The actin cortex is a contractile cytoskeletal network underneath the plasma membrane. Cortex contractility drives cell shape deformations during many cellular processes, including cell migration, cell rounding and cell division. When a cell divides, it forms an equatorial actomyosin ring that ingresses to separate the two prospective daughters, or poles, of the cell. The cortex of both poles is contractile, and a contractile imbalance between poles can result in cytoplasmic flows from one pole into the other, destabilising the position of the cleavage furrow and potentially leading to division failure. To ensure a stable cytokinetic cell shape, polar contractile forces must be mechanically balanced by opposing forces that resist cortex contractions. Here, we hypothesise that resistance of the plasma membrane to cortex contractions stabilises cell shape. To test this, we aim to: (1) characterise membrane dynamics during cortex contractions in dividing cells, combining cell biology and quantitative imaging; (2) measure mechanical membrane properties, using a combination of biophysical methods; (3) develop a theoretical model of the mechanics arising from membrane dynamics during cortex contractions, and verify this model using experimental perturbations. We will then test the model in other contexts where cortex contractions play a role, such as during cell rounding and cell migration. Because cell shape deformations in general are driven by cortex contractions, resistance of the membrane to cortex contractions could constitute a universal mechanism to stabilise cell shape in many different cell biological contexts.

53BP1 is a key protein implicated in immunoglobulin class-switching and DSB repair pathway choice. 53BP1 accumulation at DSB sites occurs rapidly after DSB formation, helping to promote non-homologous end joining (NHEJ) and antagonise DNA end resection. After DSB formation histones are specifically phosphorylated and ubiquitylated, which coincides with the recruitment of 53BP1. 53BP1 has been proposed to bind to modified chromatin directly, but the molecular details of this interaction are still unclear.

I plan to research how 53BP1 binds specifically to modified chromatin, investigating how histone post-translational marks are recognised directly in the context of a full nucleosome core particle (NCP). Recent work from my host laboratory has elucidated that 53BP1 reads a bivalent histone mark: H4 Lys20 di-methylation and H2A Lys15 ubiquitylation. I plan to crystallise the nucleosome-binding portion of 53BP1 in complex with an appropriately modified NCP, in order to discover the atomic details of this interaction. If successful this will be the first crystal structure of a protein bound to a post-translationally modified NCP. Furthermore, it will illuminate the remarkable selectivity of 53BP1 for the H2A Lys15 ubiquitin mark.

I plan to probe the functional consequence of 53BP1 binding to chromatin: is it just acting as a recruitment platform, or does 53BP1 also have a direct role in ordering chromatin? Using methods developed in the Durocher laboratory these questions can be answered directly through X-ray crystallography, biophysics and biochemistry.

One of the most fundamental functions of the brain is to learn and generate movements. Most neuronal activity is, in fact, related to motor output and the brain has a remarkable ability to acquire and improve motor skills. Despite the obvious relevance for shaping our own behavioral repertoire, the implementation of the learning process in the neuronal circuitry remains very much an open question. I suggest a tutor-student relationship of two important brain areas, with the motor cortex (MC) instructing the basal ganglia (BG), as a fundamental organizing mechanism in motor learning. Evidence from the host laboratory shows that while the execution of certain complex and precise motor sequences can be MC independent, their acquisition depends on both the BG and the MC. Together with preliminary data indicating that MC lesions lead to a loss of the suppression of disadvantageous actions, this suggests that MC tutors the BG during motor learning. To demonstrate this tutor-student relationship and to identify its means, I will employ state-of-the-art techniques. I will combine the host lab’s sophisticated motor skill learning paradigms with electrophysiological recordings and manipulations of neuronal activity. Key for this is a novel viral approach, developed during my PhD, for the specific targeting of cortico-striatal neurons, which provide MC’s input to the BG. Based on this approach, specific ablations of these projections, correlations of their activity with behavior and temporally precise optogenetic manipulations will identify MC as a tutor and characterize the nature of the tutoring signals. The proposed work will not only identify mechanisms underlying the interplay of defined neuronal circuits, but also reveal fundamental principles of motor skill learning and brain function.

