The molecular chaperone Hsp90, a dimeric ATP-driven molecular machine, is one of the most abundant proteins found in eukaryotic cells. This specialised chaperone is known to function as part of several complexes to assist in several essential cellular functions including the folding, maintenance and degradation of client proteins, although the exact mechanisms by which these functions occur is not yet known. In order to elucidate the function of this crucial molecule, the mechanisms and roles of the large conformational changes it undergoes and the forces involved must be studied. To achieve this, experimental techniques will be used that provide excellent force sensitivity and high spatial resolution. In combination with in vivo experiments, I will use custom-built optical trapping and fluorescence setups together with biochemical approaches and mutagenesis, which will provide me with a unique set of tools with which to delineate the specific domain orientations and forces that are required for the function of Hsp90. This will enable me to address the timely question of how the conformations and mechanics of Hsp90 are influenced by co-chaperones and client proteins.

Follicular helper T (Tfh) cells play a key role for inducing antigen-specific responses. Although substantial numbers of Tfh cells reside in the gut-associated lymphoid tissues, it is not fully understood how Tfh cells develop and whether Tfh cells enter the memory pool. To clarify the mechanisms for Tfh differentiation and maintenance in the gut, I will investigate commensal-specific CD4 T cells throughout the course of bacterial colonization. For this purpose, I will focus on segmented filamentous bacteria-(SFB) induced responses, because SFB preferentially induces both Th17 and Tfh cells. Since Th17 cells show plasticity into Tfh cells, I will examine the role of RORgt in Tfh cell development and memory T cell formation, using our established transgenic mice bearing SFB-specific T cell receptor. Next, to identify genes responsible for Tfh cell development, genome-wide analyses will be performed comparing gene expression profiles between wild type and RORgt-deficient Tfh cells. In addition, a proteomic approach will be used for identification of Bcl6 binding partners. For this analysis knock-in mice bearing tagged Bcl6 will be newly generated using the CRISPR-Cas9 system. In addition, based on these data, knockout-based screening will be done in primary CD4 T cells. Focusing on the genes most strongly associated with Tfh cell development, I will generate mutant mice using the CRISPR-Cas9 system. Using these mice, I will investigate molecular mechanisms by which the targeted gene regulates the Tfh program in coordination with Bcl6 in vivo. Through this proposed project I will elucidate how Tfh cells develop and are maintained in response to luminal bacteria.

A wide range of microbes can synthesize and secrete toxic compounds or protein-based virulence factors to inhibit the competing organisms by disrupting core cellular activities. To counteract this, eukaryotes have evolved elaborate mechanisms to surveil cellular and organismal integrity and defend against the xenobiotic attacks. Previous studies have revealed some xenobiotic-related surveillance and defense pathways in C. elegans. However, the potential memory of such responses in these pathways has not been studied. Since naturally an organism can be challenged by the same xenobiotic insults repeatedly within a specific habitat, and the xenobiotic threats to a parental generation stand a good chance of also threatening the next generation, it is reasonable to hypothesize that the surveillance and defense system could be sensitized by continuous exposures to particular toxins and for a short generation time organisms like C. elegans, perhaps even “remembered” across generations. To test this, I will establish appropriate systems to investigate whether the mitochondrial surveillance and defense pathways activated by xenobiotic attacks or disruption of mitochondrial genes can be sensitized both within one generation and between generations in C.elegans. Further, I will perform large-scale genetic screens to identify genes involved in sensitization processes and dissect the functions and underlying mechanisms of the identified genes. This research will contribute to the understanding of the eukaryotic innate immune system and provide potential new pharmaceutical strategies to treat mitochondrial dysfunction diseases and innate immunity disorders.

Traditionally, enzyme catalytic power has been assigned to active site residues that make and break chemical bonds and provide electrostatic interactions to stabilize a reaction’s transition state. However, simply adding these residues to water does not provide significant catalysis -these residues allow rate enhancements only in the context of the folded protein matrix -the enzyme scaffold. Furthermore, electrostatic interactions are not localized to the enzyme active site, as the enzyme scaffold has many charged and polar groups in the regions surrounding the active site. Despite the clear importance of the enzyme scaffold for catalysis, the general properties of the scaffold and how it contributes to enzymatic rate enhancements have been little studied and are not well understood. I propose to determine the properties of the enzyme scaffold and how they promote catalysis, with a focus on the well-behaved model enzyme Ketosteroid Isomerase (KSI). I will use a multidisciplinary approach, combining room temperature crystallography and solution NMR to characterize the dynamic and structural properties of the enzyme scaffold in complexes representative of the distinct species along the KSI catalytic cycle. Electrostatic properties of the scaffold will be assessed, and the range at which they have influence will be tested. The research proposed here subscribes to the overall goal of developing a predictive understanding of how specific properties of an enzyme scaffold contribute to catalysis and to the ambitious long-term goal of designing enzymes that rival natural enzymes in efficiency and specificity.

