranslational modification of proteins, and DNA damage. Among the biological radicals, superoxide radical anion (O2 •– ) is the primary radical species that can initiate the reactive oxygen species (ROS) cascade. There are several recognized pathways of cellular O2 •– production, both enzymatic (e.g., from mitochondrial OXPHOS proteins, NADPH oxidases) and non-enzymatic (e.g., radiation, photochemistry, xenobiotics).1 ,2
Increased generation of mitochondrial O2 •– has been implicated in numerous diseases, including neurodegeneration, cardiovascular diseases and cancer. However, in most cases, it is experimentally challenging to define the actual mechanistic role of O2 •– . The inability to selectively detect O2 •– or to modulate its production are clear limiting factors. In fact, for most pathologies the mechanisms involved and the role of ROS remain not fully understood. The discrepancy between many reports on the role of ROS in a specific disorder may be attributed to differences in: (i) the species detected (identity ); (ii) the amount (level ), and (iii) the localization of ROS production at the subcellular level (location ). Due to the transient nature of ROS, addressing those three variables is not trivial and requires the development of novel tools for rigorous detection, identification, and quantitative analyses of ROS at the subcellular level. Despite the advances in the understanding of the chemical biology of ROS and tremendous effort devoted to the development of sensors for ROS, reliable detection and quantification of O2 •– remain a challenge.
Objectives: There are several scientific and technical barriers in the detection of subcellular O2 •– . Mitochondrial O2 •– detection and quantification remain a significant conundrum in redox biology which hamper the progress in our understanding of mitochondrial redox biology. New tools for modulation of mitochondrial O2 •– production and for the specific detection of O2 •– at the subcellular level are desperately needed to understand its role in cancer cell proliferation and for the development of novel redox-based anticancer strategies.3 Rigorous detection of superoxide requires the use of probes that produce specific, marker products formed only in the presence of superoxide.4,5 These include cyclic nitrone spin traps (e.g., DIPPMPO) and hydroethidine (HE) fluorogenic probe. Laboratories of Drs. Hardy and Zielonka have been involved in the development and characterization of mitochondria-targeted probes, including mitochondria-targeted spin traps or HE, as evidenced by numerous joint publications. This PhD thesis project will also benefit from both labs’ experience gained in previous studies on the development of novel mitochondria-targeted agents, to delineate the role of mitochondrial O2 •– in cancer cell proliferation and anticancer therapies.6-8
This proposed thesis is aimed at developing new chemical biology tools to detect mitochondrial superoxide in cell-free and cellular systems. The developed chemical probes will be applied to the studies of the role of mitochondrial superoxide in cancer cell proliferation and in anticancer strategies.
Methodology
- Aim 1. Preparation of nanoparticles and mitochondria-targeted nanoparticles precursors
Aim 1 of the thesis will focus on the synthesis of biologically compatible bis-functionalized hybrid mesoporous silica nanoparticles (MSNs) allowing the grafting of the probes (Aims 2 & 3) knowing the accessibility to the porosity. MSNs have been used previously as nanocarriers for mitochondrial drug delivery, 9 but few studies proposed their use for ROS detection. The triphenylphosphonium (TPP) cationic moiety is one of the most adaptable targeting group used to conjugate molecule of interest to lipophilic cations to target the molecule/particle to mitochondria.10 Aim 1 will involve preparation and rigorous characterization of MSNs with the spin traps and redox probes grafted inside and mitochondria-targeted MSNs decorated with mitochondria targeting TPP moieties outside (Fig. 1), to be used in Aims 2-4 of this project.
- Aim 2. Develop Nano-SpinTrap and the Nano-BOOST technique
Aim 2 will involve preparation of mitochondria-targeted nanoparticle where the spin trap is anchored inside the pores of the silica (Fig. 2) and evaluation of its performance by EPR spectrometry in cells stimulated to produce O2 •– . Next, the Nano-BOOST technique will be developed, to extend the applicability of the BOOST (boronate oxidation-spin trapping) assay to intact and permeabilized cells and isolated mitochondria. One of the limitations of the BOOST assay is the reduction of the O2 •– adduct (DIPPMPO-OOH) into the • OH adduct (DIPPMPO-OH) by GSH before its reaction with the boronate probe. Nano-spin trap (prepared in Aim 1) will be used to stabilize DIPPMPO-OOH. Combination of the Nano-spin trap with the boronate probes will enable the extension of the BOOST technique to intact cells, where the reduction of ‘free’ DIPPMPO-OOH to DIPPMPO-OH limits the utility of the technique. The combination of the particle-bound spin traps with boronate probes for O2 •– detection will provide an exciting opportunity to selectively monitor cellular O2 •– using fluorescence-based detection.
- Aim 3. Develop a Superoxide kit
Aim 3 will focus on modification of the chemical structure of ethidium-based probes for O2 •– . Embedding the probes in biologically-compatible nanoparticles to overcome their limitations related to their reactivity with heme proteins is part of the strategy. Heme proteins are responsible for the rapid consumption of HE, leading to the formation of E+ and dimers. Aim 3 will explore the potential to protect HE from heme proteins and extend its applicability to heme-rich cells (e.g. cardiomyocytes) and organelles (mitochondria) by grafting the probe in biocompatible nanoparticles (Nano-HE) to block the interaction of HE with the protein heme centers. Aim 3 will also explore the potential to design novel HE-based probes for O2 •– based on self-immolation chemistry. The nanoparticle-embedded HE molecule will be derivatized with a fluorophore, which will be released selectively when HE is converted into 2-OH-E+ . The hypothesis is that the extent of the release of free fluorophore will be a specific measure of O2 •– production (Fig. 3).
