Photosynthesis is the major process used by plants, algae and cyanobacteria to generate energy from sunlight, being indispensable to the existence of all the advanced forms of life on Earth. In this process, with the consumption of the inorganic CO2, O2 is formed together with different organic compounds, mostly sugars and other carbohydrates. This maintains the level of O2 in the atmosphere, and also captures CO2 - with important ecological and environmental effects. These processes occur largely in light-harvesting complexes, which are groups of interacting subunit proteins that may be part of a larger supercomplex of a photosystem - the functional unit in photosynthesis. In this project we will investigate the coupling between the protein environment and the excitation states of the reaction center (RC) and the CP43 and CP47 “antennae” pigment subunits of the Photosystem II (PSII) complex. The main aim is to combine (i) the recent experimental data available on high-resolution PSII structures and energetics, with (ii) the latest developments in simulations and analysis of atomistic systems with both quantum and classical, atomistic levels of theory. In particular, we will use semi-classical (SC) and quantum mechanics / molecular mechanics (QM/MM) methods to probe and characterize quantitatively the role played at multiple scales by the specific molecular environment provided by PSII in mediating and controlling the excitation energy transfer between the different PSII subunits.
This work should lead to the identification of the most important molecular-mechanistics steps and factors that control the efficiency of energy transduction within PSII.

Determining how sensory information is detected and processed in the brain to produce adaptive behaviors is a fundamental issue in neuroscience. Although sensory inputs have been comprehensively-described in many systems, the central elements of sensory circuits are often much less well understood. The Drosophila gustatory system is an attractive model to investigate the neurobiology of sensory perception, because individual taste stimuli evoke robust and stereotyped behavioral responses under the control of neural circuitry that is amenable to molecular genetic perturbation, electrophysiological analysis and high-resolution imaging. Gustatory sensory neurons (GSNs) project from the peripheral taste organs to the primary gustatory center in the brain, the suboesophageal ganglion. Here, they are presumed to synapse with different types of interneurons, which integrate the information and send their projections to either higher brain regions and/or to the motor output. Although such interneurons have been well characterized in other brain regions of Drosophila, such as the olfactory system, almost nothing is known about gustatory interneurons. The goal of this project is to develop activity-dependent genetic reporters that will unequivocally label gustatory interneurons and to use these to characterize their morphology, neurochemistry and physiology to determine how these neurons transduce and transform taste evoked-sensory information.

To address the cellular and molecular requirements for Th17 differentiation during commensal colonization, I will focus SAA1 protein, which is strongly induced by SFB colonization. To monitor SFB colonization easily and surely, I propose to generate SAA1 luciferase mCherry knock in reporter mice and to use an ileal looping surgical approach that does not impair host circulation. Using this approach, we can understand when and where SAA1 is induced in response to SFB colonization. Although SFB may attach intestinal epithelial cells (IECs) and CX3CR1+ cells in intestine, which cells recognize SFB are not known. To determine the cellular requirement for SFB recognition, SAA induction and commensally driven Th17 differentiation, I will use specific cell linage depletion strategy that is well established. Using CX3CR1+ cells depleted mice, we can understand whether CX3CR1+ cells are necessary for SAA1 induction and Th17 differentiation or not.
To identify the genes which are required for Th17 differentiation in response to SFB colonization, I will try identifying the genes which are strongly induced by SFB. To eliminate secondary effects, I will purify IECs, DCs and CD4+ T cells from ileal loops of mice that are depleted CX3CR1+ cells and CD103+ DCs. Comparing these all gene profiles, we can understand the genes that are directly induced by SFB colonization in each cell linages. Thus, these genes are strong candidate that affect Th17 differentiation. In the last part of my study, I will elucidate whether the candidate genes are required for Th17 differentiation in response to SFB colonization by using conditional knock out mice.