The faithful transmission of epigenetic information is an essential process during development and cell differentiation. The mechanisms by which histone modifications and chromatin-associated complexes are re-deposited after DNA duplication, however, are not well understood. In the fission yeast Schizosaccharomyces pombe, repressive heterochromatic structures are assembled through the methylation of histone H3 at lysine 9 (H3K9) and the subsequent binding of HP1 proteins. At the centromeres, small non-coding RNAs guide the formation of heterochromatin and mediate silencing of centromeric transcripts. While the contributions of each of these components to the establishment of heterochromatin have been studied extensively, how these states are inherited remains an open question. Here we propose to analyse this process by using Chromatin in vivo Assay (CiA), a recently developed small molecule-based method to initiate heterochromatin formation in a reversible way. The key property of this approach is that the maintenance of silencing can be analysed in the absence of the initial trigger. This will allow us to perform a genetic screen to identify the components required for this process. Our goal is to identify determinants for epigenetic inheritance in this system and to use biochemistry to uncover the mechanism by which they function. We aim to be able to confront existing models on how epigenetic states are maintained.

Axon degeneration is a tightly controlled process of axon self-destruction that plays both a physiological role during development and pathophysiological role during neurodegeneration. Following a physical insult, such as a nerve transection or crush, the axon segment distal to the injury site undergoes rapid degeneration, a process that is called Wallerian degeneration. In addition to its role in axon degeneration distal to an injury site, Wallerian and Wallerian-like degeneration are also prominent features of numerous neurodegenerative diseases. However, the molecular mechanisms underlying Wallerian degeneration are poorly understood.
To identify molecules that are critical for Wallerian degeneration, I will perform a genome-wide RNAi screen. The physiological significance of the most promising candidates will be evaluated using cultured sensory neurons and genetically modified mice. I will further characterize functions of the candidates with biochemical and cell biological methods and define where the candidates reside in the Wallerian degeneration pathway. Previously, Prof. Tessier-Lavigne team found that the genetic deletion of SARM1 protects against Wallerian degeneration. However, the molecular function of SARM1 remains elusive. As such, SARM1 will also be analyzed using these methods. Finally, I will examine the function of SARM1 and the other Wallerian degeneration molecules in animal models of neuropathy. I expect that these studies will advance our understanding of the mechanisms regulating axon degeneration and will also provide insight into how to therapeutically target this pathway for the treatment of neurodegenerative diseases.

Recent microbiome studies open new opportunities to treat flora-related diseases. Here I propose to use evolutionary dynamics principles to reshape flora from a disease to a healthy state by altering expression patterns of the microbiome. Notably, this alteration of the flora can occur both at the level of species composition and at the level of genetic changes in each of these species. As a model system for microbiota reshaping I will use the lactose-intolerance (LI) condition in mice. I designed an unfavorable regime that would select against the bacteria that cause LI by fermenting lactose in the gut. This regime disfavors the bacteria that express lactose genes by putting a cost on activation of the bacterial lac-operon. By feeding mice a lactose analog that cannot be used to produce cellular energy, the cost of expressing lactose genes is expected to reduce lactose fermentation in the gut and thus to relieve LI symptoms. In parallel, I plan to apply similar evolutionary regimes on the bacterial lac-operon, yet in a more controlled system of lab-evolution experiments in E.coli cultures. These lab-evolution experiments will elucidate how promoter sequences evolve towards altered gene expression. Specifically, these experiments will follow, in real-time, the evolutionary process of de-novo promoter formation, from a random sequence to an active promoter and conversely from an active to an inactive promoter. To this end, cells with a randomized lac-promoter will be evolved in the presence of lactose, and in the opposite direction, cells with a native lac-promoter will be evolved in the presence of a lactose analog. Together, the proposed study will shed light on the evolution of transcription regulation, and on ways to reshape our flora by altering the microbiome transcriptome.

The endoplasmic reticulum (ER) constitutes a dynamic network that extends from the nuclear envelope to the plasma membrane and is involved in various contacts with other membranes. Among them, ER-mitochondria interaction sites have received special attention given their critical role in lipid homeostasis, calcium signaling and apoptosis. In particular, several reports indicate that ER stress-induced apoptosis involves direct signaling to mitochondria, likely (yet not proven) through ER-mitochondria interaction sites. Although several proteins have been found to mediate tethering and other functions in ER-mitochondria interaction sites, the description of these domains is still far from exhaustive.
This project is aimed at obtaining a detailed structural characterization of ER-mitochondria interaction sites and understanding the underlying structure-function relationships under different metabolic conditions. Specifically, given that their lipid biology is a mostly unexplored aspect, the main goal of this project is to evaluate the structural and functional aspects of the lipid composition of ER-mitochondria interaction sites. That is to test whether lipids determine selective protein sorting to these domains impacting on their function, signaling to mitochondria and ultimately cell fate. The effects of ER stress on ER-mitochondria interaction domains structure and function will also be studied and the potential role of lipids mediating these effects will be assessed. The experimental design includes biochemical purification of membrane fractions, extensive mass spectrometry-based lipidomics analyses and evaluation of structural and functional readouts of interest.