Autonomic innervation has been implicated as an important modulator of pancreas development and glucose homeostasis. Despite efforts to characterize the role of sympathetic/parasympathetic nerves, whether innervation directly controls endocrine function remains controversial. I propose to study the role of innervation in endocrine pancreas development and maintenance by using the zebrafish model. Pancreatic islet innervation has been observed in human, mouse and zebrafish with differences in the extent of innervation and specific cell types that are innervated. The zebrafish is well suited for the in vivo study of islet development with time-lapse imaging and has several advantages over other vertebrate models, including rapid embryogenesis, transparency during embryonic and early larval stages, high regenerative capacity and genome editing tools. Innervation studies in rodents and humans have been limited to specific snapshots in time and depth of the tissue, not allowing one to capture the temporal and three-dimensional spatial interactions that define the dynamic nature of innervation as the endocrine pancreas develops and matures. With reporters labelling specific neuronal and endocrine cells and tools for temporally controlled tissue specific ablation of neurons and knockdown of genes encoding specific neurotransmitter receptors, I plan to characterize innervation during pancreas development and elucidate the role of innervation on the maintenance of islet architecture, regeneration and function in response to metabolic demand. The results of this research can have a major impact on our understanding of pancreatic innervation dynamics, signalling and function.

An important aim of neuroscience is to decipher how neurons process environmentally and internally generated signals. In vivo, neocortical microcircuits are spontaneously active, providing an ongoing source of intrinsic excitatory and inhibitory network activity. This dynamic activity is integrated with sensory information and then transformed into an appropriate output. Neuronal computations have mostly been characterized in vitro, meaning our current knowledge of how neurons process information under realistic network conditions in vivo is incomplete. Recent theoretical studies indicate that in vivo network activity fundamentally alters the way in which neocortical excitatory pyramidal cells integrate synaptic input. The purpose of my proposal is to experimentally bridge this knowledge gap using an “all optical” method to non-invasively interrogate neuronal computations rapidly in 3D. This approach will be complemented by optogenetics, electrophysiology, and biologically detailed modeling. I will focus on the mouse visual and somatosensory cortical microcircuit formed by layer 5 pyramidal cells, the principal cortical output, and Martinotti interneurons, which provide strong feedback inhibition to the dendrites of pyramidal cells. L5 pyramidal cells express nonlinear mechanisms that are thought to affect spiking output during multimodal sensory integration. In contrast, virtually nothing is known about sensory computations performed by Martinotti interneurons or how they modulate L5 pyramidal cell activity. The potentially paradigm-shifting results derived from this research may transform our understanding of how neurons compute sensory information in vivo.

Humans and mice originate from pluripotent cells in the inner cell mass (ICM) of the embryo. This ICM is traditionally assumed to form after the 8-cell stage from a process termed ‘asymmetric’ cell division, which cleaves cells in parallel to the embryo surface and directly allocates one daughter cell inside. Further, it is believed that asymmetric division segregates polarity proteins and putative cell fate determinants (‘cell polarity’ hypothesis). Although asymmetric division is thought to be the primary morphological mechanism forming the ICM, it has not been directly demonstrated and it remains unclear whether biomechanical forces acting within the embryo would even allow it.

The Plachta lab has recently designed an in silico model demonstrating how adhesion and tension forces can generate virtual embryos with normal number of inner cells, faithfully recreating mammalian development. But unlike previous assumptions, these embryos feature very few asymmetric divisions. Instead, most inner cells originate from ‘symmetric’ divisions and subsequently internalize by apical constriction. Importantly, combining advanced imaging technologies of living mouse embryos and a novel custom membrane segmentation technology confirmed that asymmetric divisions are uncommon and unnecessary, and that apical constriction is the prime cellular mechanism allocating cells to the ICM in vivo.

