Vladana Vukojevic

Associate Professor of Biochemistry

We study cellular and molecular mechanisms underlying alcohol use disorder (AUD), focusing on the opioid system role in its development and management. To this aim, we use and further develop quantitative time-resolved analytical methods with single-molecule sensitivity and integrative approaches from dynamical systems theory.

My research interest, at the overarching level, lies in understanding self-organization in chemical and biological systems. Self-organization is a natural phenomenon that is inherent to living organisms and can be observed at all levels of their organization, from the cellular, tissue, organ, organism to the supra-individual, i.e., societal level. Examples of self-organization in biochemical systems include: pattern formation in developmental biology, bacteria quorum sensing and biofilm formation, metabolic oscillations (e.g., glycolytic oscillations), genetic switching, i.e., dynamic gene expression regulation via bistability or glucocorticoid pulsatility, dynamic lateral organization of plasma membrane proteins and lipids, rapid cytoskeleton reorganization via actin protein polymerization/depolymerization, to name but a few.  

In my research, I use experimental and theoretical approaches to further our understanding of self-organization in biochemical systems. To quantitatively and non-destructively characterize the kinetics of fast dynamical processes in inanimate chemical systems or in live cells/tissue ex vivo, I use and further develop fluorescence microscopy and fluorescence correlation spectroscopy (FCS) methods. To understand how reaction-diffusion processes are integrated to yield a coherent response at the system/organism level, I am using numerical simulations and approaches from dynamical systems theory.

One key focus of my current work is the role of the opioid system in the development of alcohol use disorder (AUD). The opioid system consists of opioid receptors (mu-, kappa-, delta-, and nociception, the latter being a non-classical member of the opioid receptor family) and their corresponding endogenous peptide ligands, such as endorphins, dynorphins, enkephalins, and nociceptin. Opioid receptors, which belong to the G protein-coupled receptor (GPCR) superfamily, are embedded in the plasma membrane, the first cellular structure affected by ethanol. The endogenous peptide ligands, primarily produced and processed in the nervous and endocrine systems, include molecules like beta-endorphin, which is the endogenous peptide ligand of the mu-opioid receptor. Beta-endorphin, for example, is produced as part of the hormonal cascade in the hypothalamic-pituitary-adrenal (HPA) axis. In my research, I use analytical techniques with single-molecule sensitivity to examine how ethanol influences the dynamic lateral organization and functioning of opioid receptors. I also apply concepts from dynamical systems theory to emulate ethanol's impact on biochemical transformations within the HPA axis. 

CONTRIBUTION TO SCIENCE

Functional Fluorescence Microscopy Imaging (fFMI)

My work on the advancement of fluorescence microscopy and correlation spectroscopy approaches for quantitative characterization of fast dynamical processes in live cells/tissues, started by contributing to the development of an instrumental setup for confocal laser scanning microscopy with improved sensitivity 

1. Vukojević V, Heidkamp M, Ming Y, Johansson B, Terenius L, Rigler R. Quantitative single-molecule imaging by Confocal Laser Scanning Microscopy. Proc Natl Acad Sci USA, 2008, 105:18176-18181. doi: 10.1073/pnas.0809250105
2. Vukojević V, Papadopoulos DK, Terenius L, Gehring W, Rigler R. Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells. Proc Natl Acad Sci USA 2010, 107:4087-4092. doi: 10.1073/pnas.0914612107
3. Krmpot AJ, Nikolić SN, Oasa S, Papadopoulos DK, Vitali M, Oura M, Mikuni S, Thyberg P, Tisa S, Kinjo M, Nilsson L, Terenius L, Rigler R, Vukojević V. Functional Fluorescence Microscopy Imaging (fFMI). Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells. Analytical Chemistry 2019, 91(17): 11129-11137. doi: 10.1021/acs.analchem.9b01813
4. Oasa S, Krmpot AJ, Nikolic SN, Clayton AHA, Tsigelny IF, Changeux JP, Terenius L, Rigler R, Vukojevic V. Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM). Anal Chem. 2021, 93(35):12011-12021. doi: 10.1021/acs.analchem.1c02144
5. Nikolić SN, Oasa S, Krmpot AJ, Terenius L, Belić MR, Rigler R, Vukojević V. Mapping the Direction of Nucleocytoplasmic Transport of Glucocorticoid Receptor (GR) in Live Cells Using Two-Foci Cross-Correlation in Massively Parallel Fluorescence Correlation Spectroscopy (mpFCS). Anal Chem. 2023 95(41):15171-15179. doi: 10.1021/acs.analchem.3c01427