Microtubules are cytoskeletal polymers essential for cell division, motility, neuronal development and intracellular transport. Microtubule function relies on active remodeling of microtubule network through a process known as ‘microtubule dynamic instability.’ In this tightly regulated process, individual microtubules switch from growth to shrinkage and back. Although discovered almost thirty years ago, the molecular mechanisms of microtubule dynamic instability and its regulation remain largely unknown. My goal is to combine my PhD background in theoretical physics with my postdoctoral experience in experimental biophysics to elucidate the mechanisms regulating this molecular switch. The project involves development of a novel reconstitution setup, using microfluidics and single-molecule fluorescence imaging to achieve rapid controlled changes of reaction solutions. This setup will allow investigation of a whole range of previously unattainable experimental conditions, critical for rigorous testing of existing theoretical models. In addition to the fast-growing, plus microtubule ends, detailed characterization of minus end dynamics, which to date remains largely unstudied, will be performed. Furthermore, the project will use microfluidics, multi-color, single-molecule fluorescence imaging, quantitative image analysis and theoretical model development to address how ensembles of microtubule associated proteins collectively regulate microtubule dynamics. This unique blend of theory and experiment will ultimately lead to reconstitution of the key functional properties of the intricate network of proteins regulating the behavior of dynamic microtubule ends.

The brain is one of the most complex systems known and scientists are far away from a complete understanding of brain function. The brain consists of a complex network of neurons that communicate using electrochemical signals. Several large scale projects such as the Human Brain Project and the Human Connectome Project focus on mapping and computer simulation of these networks. Much less systematic effort is invested in the characterization of the neuronal population. Neurons are a heterogeneous population and according to current knowledge give rise to more than hundred cell types. We propose that deep knowledge about neuronal cell types is crucial for the interpretation of these networks. Trying to understand connectivity maps based on incomplete knowledge about the building blocks is similar to the challenge of understanding electronic circuitry function without distinguishing between resistors, capacitors and diodes. Cells within an organism went through distinct differentiation paths during development which we term the cell lineage. These differences in cell lineage are reflected in the epigenetic cell-state. We hypothesize, that knowing for each cell its lineage and its transcriptome will provide a solid ground for molecular definition of cell-types. Here, I describe a framework aiming to obtain such definition by combining methodology from genome editing and single-cell transcriptomics. I propose to use genome editing tools for dynamic genome manipulation which has the potential to enable cell lineage tracing for millions of cells. Importantly, I describe how to allow for the coupling of lineage with biological context using single-cell transcriptomics.

Given its anatomical organization, it is clear that Layer (L)1 of the neocortex plays a key role in cortical function. L1 is composed almost exclusively of afferent axons originating from distal brain areas and the dendrites of their postsynaptic targets. Thus, L1 acts as an integration hub of distant and local activity. Despite the vast amount of neocortical recordings amassed over the past decades, L1 remains largely uncharacterized. Existing methods are unable to record afferent activity from the acellular L1 neuropil. Genetically encoded calcium indicators (GECIs) allow recording activity from presynaptic structures of defined neuronal populations. However, current sensors diffuse poorly to distant synapses or photobleach significantly when imaged in small compartments, reducing the signal-to-noise ratio. Here, we propose to develop novel specialized sensors for studying the connections linking distant brain regions, such as L1 afferents. First, we will develop genetic strategies for the efficient targeting of GECIs to presynaptic compartments while preserving their sensitivity and photostability during synaptic optical recordings. Second, we will develop methods to optically record activity from synapses with known connectivity. Combining the expertise of each team we will rapidly test these novel sensors in vivo and ex vivo in axons representing the three main classes of L1 inputs. The sensitivity of the different sensors is going to be measured by imaging L1 axonal boutons in acute brain slices. The performance of the synaptic sensors in vivo is going to be characterized by imaging L1 axonal boutons in awake behaving mice. By applying these novel sensors we will describe the basic functional organization of L1. We will characterize afferent activity from cholinergic and thalamic projections to L1 in behaving animals with synaptic resolution. In addition we are going to determine whether functionally diverse cortico-cortical signals intermingled in L1 target different pyramidal neurons. Our project will shed light on the functional organization of L1. Importantly, the tools to be generated will have wide applications in neuroscience by allowing recordings from afferents of any length scale and relating their function with connectivity.