- Aim 4. Assess the applicability of the probes for O2 •– detection in model biological systems.
After having characterized their chemical and biochemical reactivity, all new synthesized probes will be evaluated in model cellular systems to assess their applicability for O2 •– detection, including tolerability of the synthesized nanoparticles. The Zielonka lab routinely uses the following models: (i) activated neutrophils; (ii) activated macrophages; (iii) endothelial cells stimulated with a redox cycling agent; and (iv) isolated mitochondria in the presence of electron donors. The experiments on model cellular systems will help establish the performance of the probes in the detection of O2 •– and their response to other oxidants. The experiments on isolated mitochondria using different energetic substrates will help establish the ability of the probes to accumulate in mitochondria and detect O2 •– from different mitochondrial complexes. The ultimate goal of this thesis is to develop a fluorescence-based assay(s) to selectively measure mitochondrial superoxide.
Complementarity
This PhD thesis will take advantage on the ongoing collaboration between both AMU and MCW labs, focused on the development of new redox probes and modulators, to tackle the basic challenges in the redox biology field. Both labs use their chemical backgrounds to develop, characterize, and validate the new probes and tools in the field of redox research, they have strong, complementary expertise that results in strong collaboration and synergistic interactions, as evidenced not only by the exchange of scientific visits (>20) but also by numerous high-impact joint publications (47 articles) and patent applications (7).
This thesis is innovative as it proposes to develop novel tools for redox biology/medicine and will take advantage of the SuperO2 International Research Project (IRP) recently awarded to Dr. Hardy to support the collaboration between the research teams from ICR and the Medical College of Wisconsin (MCW) to address current challenges in the studies of the role of superoxide in cancer cell proliferation and the mechanism of redox-based chemotherapies.
REFERENCES
1) Winterbourn C. C. Nat Chem Biol 2008, 4, 278. 2) Halliwell B.; Gutteridge J. M. C. in Free Radical in Biology and Medicine New York 4th edition 2007. 3) Cheng, G. et al. J Biol Chem 2018, 293, 10363. (4) Hardy, M. et al. ARS 2018, 28, 1416. (5) Zielonka, J.et al. Free Radic Biol Med 2018, 128, 3. (6) Cheng G. et al. Nat Commun 2019, 10, 2205. (7) Huang, M. et al. Advanced Science, 2022, e2101267. (8) Cheng, G. et al. Free Radical Biol. Med. (2023), 205, 175-187. 9) Pan, J. et al. iScience 2018, 3, 192. (10) Zielonka, J.et al. Chem Rev 2017, 117, 10043.
Keywords and thematic
Organic chemistry, reaction kinetics, mitochondria, reactive oxygen species (ROS), superoxide, redox-biology, cancer, probes for ROS detection, spin trapping, fluorescent probes, hydroethidine.
Required profile and skills
The applicant will have obtained a master's degree in molecular chemistry (organic, bioorganic) or equivalent and must be motivated to get involved in a project at the chemistry / biology interface. A good knowledge of classical organic synthesis techniques as well as purification and analytical methods is essential. Notions in biochemistry, electron paramagnetic resonance and inorganic chemistry will be appreciated. The candidate should be organized, autonomous, have good communication skills and a team spirit.
English level.
Doctoral supervision
The PhD student will be in direct interaction with his thesis supervisors (Dr. M. Hardy) at the SREP team and Dr. J. Zielonka at the MCW. He/she will present his work during the weekly group meetings.
Annual monitoring committee is organized by the doctoral school to give the opportunity to the doctorant to present his/her work.
Funding. The funding of the thesis will be provided by the CNRS.
Valorisation
The results will be published in peer review journals in the domain of chemical biology. Patens may be deposit as well.
International
Possibility for an international mobility at the Medical College of Wisconsin (USA) for the cell-based studies and at Lodz University of Technology (Poland) for mechanistic and kinetic studies.
Collaborations
Medical College of Wisconsin (USA)
Lodz University of Technology, Institute of Applied Radiation Chemistry (Poland)
Funding category: Autre financement public
The funding of the thesis will be provided by the CNRS
PHD title: chimie
PHD Country: France
Requirements
Specific Requirements
The applicant will have obtained a master's degree in molecular chemistry (organic, bioorganic) or equivalent and must be motivated to get involved in a project at the chemistry / biology interface. A good knowledge of classical organic synthesis techniques as well as purification and analytical methods is essential. Notions in biochemistry, electron paramagnetic resonance or nanomaterials will be appreciated. The candidate should be organized, autonomous, have good communication skills and a team spirit.
Possibility for an international mobility at the Medical College of Wisconsin (USA) for the cell-based studies and at Lodz University of Technology (Poland) for mechanistic and kinetic studies.
Additional Information
Work Location(s)
- Number of offers available
- 1
- Company/Institute
- AMU Institut de chimie radicalaire
- Country
- France
- City
- Marseille
- Geofield
Where to apply
- Website
https://www.abg.asso.fr/fr/candidatOffres/show/id_offre/123668
Contact
- Website
https://icr.univ-amu.fr/fr/equipes/srep/
STATUS: EXPIRED