Formation of beta-sheet-rich protein aggregates is linked with several functional and many pathological biological processes. In the latter case, the small early aggregates, rather than the fully-grown fibrils, have been implicated in cellular toxicity, but it is difficult to probe the pathway for aggregate formation in experiments due to the heterogeneity inherent in the aggregation process and the small concentration of such toxic oligomers present in the system at any given time. Yet, a good understanding of the factors that facilitate or inhibit to primary and secondary nucleation of protein aggregates is of key importance for controlling and inhibiting the formation of toxic species. Computer simulations can provide invaluable insights to that end since they allow input from experimental data relating to different length and timescales to be integrated. The aim of the proposed research is to explore the nucleation pathways of early oligomeric species, using a simple coarse-grained model that takes into account that aggregation prone peptides can occur in different states with different interaction properties. We will investigate how sensitive the kinetics of the fibril-growth process is to the interaction properties of the states that the peptide can acquire and the oligomeric species they produce. Furthermore, we will explore how the surface of existing fibrils can catalyze the formation of new oligomers, a process called secondary nucleation. Finally, we will investigate the effect of one particular external factor- mechanical stress- on the aggregation mechanism.

Tight regulation of cell size is essential for both multicellular and unicellular organisms to achieve an optimal size for specific physiological functions – for example, to balance nutrient uptake and cell growth. In principle, size regulation requires coordination of cell growth and division. In the budding yeast Saccharomyces cerevisiae, this occurs at a size check-point for cell cycle progression within the G1 phase of the cell division cycle prior to DNA replication. Although extensive studies have identified several regulatory proteins that critically affect size control, the molecular mechanism that allows cells to measure their own size remains elusive. One major candidate for a size sensor is the unstable G1-cyclin Cln3, which during G1 triggers irreversible commitment to cell division. Since the Cln3 concentration remains constant through the cell division cycle, we hypothesize that cells may titrate the number of Cln3 molecules, which increases proportionally with cell size, against their DNA, which remains constant in G1. Such a mechanism could result in an activity proportional to the number of Cln3 molecules rather than concentration. Here, we propose to directly test this hypothesis using a reconstituted, single-molecule imaging approach combining Total Internal Reflection Fluorescence (TIRF) microscopy and microfluidics to examine the interaction of single purified Cln3 molecules with its downstream target proteins on flow-stretched DNA. The observation of Cln3 binding non-specifically and sliding on DNA would provide strong support for the titration model of size control, thereby solving a longstanding fundamental problem in cell biology.

Transcriptional co-repressors are essential components of the highly complex gene regulatory system. Despite their importance in ensuring cell type and state-specific gene expression, we still have little understanding of their precise molecular mechanisms of action. The goal of this integrative genomics project is to provide a comprehensive understanding of co-repressor action using adipogenesis as a model system. Specifically, I aim to (1) determine the genome-wide DNA binding dynamics of the co-repressor KAP1, (2) integrate these data with available binding profiles of the co-repressors SMRT and NCor1; and (3) derive the regulatory logic of co-repressor complexes. This will be achieved through explorative computational investigation of the dynamic relationship between co-repressor complex formation, transcription factor DNA binding, gene expression, and chromatin structure during differentiation, and by experimentally validating emerging hypotheses through knock-down or over-expression. The choice of adipogenesis is motivated by the established importance of co-repressors in this system, its medical relevance, the availability of well-established in vitro models and the wealth of molecular data and tools available for downstream validation. The proposed approach will uniquely integrate complementary genome-wide datasets in a controlled differentiation set-up, aiming to unravel the dynamic gene regulatory properties of co-repressor complexes. A thorough understanding of co-repressor function may also lead to translational applications, including the development of new strategies to tackle fat mass excess-related health problems.

The symbiosis between the calcifying marine microalga Emiliana huxleyi (E. huxleyi) and the marine bacterium Phaeobacter gallaeciensis (P. gallaeciensis), is unique as it shifts from mutualism into parasitism. Although various data suggest that this symbiosis is highly prevailing and significant in oceanic habitats, little is known regarding this association and its affect on the single organism and the entire ecosystem.
By merging molecular and cell biological approaches, microscopy and geochemical techniques, I will characterize various aspects of the algal-bacterial interaction and address the following issues:
The colonization pattern of E. huxleyi by P. gallaeciensis: What algal and bacterial life modes are presented during colonization? Is a bacterial biofilm created during the process? Do algal-derived molecules, known to be utilized by bacteria, promote the establishment of symbiosis? Exploring these questions will allow me to portray the molecular and physiological profile, of both alga and bacterium, in the course of colonization.
The bacterial biosignature in algal calcified remnants: The calcified disks that are produced by E. huxleyi rely on uptake of seawater for ion supply, and thus record in their chemistry various characteristics of the algal environment and physiology. By exploiting experimental approaches commonly used in Earth sciences, I will examine how the algal-bacterial relation shapes the chemical signals encapsulated in the calcified remnants of algae, known to be of importance in the reconstruction of past environments.