Metastasis is the major cause of cancer-related death. This process is realized by circulating tumor cells (CTCs) that shed from the primary tumor site and colonize distant organs. Despite the enormous clinical implications, little is known about the key genetic events that allow only certain CTCs to successfully initiate a metastatic lesion. In fact, the number of CTCs largely exceeds the number of clonal metastatic lesions in patients, suggesting a cellular hierarchy within CTCs and indicating that majority of CTCs is likely to die in the bloodstream, with only a minor CTC fraction being effectively stem-like metastatic precursors. We aim to characterize the genetic alterations accumulated by single CTCs to gain insights into CTCs heterogeneity and to ultimately identify metastasis-relevant genes in human pancreatic cancer patients. To this end, we will use a newly developed device for the isolation of CTCs from blood samples of cancer patients combined with single-cell next generation sequencing. First, we will sequence multiple single human CTCs and single cells from matched primary tumors to retrieve the mutation profile of each gene and the frequency of each mutation in CTCs and corresponding primary tumors. Second, we will use sphere-formation assay to screen all the candidate mutations which were enriched or unique in CTCs. Third, focusing on the strongest hits, we will assess whether doxycycline-inducible expression and/or knockdown of a candidate mutated gene affects CTCs number and metastasis in xenografts. Our studies will ultimately lead to the discovery of metastasis-relevant genes altered predominantly in stem-like CTCs.

Morphological synaptic changes, specifically those of dendritic spine remodeling, have been directly linked with functional aspects of learning and memory by rewiring and/or controlling the level of connectivity within the neural network. Following learning, the degree of dendritic spine remodeling correlates with behavioral performance, and is therefore suggested to represent a structural correlate of learning and memory. One factor which controls the regulation of spine and synapse turnover appears to be the excitation/inhibition balance of cortical networks. Furthermore, imbalance of excitation/inhibition is associated with cognitive disorders such as autism and schizophrenia. However, it remains unclear how exactly modulation of the excitatory/inhibitory balance can promote or reduce cortical plasticity. I therefore intend to examine how manipulating the activity of inhibitory interneurons will affect the learning and/or performance of a cognitive task, how it will affect the experience-dependent structural and functional connectivity within the network. To this end I will use newly developed genetic and optogenetic tools to activate or deactivate a specific sub group of cortical inhibitory interneurons during different stages of learning and performance. I will then examine the animals' behavior, the spine turnover (using two photon microscopy) and the neuronal connectivity (using electrophysiology) across the different perturbations. I hope these studies will shed light on the causal relationship between cortical structure and function to learning and memory processes.

We propose to elucidate the basis for positional information by hormones during plant morphogenesis. While it is known that cell fate decisions require simultaneous input from multiple hormones, to-date a precise understanding of how these signals are coordinated and act together to drive morphogenesis does not exist. Our limited mechanistic understanding is largely due to the difficulty to quantify the distribution of these small molecules in space and time. To explore this fundamental question, we will exploit recent advances in synthetic biology to engineer an RNA-based biosensor platform applicable to a broad range of small molecules and in particular to hormones. Using live-imaging technologies, we will use the sensors to obtain quantitative dynamic 3D maps of hormone distributions and relate these maps to the spatio-temporal distribution of cell identities, both during normal morphogenesis and upon perturbations of hormone levels. This analysis will be done on the shoot apical meristem, one of the best-characterized developmental systems in higher plants. In this context, mathematical approaches will be essential to analyze and establish a predictive model for how multiple hormones influence cell fate in a spatio-temporal manner.