These recent findings provide new understanding how the first embryonic structures form during mammalian embryogenesis. The goal of this proposal is to reveal how molecular mechanisms determine the polarity and fate of pluripotent cells coordinated by apical constriction-dependent ICM formation.

Non-coding RNAs that migrate between cells and organisms and act non-autonomously have recently attracted much attention. The inherent properties of RNA interference, i.e. sequence specificity, provide these “mobile RNAs” with the ability to affect essentially any gene in neighboring cells or tissues, while foreign RNA uptake can inform an organism about its habitat. In both plants and nematodes, mobile RNAs mediate cell signaling, antiviral defense, and facilitate transgenerational epigenetic inheritance. In mammals, extracellular miRNAs are highly abundant in many body fluids, making them attractive candidates for mediating trans-cellular communication. However, mammalian mobile RNAs have not yet been demonstrated definitively, and foreign RNA uptake through mammalian intestine is controversial. Major gaps in our understanding are the biochemical properties of mobile RNAs, and their mechanism of action, if any, in mammals. I propose to generate novel assays to investigate RNA uptake and mobility in mammalian “mosaic” organoid cultures. Using these assays, I will investigate how different cellular niches within the intestine respond to foreign RNA, whether small RNA can act non cell-autonomously, and what properties equip small RNAs with the ability to mediate trans-cellular communication. Ultimately, my studies are expected to generate new insights into small RNA based communication, and might resolve crucial issues in RNA-based drug delivery.

There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

The aim of this project is to understand how molecular forces ‘scale-up’ to generate biological outcomes. We focus on understanding the contractile actomyosin cell cortex, a sub-micron thick network that lies beneath the plasma membrane that supports cellular and tissue morphogenesis. Ultimately, functional outcomes and active mechanical behaviour at the larger scale must arise from the intrinsic biophysical properties of the force-generating molecules of the cortex. However, how these properties emerge is unclear. Our consortium will implement novel experimental approaches solutions to tackle this question. First, we will develop myosin mutants that allow us to modulate specific parameters of motor function (e.g. force, step-size, directionality, torque; Bryant) under control by light; we call this ‘Optomechanics’. Second, we will develop hydrodynamic active-gel theory for describing essential physical behaviors arising from molecular-scale force and torque generation in the cortex (Grill). We will apply these approaches to understand the contribution of the cortex to cellular morphogenesis during cytokinesis (Grill) and junctional tissue homeostasis (Yap). For this purpose, we bring expertise in myosin biophysics and molecular bioengineering (Bryant), cortical mechanics and theory (Grill), and cell junctions and morphogenesis (Yap). The bioengineering team will generate validated reagents that will be implemented in cells by the biological teams; results from the latter will reveal how modifications in myosin force generation at the single molecule level relate to in-vivo function. We will combine optical imaging, laser nanodissection with hydrodynamic theory to develop quantitative descriptions of cortical mechanics during cytokinesis in the C. elegans zygote. In parallel, we will define the impact of cadherin on cortical mechanics at mammalian epithelial cell-cell junctions, and how this influences mechanical coherence of populations. This combination of molecular manipulation with theory will allow us to describe how actomyosin mechanical activity at cell and tissue scales emerges from myosin motor function at the molecular scale, and how myosin molecular force drives cell- and tissue-level mechanics.