Turnover of intermediates during amyloid-β aggregation towards early diagnosis of amyloid diseases

I have contributed to laying the foundation for the development of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) imaging for simultaneous detection of amyloid-β and lipids in brain tissue preparations [1, 2], developed a new method to monitor the turnover of intermediates during amyloid-β aggregation [3], successfully used this method to measure the concentration and size of structured amyloidogenic protein nanoplaques in blood serum [4] and characterize conformation-sensitive and sequence-independent monoclonal antibodies (mAbs) [5].

1. Solé-Domènech S, Sjövall P, Vukojević V, Fernando R, Codita A, Salve S, Bogdanović N, Mohammed AH, Hammarström P, Nilsson KP, LaFerla FM, Jacob S, Berggren PO, Giménez-Llort L, Schalling M, Terenius L, Johansson B. Localization of Cholesterol, Amyloid and Glia in Alzheimer’s Disease Transgenic Mouse Brain Tissue Using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Immunofluorescence Imaging. Acta Neuropathologica, 2012 125:145-157. doi: 10.1007/s00401-012-1046-9
2. Carlred L, Gunnarsson A, Solé-Domènech S, Johansson B, Vukojević V, Terenius L, Codita A, Winblad B, Schalling M, Höök F, Sjövall. Simultaneous Imaging of Amyloid-β and Lipids in Brain Tissue using Antibody-Coupled Liposomes and Time-of-Flight Secondary Ion Mass Spectrometry. J. Am. Chem. Soc. 2014 136:9973-9981. doi: 10.1021/ja5019145
3. Tiiman A, Jarvet J, Gräslund A, Vukojević V. Heterogeneity and turnover of intermediates during amyloid-β (Aβ) peptide aggregation studied by Fluorescence Correlation Spectroscopy Biochemistry, 2015 54:7203-7211. doi: 10.1021/acs.biochem.5b00976
4. Tiiman A, Jelić V, Jarvet J, Järemo P, Bogdanović N, Rigler R, Terenius L, Gräslund A, Vukojević V. Amyloidogenic nanoplaques in blood serum of patients with Alzheimer’s disease revealed by time-resolved Thioflavin T fluorescence intensity fluctuation analysis. J. Alzheimer's Dis. 2019 68: 571-582. doi: 10.3233/JAD-181144
5. Bonito-Oliva A, Schedin-Weiss S, Younesi SS, Tiiman A, Adura C, Paknejad N, Brendel M, Romin Y, Parchem RJ, Graff C, Vukojević V, Tjernberg LO, Terenius L, Winblad B, Sakmar TP, Graham WV. Conformation-specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen. J Cell Mol Med. 2019 23(3):2103-2114. doi: 10.1111/jcmm.14119

Ethanol effects on opioid receptor lateral dynamics and spatial organization at the nanoscale level

My study of ethanol (alcohol) effects on opioid receptor lateral organization and dynamics in the plasma membrane has shown that ethanol and naltrexone exert an opposite effect on the lateral dynamics of the mu-opioid (MOP) receptor, and that pretreatment with naltrexone is protective against ethanol effects on MOP receptor lateral mobility [1, 2]. We could verify using electrophysiology that ethanol alters opioid regulation of Ca²⁺ influx through L-type Ca²⁺ channels [3] and were able to show that it also changes the spatial organization of proteins in the plasma membrane at the nanoscale level [4].