A bird that flies rapidly through dense foliage to land on a branch– as birds often do – engages in a veritable three-dimensional slalom, in which it has to continually dodge branches and leaves, and plan a collision-free path to the goal in real time. Here we aim to study how birds control the speed of their cruising flight, fly safely through narrow gaps, decelerate to a smooth landing, and how they transition seamlessly through these different flight modes. Research with insects indicates that there are neural programs for controlling specific features of flight, such as regulation of velocity and altitude, measuring distance flown, and homing. However, we do not know how these programs are organized to generate the complex flight trajectories that we observe in nature. There is an analogous deficit in our understanding of flight mechanics because most studies concern single modes of flight, such as take-off, cruising, hovering, or turning. We know little about how animals make transitions between flight modes. Our research will address these fundamental gaps in our understanding of animal flight by studying visual control of flight and wing kinematics in three bird species: two parrots, which are specialized for cruising flight (Budgerigars: Srinivasan; Lovebirds: Lentink) and a hummingbird, specialized for hovering flight (Altshuler). We focus on birds because they are visually agile, and can be trained to perform complex behaviors. The project will begin by characterizing the visual control of key flight modes: take-off, cruise, obstacle avoidance, and landing. We will then study transitions between these modes. The most effective way to study how vision is used to control flight involves the use of virtual reality environments. We will develop tools for instantaneously tracking the position and orientation of a bird’s head, and also tracking its wing position although not in real time. The head tracker will be combined with a video presentation system to create an arena in which what the bird sees can be manipulated relative to how it flies. The results from these experiments will have implications for the design of bio-inspired aircraft and of bird-friendly man-made structures, such as wind farms.

The ends of linear chromosomes are protected from degradation and fusions by specialized structures called telomeres, which are composed of repetitive DNA bound by specialized proteins. In the absence of a maintenance mechanism, telomeres shorten during each cell division, due to the inability of conventional DNA polymerases to fully replicate the DNA end. This process limits the replicative capacity of somatic cells and therefore controls the aging process: once telomeres are too short, they loose their protective properties and cells stop dividing or simply die. To circumvent telomere shortening-associated aging and senescence and to gain immortality, cancer cells must maintain their telomeres. Most cancers do so by activating telomerase, but approximately 10% use the alternative ALT pathway. However, in response to telomerase inhibition, cells react with ALT activation, stressing the need to understand the ALT mechanism for cancer therapy. ALT mechanisms rely on recombination between telomeres, giving rise to features, such as heterogeneous telomere length, presence of extra-chromosomal telomeric DNA and telomeric sister chromatid exchange events are likely the cause and/or consequence of de-regulated telomere recombination. However, what allows such recombination events and the pathways leading to them is not currently understood. The Karlseder lab has recently engineered a worm strain that exclusively relies on ALT for telomere maintenance. Here, I propose to use this unique and powerful model to understand how ALT is activated and the molecular mechanism underlying telomere elongation by ALT in nematodes and mammals.

Maintenance of life requires the preservation of genome integrity. DNA damage is a threat to genome stability and unrepaired damage such as double-strand breaks (DSB) can lead to loss of genetic information, cell death or cancer. Hence cells in all three domains of life have highly conserved mechanisms to repair DSBs. While repair via homologous recombination is most faithful, cells with only a single copy of their chromosome must employ alternative end-joining pathways, which can result in loss of genetic information or mutations. Little is understood about the molecular mechanism of action or regulation of end-joining (particularly in bacteria). This study proposes to establish Caulobacter crescentus as a model system to study DSB repair. While little is known about this process in C. crescentus, my preliminary experiments indicate that an end-joining pathway is active in this bacterium. The project aims to understand the mechanism of end-joining, and to find and characterize the proteins required for DSB repair via this pathway. I will utilize genetic tools developed in the host laboratory such as high-throughput mutagenesis screens and Chromosome-Conformation-Capture, along with traditional genetic read-outs to understand the mechanisms of DSB break repair in C. crescentus and its effects on chromosome function. Studies in this tractable system could provide insight into DSB repair in other pathogenic bacteria as well as humans.

