Engineered biomaterial systems can be used to answer mechanistic and applied questions of stem cell biology such as how to control stem cell fate decisions, generate populations of differentiated cells, or optimize the regenerative capacity of a biomaterial. Cardiac stem cells (CSCs) have recently been identified and hold significant promise. In vitro they are clonogenic and multipotent, able differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells; in vivo they can modify cardiac healing post-injury. The microenvironment (niche) surrounding CSCs in vivo is thought to provide extrinsic signals that influence CSC fate (quiescence, self-renewal, differentiation, apoptosis, motility). Cell behavior is modulated by a spatio-temporally integrated set of biophysical parameters, but rigorous study of the role extrinsic factors play in CSC fate has not been undertaken. I hypothesize that biomaterial systems with spatio-temporal control over display of matrix, cell, and soluble cues will enable systematic investigation of mechanisms underlying CSC mechanobiology. I propose a HFSP Short Term Fellowship to initiate a collaboration with Garry Duffy (RCSI, Ireland) that integrates our expertise in biomaterial systems and cardiac stem cell biology to quantify the influence of cell-cell and cell-matrix interactions on early CSC fate decisions. We will generate critical pilot data and start preparing joint extramural grant applications. CSC niche analogs will enable mechanistic study of extrinsic regulation of physiological and pathological CSC processes and will prove critical for developing CSC-biomaterial composites to induce cardiac regeneration.

Until recent times our knowledge of the microbes living within the
human body was limited to the small proportion that could be cultured.
However, within the last few years advances in sequencing technologies
have facilitated the first comprehensive surveys of the human
microbiota. These surveys have revealed not only an extraordinary level
of microbial diversity, but also suggest that there has been
significant co-evolution of host and microbiota. The goal of this
project is to carry out large-scale mapping of the host-microbe
interactions that are driving this co-evolution. To achieve this I will
use microarrays to measure host (human) gene expression and use
barcoded pyrosequencing of a portion of the 16S rRNA gene to measure
microbial abundance. These measurements will be carried out in multiple
biopsies of both healthy individuals and those with inflammatory bowel
disease (IBD). Host gene expression profiles will be used to define
disease state as well as identify features of microbial community
structure associated with patterns of host response, and then
comparisons between samples will be carried out to determine the genes
and microbes associated with health and disease. The results will be
used to shed light on the etiology of IBD and more generally on the
nature of host-microbe interactions.

Telomeres are critical for genome maintenance, as they prevent the natural chromosome ends from being recognized as damaged DNA. While telomere length decreases during the replication of normal cells, immortal cancer cells acquire the pathways that keep telomere length abnormally. Understanding the dynamics and differences of telomere regulation in normal and tumor cells is essential for comprehension of chromosome inheritance mechanism and the successful combating of cancer. Human telomeres associate with a number of factors that can influence chromosome integrity, such as the telomeric core complex and proteins involved in damage checkpoints, repair and recombination. Since cohesin, a ring-like complex that holds sister chromatids, is involved in checkpoints, repair and recombination pathways, it is tempting to hypothesize that cohesin acts in telomere maintenance in interaction with these pathways. To deepen our knowledge about telomere dynamics in both normal and cancer cells, I propose two specific aims: AIM 1 will focus on the novel role of cohesin in telomere maintenance, and AIM 2 is designated to explore telomere dynamics in cancer cells. I will examine the cell cycle dependent localization of cohesin at telomeres and the telomeric effects of cohesin suppression in AIM 1. Since SMC1, a cohesin subunit, is phosphorylated by damage checkpoint kinase, I will also investigate the importance of the phosphorylation in telomere dynamics. In AIM 2 I will study the dynamic binding of proteins and the replication pattern at telomeres in different types of immortal cells, with the goal to utilize the results to develop targeting strategies for different cancer cell types.