The crucial importance of secreted extracellular scaffolding proteins (ESPs), such as precerebellins, neuronal pentraxins, and C1q-like molecules, for the formation and function of neuronal synapses in vivo has only recently been discovered. These molecules share a modular structure, with domains that specifically interact with pre- and postsynaptic cell surface receptors to bridge neurons across the synaptic cleft. Guided by structural information of known ESPs, we aim to develop synthetic connector molecules that will expand the range and affinity of trans-synaptic interactions. These synthetic ESPs will likely become new and powerful tools to manipulate the number and properties of excitatory and inhibitory synapses, and to regulate synaptic communication and cognitive abilities in mouse models in vivo.
To achieve our goals, we will develop and implement a highly multi-disciplinary approach based on the diverse and complementary expertise and state-of-the-art techniques established in the applicants’ laboratories. Armed with structural and biophysical knowledge about physiological ESPs and their receptor interactions, we will design hybrid molecules that contain novel combinations of binding domains and thus promote a novel spectrum of molecular interactions across neuronal synapses. The synthetic ESPs will be further refined by mutagenesis or by replacing native domains with nanobodies of desired specificity. These novel neuronal connectors will be recombinantly produced, purified and delivered into subregions of the hippocampus and the cerebellum by acute or chronic injections using osmotic pumps. We will also express genes encoding the designer ESPs using viral vectors with neuron type-specific promoters. Electrophysiology, confocal microscopy, nanoscopy and behavioral analyses will be then employed to verify the targeting and functional efficacy of synthetic ESPs at the synaptic, neural network and organismic levels. Their effects on motor and spatial learning, reversal learning, pattern separation and completion will be studied in combination with recordings of neural activity oscillations and single-cell firing.
Considering the huge potential variety and versatility of these novel tools, we anticipate an immediate impact onto basic neuroscience and, in longer term, potential new avenues for the treatment of neuropsychiatric disorders.

Understanding how activity in large-scale brain networks causes behavior remains a fundamental challenge in neuroscience due to the inherent experimental limitations in the number of neurons that can be simultaneously recorded or manipulated. Unravelling complex biological systems to which we have limited access to measure or control the internal dynamics is notoriously difficult. Control engineering has solved this challenge for the control of complex man-made systems, such as aeroplanes and chemical plants, for which also only a small fraction of the state variables are accessible and controllable. Despite its clear potential, control engineering has not yet been adopted by neuroscience.
One mechanism by which information is thought to be integrated across multiple brain areas is by coherent oscillations of the local field potential at particular frequencies. Despite indirect evidence for such orchestration of cortical activity, a causal demonstration of oscillation-based information flow during behaviour has not yet been established. We will develop data-driven models of the interaction between cortical areas by using unscented Kalman filtering. These mathematical models then allow us to design control algorithms that apply optogenetic perturbations to control cortical interaction dynamics by manipulation of the temporal activity structure. We will develop mathematical models to determine the direction of information flow between brain areas by recording neural activity in auditory, visual and prefrontal cortex, during the performance of a demanding auditory-visual discrimination paradigm. Having identified the neural signatures associated with different behavioural states, we will perform closed-loop control of frequency-dependent information flow in the behaving animal to test the hypothesis that dynamic interactions between cortical areas play a causal role in perception and decision-making.
By combining control engineering and neurophysiology we will develop a new class of mathematical models which allow us to describe and manipulate neural activity, and therefore enable unique insights into brain function.

A new frontier in RNA biology has emerged in the last 5 years with the discovery that small RNAs (sRNAs) exist outside of cells and can be transferred from one cell to another in a functional form. This has been proposed as a mode of cell-to-cell communication and, in theory, could also enable communication between different organisms. Yet the basic mechanics of extracellular sRNAs are not understood, including how specificity in secretion is achieved and how sRNAs enter a functional RNA interference (RNAi) pathway in recipient cells. Without these details, it is difficult to predict the extent to which mobile sRNAs operate across biology, and to discern how to harness this mechanism in medicine. We approach these questions by examining sRNA transport in a host-pathogen system. We have found that an Argonaute protein (HpWAGO) and sRNAs, including microRNAs (miRNAs), are secreted by Heligomosoides polygyrus, a gastrointestinal nematode that infects mice. We will capitalize on combined expertise in nematode sRNA biology (Claycomb), host-pathogen interactions (Buck) and small RNA-target evolution (Abreu-Goodger) to examine the functional properties of HpWAGO and its sRNAs in both nematodes and mouse cells. Our approach integrates genetic and molecular techniques along with high-throughput proteomics and Next Generation sequencing. We exploit the nematode C. elegans for functional studies because H. polygyrus is genetically intractable and sRNA biology is well understood in C. elegans. The results from both systems will be computationally integrated to understand the mode of action, identify binding partners and their functional effects on gene expression, and explore the evolution of parasitic sRNA-related loci and their targets. Since there is no precedent for secretion of RNA or Argonaute proteins by nematodes, we will attempt to recapitulate a functional sRNA secretion system in C.elegans, which would bolster the nascent field of extracellular RNA communication and enable us to further dissect the secretion mechanism. Our overarching goal is to provide the first detailed characterization of an RNAi pathway that can mediate interactions between an extracellular pathogen and its host, which will enable us to predict the breadth of this mechanism across biology and lay a foundation for its functions in host-pathogen co-evolution.