1. Gruol DL, Nelson TE, Michaels S, Vukojević V, Ming Y, Terenius L. Ethanol Alters Opioid Regulation of Ca2+ Influx through L-type Ca2+ Channels in PC12 Cells. Alcohol Clin Exp Res. 2012 36(3):443-456. doi: 10.1111/j.1530-0277.2011.01631.x
2. Tobin SJ, Cacao EE, Hong DW, Terenius L, Vukojević V, Jovanović-Talisman T. Nanoscale Effects of Ethanol and Naltrexone on Protein Organization in the Plasma Membrane Studied by Photoactivated Localization Microscopy (PALM). PLoS One. 2014 9:e87225. doi: 10.1371/journal.pone.0087225
3. Rogacki MK, Golfetto O, Tobin SJ, Li T, Biswas S, Jorand R, Zhang H, Radoi V, Ming Y, Svenningsson P, Ganjali D, Wakefield DL, Sideris A, Small AR, Terenius L, Jovanović-Talisman T, Vukojević V. Dynamic lateral organization of opioid receptors (kappa, muwt and muN40D) in the plasma membrane at the nanoscale level. Traffic 2018 19(9): 690-709. doi: 10.1111/tra.12582
4. Tobin SJ, Wakefield DL, Terenius L, Vukojević V, Jovanović-Talisman T. Ethanol and Naltrexone Have Distinct Effects on the Lateral Nano-organization of Mu and Kappa Opioid Receptors in the Plasma Membrane. ACS Chem. Neurosci. 2019 10(1): 667-676. doi: 10.1021/acschemneuro.8b00488

Predictive modeling of the dynamic self-regulation of Hypothalamic-Pituitary-Adrenal (HPA) axis activity under normal physiology and stress

Building on my experience of working with complex chemical systems and my knowledge of dynamical systems theory, I am working on the development of mathematical models of HPA axis to better understand how self-regulation of HPA axis arises through neurochemical transformations and how it is perturbed under conditions of acute and chronic stress [1-4].

1. Čupić Ž, Marković VM, Maćešić S, Stanojević A, Damjanović S, Vukojević V, Kolar-Anić Lj. Dynamic transitions in a model of the hypothalamic-pituitary-adrenal (HPA) axis. Chaos 2016 26:033111. doi: 10.1063/1.4944040
2. Čupić Ž, Stanojević A, Marković VM, Kolar-Anić L, Terenius L, Vukojević V. The HPA axis and ethanol: a synthesis of mathematical modelling and experimental observations. Addict. Biol. 2017 22(6):1486-1500. doi: 10.1111/adb.12409
3. Abulseoud OA, Ho MC, Choi D-S, Stanojević A, Čupić Ž, Kolar-Anić Lj, Vukojević V. Corticosterone oscillations during mania induction in the lateral hypothalamic kindled rat experimental observations and mathematical modelling. PLoS One 2017, 12(5):e0177551. doi: 10.1371/journal.pone.0177551
4. Stanojević A, Marković VM, Čupić Ž, Kolar-Anić Lj, Vukojević V. Advances in mathematical modelling of the hypothalamic–pituitary–adrenal (HPA) axis dynamics and the neuroendocrine response to stress. Current Opinion in Chemical Engineering 2018, 21:84–95. doi:10.1016/j.coche.2018.04.003

Non-canonical mechanisms of dynorphin peptides action

My work on FCS application to study neuropeptides interaction with live cells has led to discovering the potential of dynorphin neuropeptides to accumulate in artificial membranes and in the plasma membrane of live cells, causing defects in lipid arrangement and facilitating content exchange with the surroundings via non-canonical, receptor independent, mechanisms of action [1-4].