During sky compass navigation migratory monarch butterflies integrate the time of day with
skylight cues to generate a correct flight vector. How this integrative
processing is achieved in the brain will be addressed in the proposed project.
Electrophysiological, anatomical and molecular tools will be used to address three aims. First, the anatomy
of the monarch butterfly brain and the organization of regions relevant for
navigation will be studied. Differences in gene transcription levels in compass
related neuropils will be examined between migratory and non migratory monarch
butterflies by microarray analysis. Second, intracellular recordings will be
developed to characterize neurons involved in the processing of compass cues,
especially polarized light and spectral gradients. Recordings will compare both
butterfly forms and focus on brain regions characterized in aim 1. Third,
influences of clock-gene expressing neurons on the compass system will be
identified to find the neural correlates of time compensation. As such cells
have been found in the central complex, and this brain region contains a map of
compass directions in the locust, it is a potential interface between the
compass and the clock system. To test this, activity of clock neurons as well
as their connection to compass cells will be characterized. RNAi-mediated gene
silencing will be used to disrupt the internal clock and identify effects on
the activity of compass and clock cells.
The development of an electrophysiological approach to the monarch butterfly will
combine this method with molecular biology and significantly advance our
understanding of time-compensated compass navigation.

Gangliosides are glyco-sphingolipids present on most vertebrate tissues, but are over expressed in several tumor cells and are fundamental for tumor biology. Their own self and glycolipidic nature makes them poorly immunogenic. The findings in healthy individuals and cancer patients of circulating anti- ganglioside antibodies (Abs) indicate that specific B cells have not been clonally deleted. Some anti-ganglioside Abs have proved to be involved in B and T lymphocyte idiotypic networks and could mediate the activation of cells able to regulate the immune response against these self antigens. Many anti-ganglioside Abs are of germline origin and encoded by genes preferentially used by B-1 cells, which suggest the involvement of B-1 cells in the immunity against gangliosides. The active mechanisms controlling the immune response against non-protein antigens like gangliosides are still unknown, but B-1 cells and natural Abs could be involved. This fellowship would allow me to acquire experience in a laboratory with high expertise and cutting-edge technology, nonexistent at the moment anywhere in my home country, to purify different B-1 cell populations and study their phenotype and cytokine secretion in the presence of gangliosides and anti-ganglioside Abs. This study could prove the existence of natural anti-ganglioside Ab networks and define the role of B-1 cells in the regulation of the immune response against glycolipidic antigens. These results could be useful not only in the development of active specific anti-tumor glycovaccines, but also in the elucidation of glycolipid responses and the function of B cell subsets.

This project aims at addressing an important question in evolutionary developmental biology: Which genes show a different expression pattern between species and what is the underlying mechanism? There is strong support that highly conserved non-protein-coding elements (CNEs) are often associated with transcriptional regulation of crucial developmental genes. I will conduct a systematic computational search for genomic regions that are highly conserved in many species but lost in another species. Since strong conservation implies function, the underlying hypothesis is that loss of such a CNE leads to changes in the expression pattern of a target gene. This hypothesis will be experimentally tested by (i) verifying a cis-regulatory role for the CNE and (ii) comparing the expression pattern of a gene having the CNE to a gene lacking the CNE. For example, the experiments might reveal that (i) a CNE is a brain-specific transcriptional enhancer and that (ii) the target gene in a species having this CNE is expressed in brain while the target gene in another species lacking this CNE is not expressed in brain. This computational and experimental hybrid approach allows to associate CNE loss with divergence in gene expression. The goal of this project is to provide insight into the evolution of CNEs, to contribute to our understanding of gene expression regulation by conserved regions and to associate diverged gene expression with lost conserved elements.

The genes in our DNA encode messenger RNA molecules, which, in turn, encode proteins. Proteins are the molecules in cells that perform most of the tasks that make life possible; they are the catalysts that make chemical reactions happen, they act as structural molecules that hold cells together, and they help other molecules such as nutrients move into and out of cells. Machines called ribosomes inside of cells read, or “translate,” the information in messenger RNAs to make the encoded proteins. When this process is not done correctly diseases such as cancer can result. In addition, when we are infected with a virus, the virus hijacks our cells’ ribosomes and associated protein synthesis machinery to make copies of itself. A deeper understanding of how protein synthesis works will facilitate attempts to cure diseases such as these. Our project aims to elucidate the structure of the protein synthesis machinery as it begins to assemble on an mRNA in preparation for making the corresponding protein. This information will help us in the same way that an automobile mechanic is helped by knowing the details of how an engine is put together; it will tell us which pieces go where, what they look like, and what each one does. These pictures will dramatically advance our understanding of this central biological process.