Stem cells (SCs) hold promise for regenerative medicine. SCs were first cultured from skin, and are now used to treat burn patients. In addition, corneal blindness in one eye can often be fully cured by culturing corneal SCs from the good eye. I propose to address whether molecular distinctions between these two related adult SCs can be exploited to improve and broaden therapies for corneal blindness. Using mice as a model, I will: (1) Devise methods to purify corneal SCs, profile the mRNAs they express, and determine how they differ molecularly from well-characterized skin SCs; (2) Culture and compare long-term passaging potentials and retention of molecular differences of corneal and skin SCs. If differences are lost in vitro, do corneal SCs reacquire and sustain their identity when engrafted to common sites (cornea, backskin, kidney capsule) on a recipient mouse? This should reveal suitability of corneal SCs for long-term tissue regeneration. (3) Focus on ~100 genes highly and preferentially expressed in corneal SCs, and use in utero RNA interference to rapidly find which are critical for maintaining corneal SC identity in vivo. Promising candidates will be further tested by cellular, biochemical and classical genetic approaches. (4) Explore whether candidates from (3) can be used in gain of function experiments to see if I can reprogram skin SCs into corneal SCs. Pending promising results in vitro, I will engraft and test the efficacy of corneal-reprogrammed skin SCs to repair and regenerate damaged corneal tissue. Long-term, I hope to apply my findings to human SCs and small molecule-based reprogramming aimed at restoring corneal tissue in cases of total blindness.

A fundamental question in infection biology is what makes some diseases chronic. The disease agent, which may be a virus, bacterium or parasite, is cleared very slowly, or never cleared, by the immune system or therapy. Instead, infection is seemingly controlled but persists at low levels. Reasons for persistence may include mechanisms that involve new cycles of pathogen replication. In HIV infection, the mechanism of persistence in the face of the immune response relies on ongoing cycles of replication, and this may also contribute to persistence in the face of therapy. We previously observed that cell-to-cell spread, a directed form of HIV transmission to adjacent cells occurring in the immediate vicinity of a virus-producing cell and involving large viral particle numbers, dramatically decreases HIV infection sensitivity to antiretroviral drugs. Here we propose to use a probabilistic model of infection and mixtures of HIV particles, each expressing either CFP or YFP upon successful infection, to understand the persistence of ongoing replication in the face of antiretroviral drugs. We will quantify numbers of infecting viruses per cell in different infection modes: cell-free infection, where viral particles find a new cell to infect by diffusion in a non-directed manner, and cell-to-cell spread. We will determine whether large numbers of transmitted viral particles per cell increase the probability that at least one escapes drug action and infects the cell. Ongoing replication may lead to therapy failure and potentially contribute to viral persistence. Understanding it is of potential high interest to future curative strategies.

Sister chromatid cohesion is essential for proper chromosome segregation. Chromosome cohesion is mediated by cohesin complexes which form a ring-like structure that embraces replicated sister chromatids and hold them together until anaphase onset. Although cohesin binds to DNA dynamically before S phase, cohesion is only established during DNA replication, which requires acetylation of cohesin complexes and subsequent recruitment of a cohesin’s regulatory subunit called Sororin. Sororin additionally promotes cohesion maintenance from S phase until mitosis by antagonizing another cohesin’s regulatory subunit named Wapl. Maintenance of chromosome cohesion is extremely important in mammalian meiosis, because chromosome cohesion needs to be stably maintained for very long periods of time. In oocytes, cohesion establishment occurs during pre-meiotic S phase, which takes place before birth, and then has to be stably maintained until completion of meiosis, which only occurs several months or years later during ovulation. The mechanism responsible for meiotic cohesin stabilization for such long periods of time remains unclear. Importantly, the increase of trisomies and other birth defects with maternal age correlates with a gradual loss of sister chromosome cohesion during oocyte growth and maturation. Here, we intend to study how chromosome cohesion is established and regulated during meiosis, and to unravel the detailed mechanism responsible for maintenance of sister chromatid cohesion for very long periods of time. We will additionally study how the dynamics of meiotic cohesin complexes affect homologous pairing and meiotic recombination.

