Vladana Vukojevic

Associate Professor of Biochemistry

EXPERIMENTAL ALCOHOL AND DRUG DEPENDENCE RESEARCH

We study cellular and molecular mechanisms that underly 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.

PERSONAL RESEARCH STATEMENT

My research, at the overarching level, focuses on understanding self-organization in chemical and biological systems using experimental and theoretical approaches. Self-organization is a universal property that is inherent to living organisms at all levels of their organization, from the cellular, tissue, organ, organism to the, supra individual, i.e. societal level. Self-organization also occurs in complex chemical and biochemical reaction systems in vitro when they proceed under conditions far from thermodynamic equilibrium. It arises through entangled networks of interconnected molecules that via molecular motion and chemical interactions give rise to positive and negative feedback loops through which specific molecules affect their own production/degradation. Such complex networks exhibit collective dynamical behavior that is a property of the network as a whole and cannot be predicted, not even in principle, by studying in isolation the kinetics of individual chemical/biochemical pathways that constitute the network, thus necessitating nondestructive studies of chemical kinetics in complex reaction mixtures in vitro and in live cells. To quantitatively and nondestructively characterize the kinetics of fast dynamical processes in complex chemical systems and in live cells/tissue ex vivo, I use and further develop advanced fluorescence microscopy and correlation spectroscopy methods. In addition, I am using mechanistic mathematical modeling and approaches from dynamical systems theory to understand how individual processes in complex biochemical networks are integrated to yield a coherent response at the organism level. My most important contributions to science are briefly summarized below.

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]. This advancement enabled us to quantitatively characterize the kinetics of transcription factor-DNA interactions in live salivary glands of Drosophila and establish that the Hox transcription factor Sex Combs Reduced (Scr) finds its target genes in the nucleus of a living cell via a purely stochastic process – the homeodomain associates/dissociates rapidly (in the ms range) with/from nonspecific binding sites, whereas it remains bound for seconds or minutes at specific binding sites [2]. Our most recent work on methods development includes the development of massively parallel fluorescence correlation spectroscopy integrated with fluorescence lifetime imaging microscopy (mpFCS/FLIM) [3-5]. This method enables quantitative, scanning-free confocal fluorescence microscopy imaging and spatial-mapping of the heterogeneous distribution of molecules i.e., their concentration and diffusion/size, directional motion, local environment sensing, detection and characterization of molecular interactions, conformational changes, discrimination of spectrally similar fluorophores [3-5].

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

Quantitative transcription factor-DNA interactions and pharmacological studies in live cells
Methodology advancements have enabled us to quantitatively characterize the kinetics of transcription factor-DNA interactions in live salivary glands of Drosophila and establish that the Hox transcription factor Sex Combs Reduced (Scr) finds its target genes in the nucleus of live cells via rapid (in the millisecond range) homeodomain association/dissociation reactions with/from nonspecific binding sites, whereas it remains bound for seconds or minutes at specific binding sites [1] and to reveal an auto-regulatory switching mechanisms for Hox-mediated transcription activation/deactivation [2]. They have also enabled us to conduct precise quantitative pharmacological studies in live cells. In particular, we have quantitatively characterized the action of allosteric modulators of oligodendrocyte transcription factor 2 (OLIG2) dimerization [3] and were instrumental in revealing that CT-179, a small-molecule inhibitor of OLIG2 dimerization that is a promising drug candidate for treating Sonic hedgehog (Shh) medulloblastoma, the most common primary central nervous system (CNS) tumor in children, acts by disrupting OLIG2 homodimerization [4].

1. 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(9):4087-4092. doi: 10.1073/pnas.0914612107
2. Papadopoulos DK, Skouloudaki K, Engström Y, Terenius L, Rigler R, Zechner C,  Vukojević V, Tomancak P. Control of Hox transcription factor concentration and cell-to-cell variability by an auto-regulatory switch. Development 2019 146(12): dev168179. doi: 10.1242/dev.168179
3. Oasa S, Vukojević V, Rigler R, Tsigelny IF, Changeux JP, Terenius L. A strategy for designing allosteric modulators of transcription factor dimerization. Proc Natl Acad Sci U S A. 2020 117(5): 2683-2686. doi: 10.1073/pnas.1915531117
4. Li Y, Lim C, Dismuke T, Malawsky DS, Oasa S, Bruce ZC, Offenhäuser C, Baumgartner U, D'Souza RCJ, Edwards SL, French JD, Ock LSH, Nair S, Sivakumaran H, Harris L, Tikunov AP, Hwang D, Alicea Pauneto CDM, Maybury M, Hassall T, Wainwright B, Kesari S, Stein G, Piper M, Johns TG, Sokolsky-Papkov M, Terenius L, Vukojević V, McSwain LF, Gershon TR, Day BW. Suppressing recurrence in Sonic Hedgehog subgroup medulloblastoma using the OLIG2 inhibitor CT-179. Nat Commun. 2025 16(1):1091. doi: 10.1038/s41467-024-54861-3 

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

Our 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 showed that it also changes the spatial organization of proteins and lipids in the plasma membrane at the nanoscale level [4, 5].

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
5.  Oasa S, Sezgin E, Ma Y, Horne DA, Radmilović MD, Jovanović-Talisman T, Martin-Fardon R, Vukojević V, Terenius L. Naltrexone blocks alcohol-induced effects on kappa-opioid receptors in the plasma membrane. Transl Psychiatry. 2024 Nov 24;14(1):477. doi: 10.1038/s41398-024-03172-8

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, we are 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.