The proposed project aims to establish a new intercontinental, interdisciplinary collaboration between the Laboratory of Integrative Neuroscience, University of Buenos Aires, Argentina, headed by Prof. Mariano Sigman, and the visiting researcher Dr. Lisa Holper, Biomedical Optics Research Laboratory (BORL), University Hospital Zurich, Switzerland. Specifically we aim to introduce a new brain imaging approach by combining our novel portable, wireless functional near-infrared spectroscopy (fNIRS) instrument developed by the visitor’s laboratory with a 3D tracking device developed by the host laboratory. To show the potential of the new approach, we will conduct a pilot study focusing on two major advantages of fNIRS, namely reduced motion restriction in freely moving subjects and the possibility to simultaneously record from two interacting subjects. The experimental design will concentrate on fundamental cognitive-motor functions, by means of human cooperative versus competitive interactions, by monitoring two players in a simplified version of an air-jockey game. The actor’s and opponent’s cortical activation will be recorded by fNIRS in combination with a 3D tracking device to trace motions of the ball and the players’ hands. The context of the proposed collaboration offers a very challenging and exciting materialization of a technological advancement which will open a new vehicle for experimentation in neuroscience. The research visit aims to contribute both to the host laboratory’s technological achievements as well as the visitor’s clinical and scientific development.

The ability of a cell to produce all cell types of the body upon differentiation is known as pluripotency. In the early mammalian embryo, cells loose this capability to become restricted to a particular lineage. This loss of developmental potency occurs despite these cells possessing full genetic capability. It is thought that epigenetic alterations of DNA play a large part in this restriction process. This project aims to examine the role of epigenetic modification, specifically DNA methylation, in the maintenance of pluripotency and how this might be modified to restrict cells to a particular lineage.

On the basis of differential methylation patterns of gene promoters between pluripotent and committed cell types, several candidates for the regulation of pluripotency and lineage restriction have been identified (Farthing et al., 2008). Using novel techniques, other sites of differential methylation that likely affect early differentiation will be identified. Specifically, genome wide insulator sites that organise the three-dimensional structure of DNA and expression of neighbouring genes will be analysed for methylation changes during early development.

Genes affected in early development by changes in methylation at their promoter (identified by Farthing et al., 2008), or at nearby insulators (this study), will be tested for an effect on developmental potency by forced expression or knockdown in pluripotent embryonic stem cells. In doing so, these experiments aim to uncover a new understanding of pluripotency and how this might be manipulated for transgenic research and regenerative medicine, as well as provide novel insight into how the mammalian genome is interpreted.

Accurate segregation of the genetic material during mitosis is crucial for the maintenance of life. Chromosome segregation requires the formation of sturdy attachments between chromosomes and a microtubule-based structure named the mitotic spindle. The site of attachment of spindle microtubules onto chromosomes is named the kinetochore. Kinetochores are composed of 60-80 evolutionarily conserved proteins that form a structure up to 500 nm in diameter in metazoan cells. This is an order of magnitude larger than the largest macromolecular complexes such as RNA polymerase and the ribosome that are currently amenable to structural and enzymatic studies.

The characterization of the organization of kinetochores and their microtubule-binding activity is hampered by the inability to purify functional kinetochores from cells. The goal of our multidisciplinary international collaboration, therefore, is to reconstitute, from recombinant proteins, a functional kinetochore-microtubule interface. The microtubule-binding activity is provided by the so-called KNL1-Mis12 complex-Ndc80 complex (KMN) network, which contains ten proteins in three distinct complexes. Specifically, we propose to use an array of synthetic and analytic approaches to (i) rebuild the KMN network from purified proteins; (ii) evaluate how the immobilization of KMN network complexes on different nano-patterned substrates influences the ability to bind microtubules; (iii) study the dynamics of these arrangements using single-molecule fluorescence resonance energy transfer, total-internal-reflection fluorescence microscopy, and real-time atomic force microscopy; (iv) study the structural organization of the KMN networks by X-ray crystallography, electron microscopy and atomic force microscopy; and (v) test the significance of our conclusions for kinetochore-microtubule attachment in the genetically tractable yeast Saccharomyces cerevisiae.