Humans, and other primates, are intensely social animals. We smoothly maneuver in our social environments by effortlessly understanding others’ interactions. Understanding others’ interactions has been found to engage several brain regions in humans [1]. In our monkey kin, though, homologous neural circuits remain unknown. This state of affairs raises fundamental questions about the biological and evolutionary foundations of this skill, which is essential for both humans and monkeys. Do monkeys rely on anatomical regions related to the human ones to read their peers’ interactions? And how is such a complex yet automatic social skill generated by the actions of single neurons? This project aims to answer these questions by using a multi-scale approach. First, those parts of the primate brain devoted to understanding others’ interactions will be charted. The use of functional magnetic resonance (fMRI) as an exploratory tool will enable me to compare monkey and human circuits. Then, the project will characterize the fMRI-identified areas, in order to understand how single neurons contribute to generating this skill. Electrophysiological and pharmacological techniques will enable fine-grained characterization of the features processed by individual neurons at a mechanistic level not achievable in human studies.

A major challenge of evolutionary biology is to understand which genes control ecologically relevant traits and how variation at these genes affects the fitness of organisms in their natural environment. In order to achieve this goal, a large degree of integration between different disciplines is required. In this project I propose to thoroughly analyze and dissect an adaptive trait, seed color variation, from its role in ecological interactions to its molecular basis. In desert sunflowers, seed color often matches the color of the soil, suggesting the presence of a camouflage mechanism reducing predation. This is an extremely tractable system for ecological studies in which genetic, phenotypic and environmental factors can be manipulated independently. Together with the ample availability of genomic resources for sunflower and the generally simple genetic basis of color polymorphism in plants, this makes seed camouflage an ideal system to ask general questions about the evolution and genetic bases of adaptation. I will use a combination of field experiments, landscape resequencing, QTL mapping and molecular biology to characterize the genes controlling seed color variation in wild sunflower and its effects on predator preferences and plant fitness. This information will be complemented by association studies aimed at identifying the genetic bases of seed color variation in cultivated sunflower. Together, these data will be used to understand the evolutionary dynamics of the identified genes in natural and cultivated environments. This multi-disciplinary approach will enable a more comprehensive understanding of the genetic mechanisms underlying adaptive processes.

Lysine (K) acetylation was first discovered on histones over four decades ago. Although histones have been the primary focus of acetylation research, recent studies showed that many proteins, also outside of the nucleus and implicated in a diverse set of cellular functions, could be acetylated. The specific role of acetylation and how this posttranslational modification is regulated is largely unknown. Circadian rhythms control physiological and metabolic processes. The core circadian protein CLOCK has acetyltransferase activity counterbalanced by the deacetylase SIRT1. SIRT1 activity is regulated in a circadian manner and also the levels of its cofactor, NAD+, oscillate, suggesting a link between cyclic rhythms and energy metabolism. In this project I propose to study the circadian acetylome of neocortex in wt and tissue-specific sirt1 -/- mice, through liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis and to compare it with liver acetylome. SIRT1 is involved in diverse brain functions (ageing, neurodegeneration, learning and memory) and it is implicated in regulation of different metabolic pathways in the liver by deacetylating key enzymes implicated in glucose and lipid metabolism. By analysing the circadian acetylome in liver and cerebral cortex, I expect to identify which proteins are characterized by oscillation in lysine acetylation and the function and regulation of this rhythmic modification.