Cortical pyramidal neurons (CPNs) are the basic computational units of the cortex. CPNs integrate local and distant inputs, and innervate various targets. Our knowledge about processing of these inputs by CPNs, and on the identity of CPN's targets, is limited due to the difficulty to record intracellularly from multiple spatial locations. We propose to apply a novel protein-based voltage imaging technology combined with optogenetic actuation ('optopatch') to record and manipulate CPNs in brain slices. Using optopatch we will study the details of voltage propagation in CPN dendrites following focal optogenetic stimulation in different compartments. Then we will describe voltage propagation and spiking output in response to synaptic inputs, by stimulating local and thalamic cells while imaging voltage propagation in single CPNs. This will provide a detailed map of inhibitory and excitatory local and distant connections into CPNs. Next, we will map the functional connections within a cortical column using simultaneous activation and recordings of multiple identified cells. Together, we will provide detailed description of computations performed by single cortical cells, and of the functional connections between them. Due to the high-throughput nature of these experiments, they could be readily repeated with other cell types, different cortical regions and various disease models. Success in implementing this approach in brain slices will be an important step toward in-vivo 'all-optical electrophysiology', a technological leap that may revolutionize neuroscience research.

The tiny nematode worm C.elegans has been instrumental for identifying genes that increase lifespan. Although most of the genes increasing lifespan in C.elegans are also present in humans, only very few have been associated with human longevity. This is mainly due to the fact that humans are more complex than worms. Instead of studying single genes we will investigate a multi-gene approach where we assume that lifespan in the worm can be predicted by the behaviour of all these genes together. We do this by viewing the worm genetic system as an ecological system. Similar to the fact that ecological systems display characteristic behaviors before they can collapse, we will test if the gene-regulatory network of worms displays characteristic behavior when approaching a collapse leading to death. This will provide insight in the mechanisms of aging and might help to translate the results to humans.

1. Astrocytes, classically considered as simply supportive cells for neurons, are emerging as relevant elements in brain information processing through their ability to regulate synaptic activity. Indeed, the tripartite synapse formed by pre- and post-synaptic neurons and surrounding astrocyte structures has been proposed as a functional unit of brain processes. These novel and important functions of astrocytes are under control of G protein signaling-dependent processes, which trigger astrocyte Ca2+ signals eventually leading to the release gliotransmitters and other mediators regulating synaptic functions.
2. Recent evidence indicates that the roles of mitochondria in the brain may go beyond the mere needs of energy supply for cell survival and maintenance, being possibly involved in the regulation of synaptic functions. It is conceivable that mitochondrial G protein signaling participates in these processes. Various subtypes of G protein-coupled receptors (GPCRs) and the associated signaling molecular elements are present within mitochondria, suggesting the existence of mitochondrial G (mtG) protein signaling pathways.

Thus, astroglial mtG signaling potentially plays an important role in the regulation of tripartite synapse and hence brain functions. However, no studies have addressed this issue so far.
The present project proposes to investigate the consequences of the activation of astroglial mtG signaling pathways in brain physiology, identifying the underlying mechanisms at cellular, network, behavioral and theoretical modeling levels. We propose to generate and develop novel pharmacogenetic tools (DREADDs specifically expressed by astroglial mitochondria, mtDREADDs) that will allow the specific control of astroglial mtG protein activity. We will experimentally and theoretically analyze the consequences of activation of different mtG proteins (via mtDREADDs) on neuronal, synaptic, and network activity as well as brain functions in living animals.
The expected results will reveal novel processes of cellular signaling in the CNS, and will identify new regulatory mechanisms mediated by astroglial cells in brain function.