This project combines our collective expertise in developmental biology, neuroscience, engineering, sleep biology and in-vivo molecular imaging to create an innovative chick embryo model for advancing scientific understanding of the developmental origins of sleep, waking and brain rhythms in higher vertebrate animals. Using a novel extension of embryo microsurgical techniques, an accessible port to the embryo head through the eggshell and respiratory membranes will be made at 1/10 of embryonic development (day 2 of a 21-day incubation period), and microscopic devices engineered to measure and wirelessly report brain temperature will be surgically implanted in the brain ventricles of individual embryos, to learn when rhythmic changes in brain temperature first emerge. Eggs are then sealed until 16 days of incubation, when the embryo head will be accessed, and skull electrodes implanted. The electrodes are connected to a miniature electroencephalography (EEG) system specifically engineered for continuous wireless recording from the eggs. Individual eggs will also be attached to a device for recording embryo heartbeat and behavior from vibrations on the eggshell. During continuous EEG and behavioral recording, eggs will have periodic PET/CT images taken of their brains to relate EEG patterns to brain metabolic activity patterns using a novel dynamic imaging procedure with sub-millimeter spatial resolution and a temporal resolution of between 30 seconds and 5 minutes (CT images will be used to developmentally stage individual embryos). This information will allow us to identify characteristics reliably associated with different embryo brain states. This “phenotyping” procedure permits embryo brains to be collected when they either have been in a particular brain state for a certain period of time, or at the transition between brain states, greatly facilitating the histological and molecular characterization of the role of changes in immediate-early gene expression and neurotransmitter system function that previous work has implicated in the control of adult sleep states and brain state changes, and permitting manipulative experiments. This system will significantly accelerate the identification of the neural systems organizing the initial emergence of these important systems brain functions, and of the mechanisms through which they operate.

The exquisitely complex mechanism of the human feet encompasses over a quarter of the body’s bones. How and why did the foot structure evolve to this current form? How do the demands of walking and running stably with low energy consumption affect foot evolution? The arched human foot reduces energy consumption by storing and releasing elastic energy like a bow and string. But does the foot’s elasticity also aid in maintaining stability of gait on uneven terrains? We have assembled an international team of researchers spanning the fields of physics, mathematics, and biomechanics to address how the foot has evolved and how it is controlled to balance the needs for stability and energy consumption during locomotion.
A soft foot smoothes out the ground’s uneven features, which may help stabilize running on realistic, uneven terrains. We expect that the neural system actively controls the softness of the foot by modulating the geometry of its arched form. We verify this hypothesis using a combination of measurements on human subjects running on uneven terrains and mathematical analysis of the foot’s elasticity. Our experiments will measure the spatial distribution of 3D forces under the foot using a newly developed high resolution photoelastic technique. These simultaneous and detailed physiological and mechanical measurements give us the ability to detect subtle changes in the mechanics of the foot, thus enabling the development of an anatomically faithful, mathematically rigorous model of foot function. These models then help us glean invariant principles of foot function used to mathematically design feet that optimally balance stability and energy consumption based on the terrain unevenness. We use these theories to also augment physical replicas of ancestral foot skeletons with muscle-like springs and motors in order to assess their functional capabilities against human performance. We hope, this comparative analysis will explain the time course of foot evolution, from the advent of regular walking 3.6 million years ago, to regular endurance running and emergence of a human-like form 1.5 million years ago. Our results also have implications to the design of prosthetic and robotic feet that could help in striking the right balance between energy storage and stability on real world uneven terrains.

The main goal of this proposal is to develop DNA-origami nanoparticles as immune adjuvants, modulating shape and site-specific modification with various ligands and chemical functionalities that can elicit potent and specific immune responses from targeted subsets of dendritic cells (DCs). Towards the development of vaccines with enhanced potency and specificity of DC activation, we propose to study how nanoscale shape and site-specific ligand functionalization of DNA-origami virus-like nanoparticles impact the magnitude and specificity of DC activation. By systematic screening of immune-pathway activation of DNA-origamis combined with antigens and danger-signals, our adjuvants will provide valuable insights into the complex mechanisms of our immune system.

Monitoring enzymatic DNA repair activity is important for cancer prognosis and treatment. Activity levels can be used to predict a patient’s response to a given drug and assess any resistance that is acquired during therapy. In addition, new DNA repair enzymes as potential drug targets may be discovered by correlating their activity levels within various cancer types. Current methods to measure DNA repair activity however are slow, laborious and often indirect in reporting repair activity. These tedious approaches prevent clinical researchers in measuring activity despite their high relevance to cancer. In this proposal, I discuss the design, synthesis and characterization of fluorescent chemosensors that will allow for a fast and simple assay for DNA repair activity. These sensors are built as small DNA oligomers containing a fluorescent nucleobase and the damaged base of interest, which itself will act as a quencher. Enzymatic repair of the damaged base will then lead to an increase in fluorescent signal, thereby providing a direct read-out of activity. The fluorescent probes will allow for in vitro and in vivo detection of the clinically important O6-methylguanine-DNA-methyltransferase (repair of guanine) and the newly studied ABH3 enzyme (repair of adenine). These probes will be valuable to clinicians who are evaluating the association of these enzymes to cancer therapy and medicinal chemists who are in search for new drug targets.