The transcriptional induction of many biosynthetic pathways follows a defined temporal order, termed “just-in-time” (JIT) activation, where upstream acting enzymes are activated first, followed by downstream enzymes. Mathematical models predict that JIT activation is beneficial in unstable environments, but is suboptimal when the environment is constant. This suggests that the transcriptional regulatory networks controlling metabolic genes face an evolutionary trade-off between optimality under stable conditions and their ability to adapt to environmental changes. This prediction has, however, never been experimentally verified, nor has it been studied how JIT activation emerges during evolution. The proposed project will address this by quantitatively assaying the evolutionary cost and benefit of the JIT activation of amino acid (AA) synthesis pathways in E. coli.
I will genetically engineer strains that activate all enzymes of a pathway at once and compare their growth with wild-type JIT activation. If mathematical models hold true JIT activation will be beneficial specifically when the concentration of externally provided AAs is fluctuating. To examine the evolutionary plasticity of the regulatory network controlling JIT activation, I will evolve the engineered all-at-once strain under stable or fluctuating laboratory conditions. By characterizing the evolved strains, as well as evolutionary intermediates, I will study how regulatory networks controlling JIT activation arise during evolution and whether there is a predictable order of evolutionary adaptations. I will thereby study how environmental fluctuations influence the evolution of metabolic pathway control.

Within the visual system, directional selectivity (DS) is a well-established characteristic of many retinal and cortical neurons. Remarkably, there is no direct evidence indicating that retinal DS is responsible for cortical DS, and it is widely held that cortical DS is generated within the cortex from non-DS inputs. Nonetheless, as recent evidence has revealed that retinal ON-OFF DS ganglion cells (DSGCs) project to subcortical pathways, I propose to test whether some forms of cortical DS responses are driven by retinal DS outputs. To test this, I will alter activity of retinal DS neurons and monitor for changes in cortical DS. To modulate retinal DS neurons, I will make genetic manipulations to a mouse line in which Cre recombinase is selectively expressed in ON-OFF DSGCs, allowing me to ectopically express light activated ion channels (channelrhodopsins) in these neurons. To measure signals in visual cortex, I will use a state-of-the-art in-vivo 2-photon calcium imaging system to monitor cortical signals while I manipulate activity of retinal DS neurons. Finally, to show that these manipulations are relevant to behaviour, I will put mice into a virtual reality system, and monitor for changes in visually guided behaviour while I modulate activity of retinal DS neurons. The findings from this work will provide important insights into the detailed workings of the visual system, and reveal to an unprecedented level of detail how the cortex processes retinal inputs.

DEAD-box proteins are highly conserved enzymes carrying out fundamental functions in gene expression. They utilize ATP to unwind short double-stranded RNAs or to remodel ribonucleoprotein particles. Mss116 is a yeast mitochondrial DEAD-box protein required for efficient splicing of group II introns in vivo, rendering it a crucial component of the mitochondrial gene expression machinery. The precise mechanisms of Mss116 function are yet to be determined. Furthermore, the important question if Mss116 and DEAD-box proteins in general are able to translocate on RNA remains insufficiently answered, so far. Single-molecule methods are well-suited to approach such mechanistic questions.

Starting from unpublished data from the Ha lab indicating that Mss116 may indeed move on single-stranded RNA, I propose a detailed and elaborate single-molecule study addressing the precise mechanisms of Mss116 function. The first goal is to demonstrate, for the first time, translocation of Mss116 on single-stranded RNA using a "fleezer", a cutting-edge single-molecule instrument combining fluorescence detection with an optical tweezer that allows the observation of single fluorescence-labeled protein molecules in a space and time resolved manner at high resolution. Further, I aim to monitor Mss116 function on an endogenous substrate, the group II intron aI5gamma, allowing for detailed mechanistic insights of how Mss116 facilitates the folding process of a group II intron. Together with studies of mutant proteins and related DEAD-box proteins this study aims to provide highly significant mechanistic insight into the function of DEAD-box proteins in general.