One of the main challenges of this century is to better understand the function of the brain in order to solve its devastating disorders. We have learned a great deal about the basic components of the brain: the different types of neurons, each with its unique arborization, and the connections, termed synapses that enable neurons to communicate with each other. We also have a basic understanding of how information is transmitted through electrical and chemical signals. However, we know little about how individual neurons integrate the multiplicity of inputs, both excitatory and inhibitory, to compute a single output. Nor do we understand much about how individual neurons work in concert within a specific circuit to perform complicated computations. A major limitation has been our inability to monitor and control the electrical and synaptic activities of neurons inside the brain of a living animal. Overcoming this limitation requires the combination of different technologies to enable neuronal activity to be monitored with high sensitivity and fidelity while minimally interfering with brain function.
This grant exploits advances in optical methods, molecular biology, genetics, protein engineering, and electrophysiology and brings together tools developed in three laboratories, each with distinct expertise. These include a fiber-optic-based "micro-optrode" that can be inserted into the brain of a living animal in order to simultaneously record the electrical activity and biochemical signals. In particular, this instrument can detect and decode optical signals transmitted by probes that respond to the microenvironment inside the neuron. Such molecular probes, which are introduced into neurons through genetic manipulations, are designed to reveal how the neuron decodes synaptic messages that it receives from its connected partners in the brain of an animal receiving sensory stimulation. To dissect how signal processing within the various compartments and arbors of a neuron contributes to its function, we are exploiting a novel strategy to locate the molecular probes in specific neuronal microdomains. The combination of innovative approaches that we propose in this grant is expected to produce new knowledge about the contribution of specific neuronal subtypes and circuits to the complex processing of the brain in normal and pathological conditions.

Adolescence is marked by high onset and incidence of mental disorders and intense changes in specific brain areas. Prefrontal cortex (PFC) and basolateral amygdala (BLA) form a circuit involved in emotional learning believed to underlie many of these disorders. Anatomical and morphological changes occur in PFC, BLA and their connections during adolescence; nevertheless, how these changes affect PFC-BLA function and its consequences to sensitivity to stress and onset of mental illness is currently unknown. Aiming to achieve a more functional understanding of how the maturation of these sensitive areas might underlie normal and pathological brain function, we propose to investigate how their synaptic connectivity changes during adolescence. In aim 1 we will characterize the maturation of PFC-amygdala synaptic responses at different time points during adolescence and adulthood. In aim 2 we will examine whether developmental changes in plasticity underlie sensitivity to stress. Lastly, we will test whether selectively manipulating the activity of PFC projections can rescue the deleterious effects of stress in behaving animals. Understanding how these emotional regulatory circuits functionally mature will help identify the mechanisms responsible for the high incidence of mental disorders during adolescence and provide tools for earlier and more targeted interventions.

Although strong data from mouse models show that HMP deregulation causes AML development, its role in the human disease has not been demonstrated yet. Using the most advanced techniques to target gene expression in primary human cells as well as by taking advantage of the novel conservative culture method developed by the host laboratory for maintaining leukemia patient samples in vitro, this research proposal aims to investigate the role of HMP genes in the regulation of human LSC activity. More specifically, benefiting from the advanced expertise of the host laboratory in lentiviral overexpression and shRNA approaches in human primary cells, I will investigate the role of each one of the HMP genes in the regulation of patient LSC frequency, self-renewal activity and survival to chemotherapeutic treatment. The read-out of this study will rely on a state-of-the-art functional xeno-transplantation assay in immuno-compromised mice. This work will lead to a deeper understanding of the molecular biology of human AML, possibly revealing new options for therapeutic targeting of the disease in patients.

The functionalization of O2 within Fe containing enzymes will be investigated to obtain a detailed understanding of their geometric and electronic structure at intermediate stages throughout catalysis. Fe Intermediates are important in many human drug interactions and disease states but heme Fe (HFe) intermediates have not been accessible at an insightful level by more generally applied methods such as NRVS or VTVH MCD, while the experimental condition and energy range of L-edge XAS cause ligation damage. The development of resonant inelastic x-ray scattering (RIXS), a high resolution probe of 3d metal-ligand orbital covalence, will be used to investigate HFe and non heme Fe (NHFe) intermediates.
The research requires; (1) the development of the selection rules, drawing information content of RIXS relative to the established method of L edge XAS. (2) Following which RIXS measurements can be performed for heme model complexes where there is already K and L edge XAS data available. (3) RIXS measurements will then be performed on simple heme proteins where L edge XAS information is inaccessible. (4) This will be developed to the investigation of oxyhemoglobin on both the protein and the model complex. (5) With the acquired means of accurate interpretation of RIXS measurements (obtained from steps 1 to 4) the high valence Fe(IV) intermediates compound I & II and equivalent enzyme systems will be investigated and compared to RIXS results for equivalent non-heme complexes.