1. Marinova Z*, Vukojević V*, Surcheva S, Yakovleva T, Cebers G, Pasikova N, Usynin I, Hugonin L, Fang W, Hallberg M, Hirschberg D, Bergman T, Langel U, Hauser KF, Pramanik A, Aldrich JV, Gräslund A, Terenius L, Bakalkin G. Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. J. Biol. Chem. 2005 280:26360-26370. doi: 10.1074/jbc.M412494200
2. Hugonin L, Vukojević V, Bakalkin G, Gräslund A. Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides. BBA-Biomembranes, 2008 1778:1267–1273. doi: 10.1016/j.bbamem.2008.02.00
3. Hugonin L, Vukojević V, Bakalkin G, Gräslund A. Membrane leakage induced by dynorphins. FEBS Lett. 2006 580:3201-3205. doi: 10.1016/j.febslet.2006.04.078
4. Maximyuk O, Khmyz V, Lindskog C-J, Vukojević V, Ivanova T, Bazov I, Hauser KF, Bakalkin G, Krishtal O. Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction. Cell Death Dis. 2015 6:e1683. doi: 10.1038/cddis.2015.39

Oscillatory reactions: Advancing mechanistic insights and harnessing autocatalytic feed-back loops for quantitative analyte analysis with improved sensitivity
Experimental studies of detailed kinetic mechanisms underlying oscillatory chemical and biochemical reactions are challenging. Dedicated methods are therefore needed that enable us to obtain quantitative information that can be used to understand the causal connectivity and correlations among chemical species that constitute the investigated system. One such method that I have adopted since its insception is Quenching of Small-Amplitude Limit Cycle Oscillations, i.e., Quenching Analysis (QA) [1, 2]. QA is specifically designed to take the advantage of the oscillatory dynamics that emerges in the vicinity of a supercritical Hopf bifurcation and harness it to acquire unique quantitative information about the investigated system that can easily be compared with model predictions. By comparing the experimentally derived quenching concentrations and quenching phases with results obtained from mechanistic models, one can determine whether key reaction pathways are correctly identified and incorporated in the model of the reaction mechanism and assess how realistically the model reflects the true mechanism of the investigated system. Furthermore, one can harness the power of autocatalytic feedback loops in non-linear chemical systems to achieve quantitative analyte analysis with improved sensitivity [3].

1. Vukojević V, Sørensen PG, Hynne F. Quenching analysis of the Briggs-Rauscher reaction. J. Phys. Chem. 1993 97 (16):4091-4100. doi: 10.1021/j100118a027
2. Vukojević V, Graae Sørensen P, Hynne F. Predictive Value of a Model of the Briggs−Rauscher Reaction Fitted to Quenching Experiments. J. Phys. Chem. 1996 100 (43):17175-17185. doi: 10.1021/jp960785o
3. Vukojević V, Pejić N, Stanisavljev D, Anić S, Kolar-Anić Lj. Determination of Cl-, Br-, I-, Mn2+, Malonic Acid and Quercetin by Perturbation of a
   Nonequilibrium Stationary State in the Bray-Liebhafsky Reaction. The Analyst, 1999 124:147-153. doi.org/10.1039/A807608A

As an educator, I aim to significantly contribute to the advancement of individually tailored doctoral education at KI and its internationalization. By teaching advanced analytical methods, I enable PhD students to conduct ambitious and innovative research that pushes the frontiers of knowledge and prepares them to address complex global challenges in their respective fields using these experimental techniques.

• I have designed and teach at KI the doctoral course "Functional Fluorescence Microscopy Imaging in Biomedical Research", which has been running since 2010.
• I have been engaged as an Invited Lecturer at the Hokkaido Summer Institute since its inception in 2016. This program brings together world leading researchers with proven educational and research records to provide educational courses in cooperation with faculty members from Hokkaido University, Sapporo, Japan.
• I am a Visiting Professor at the Faculty of Physical Chemistry, University of Belgrade, Serbia since 2011.
• I work with pedagogical development and support course organizers within the doctoral program Allergy, Immunology and Inflammation (Aii) to further improve their educational practices by implementing pedagogical approaches and effectively designing their courses using the long-distance teaching and learning platform Canvas to develop a student-centred didactic environment that facilitates learning.