What can a honeybee ‘understand’ with a miniature brain containing only 960,000 neurons? What visual features can she detect innately and how complex are the visual categories she can learn in her lifetime? Despite the wealth of experimental data on bee learning behaviour, there is a remarkable disagreement in this question, due to the general lack of understanding of the neural computations underpinning visual cognition in insects.
In this project I propose to generate and test computer models of the neural basis of visual category learning in bees using advanced techniques from computational neuroscience. I will (1) integrate the currently available information on the neurobiological background of bee vision in a computer simulation; (2) use in silico evolution to find possible neural architectures that satisfy the neurobiological constraints and are consistent with behavioural data; (3) use my model to design experiments that can maximally discriminate between competing hypotheses.
Bees in particular offer an excellent model system for such studies, as their behaviour and cognitive abilities, as well as the neurobiology of their sensory systems, are extensively studied. It is not surprising therefore that bees have become the model for studying seemingly advanced cognition with minimal neuronal hardware, and therefore a benchmark for all comparative cognition research. Thus, the implications of the proposed project will not only concern insect psychophysics, but will alter our understanding of computer vision, neuroscience and behavioural ecology.

Attention is well known to enhance sensory processing in the cortex. There is strong evidence that such effects are partly mediated by the release of acetylcholine (ACh) by cholinergic neurons arising in the basal forebrain (BF) and projecting throughout the cortex. Direct stimulation of the BF has been shown to improve sensory processing. However, what neural mechanisms control the BF activity and how it is influenced by the state and expectations of the behaving animal are still largely unknown. Here, I aim to unravel mechanisms that translate reward expectations into enhanced sensory processing and behavioral performance.

Due to the availability of new molecular and genetic tools that allow recordings and manipulation of specific cell types, all the experiments will be performed with mice. First, I will develop a visual discrimination task with varying reward sizes to test how visual cortical processing and the animal’s performance depend on the expected reward. Second, I will perform cell-type specific recordings from cholinergic and GABAergic neurons in the BF to determine whether and how these cells encode the expected reward size. Finally, I will employ mono-synaptically restricted, viral tracing techniques to record from dopaminergic neurons in the ventral tegmental area (VTA) that project to the BF and test whether these projections transmit information about the expected reward size. Manipulation of projections from the VTA to the BF will allow me to disentangle their impact on sensory processing and behavior. With this approach I will unravel the specific role of VTA neurons projecting to the BF in the reward dependent top-down control of visual processing.

Meiosis is a specialised form of eukaryotic cell division that diploid cells undergo to generate haploid gametes. Central to meiosis is a set of processes involving the physical interaction of homologous chromosomes at sites of programmed DNA double-strand breaks. The physically joined chromosomes are resolved in a manner that either results in the reciprocal exchange of DNA between the homologous chromosomes (a crossover product) or not (a non-crossover product). Crossover products are necessary to create genetic diversity and prevent the mis-segregation of chromosomes, a source of infertility and birth defects in humans. Both the number and physical distribution of crossover products along chromosomes are tightly regulated. The mechanism of this regulation is unknown. However, it has been shown that the SUMO E3 ligase protein Zip3 binds to both the precursors of crossovers and non-crossovers at early stages of meiosis, and specifically to the sites of crossovers at late stages of meiosis. I propose to exploit this for development of a method for enriching for chromatin designated for both crossover and non-crossover formation and for chromatin marked for crossover formation specifically. Proteomic analysis of these chromatin samples will provide new insight into crossover regulation by identifying key players that will be verified by classical genetic and molecular biology approaches.

Plants are sessile organisms and highly dependent on light as their energy source. This means competing vegetation is an enormous threat for the plant´s survival. The change in light quality produced by surrounding plants is sensed by the phytochrome family of photoreceptors. Canopy shade inactivates phytochromes and thus induces an escape program to improve the plant´s access to light. If this strategy fails flowering is accelerated to enhance the chance for reproductive success. At the same time, this early flowering is accompanied by reduced biomass and seed yield. However, it is very poorly understood how premature flowering is induced by canopy shade and repressed by phytochrome activity. Some downstream genes necessary for acceleration of flowering in canopy shade are already known, but it is not understood how they are regulated by phytochrome signaling. Furthermore, these genes only execute a subset of the shade-induced flowering pathway.
My aim is to investigate the mechanisms regulating flowering through phytochromes and canopy shade using two candidate gene approaches to analyze different signaling levels. Furthermore, as an unbiased approach, I will conduct a forward genetic screen to identify novel regulators of the phytochrome-dependent flowering. The combination of genetic, molecular biological and biochemical tools will allow me to examine until now neglected levels of this signaling pathway and aims to further our understanding of this essential response that controls the plants´ fitness.