We believe that our synthetic kinetochore-microtubule interface will eventually become accessible to the type of structural and enzymatic studies that continue to provide so much new understanding of the workings of other smaller biomolecular machines such as polymerases and the ribosome. Our studies will illuminate the process of kinetochore-microtubule attachment and will greatly advance the comprehension of the molecular bases of chromosome segregation.

Sensation of pain is triggered by specialized sensory neurons or nociceptors that display net-like processes directly beneath the skin. Unfortunately, the subjective nature of pain sensation, as well as the complexity of the underlying neuronal circuits in higher organisms makes it extremely difficult to study. Yet, essential principles of pain sensation could be analyzed in “simple” model systems such as Caenorhabditis elegans. This tiny nematode offers an unrivalled potential to use state-of-the-art technologies, as it displays just 302 neurons of which all synaptic connections are known. The sensory FLP and PVD neurons envelop the nematode with highly branched dendritic arbors and preliminary evidence indicates that conserved ion channels mediate specific responses of these neurons to “harsh touch” nociceptive stimuli. In this project, I will employ novel light-dependent regulators of neuronal activity (optogenetic tools), i.e. blue light-activated depolarizing Channelrhodopsin (ChR2), and yellow light-driven hyperpolarizing Halorhodopsin (HR), to define how the behavioural output of this FLP-PVD nociceptor network is evoked in living animals, and how it depends on physiological properties of the neurons involved. Furthermore, I will characterize putative nociceptive genes, as revealed by genetic analysis of PVD microarray profiles, by combining optogenetics, electrophysiology, optical Ca2+ imaging and (automated) behavioural assays using video tracking. My findings can provide key insights in pain sensation that are likely to apply also to higher organisms.

Most cancers originate from epithelial cells that have pronounced apico-basal polarity. Loss of cell polarity in these cells triggers tumor development. Against the tumor development, epithelia have an intrinsic mechanism that eliminates oncogenic polarity-deficient cells from the tissue, which acts as an “intrinsic tumor suppression”. In Drosophila imaginal epithelia, clones of cells mutant for evolutionarily conserved tumor suppressors, such as scribble or discs large, lose their polarity and are eliminated from the tissue. This elimination of polarity-deficient cells is caused by ‘cell competition’, the cell-cell communication-based cell elimination mechanism. When cell competition is genetically inactivated, polarity-deficient cells not only escape the elimination but grow aggressively and develop into tumors. Cell competition is thought to play an important role in organ size control, tumorigenesis, cancer progression, and maintenance of stem cell in the niche, but the underlying mechanism is poorly understood. The phenomenon in which ‘pre-malignant’ polarity-deficient cells are eliminated from epithelia by cell competition can be used as a ‘sensitized’ cell-competition assay system that has never been established. Using this system, I have developed a novel “non-cell autonomous” genetic screen system for identifying factors that regulate cell competition. In the proposed study, I will first take two parallel approaches, non-cell autonomous genetics and live-imaging approach, to dissect the molecular basis of cell competition, followed by a mathematical modeling to elucidate the universal principle of epithelial intrinsic tumor suppression governed by cell competition.

The unicellular eukaryote Plasmodium establishes malaria infection when parasites delivered by a mosquito bite invade and expand in the host liver. The onset of disease follows when these parasites spread to the blood. The initial parasite expansion within hepatocytes of the liver produces merozoites, the developmental stage of Plasmodium that infects erythrocytes and leads to disease symptoms. The infection of erythrocytes also results in the production of merozoites, thus propagating the blood stage of infection. The host cell pathways that are required for merozoite formation in either cell type and the mechanisms used by the parasites to manipulate these host pathways are largely unknown. The objective of this proposed research is to understand the molecular mechanisms by which Plasmodium parasites are able to create an environment that supports replication inside mammalian cells. To identify host pathways involved in merozoite formation, we will target candidate proteins with small interfering RNA in infected hepatocytes. In parallel, we will examine the association of host organelles with the intracellular Plasmodium-containing vacuole and determine the functional significance of any associated host compartment. To identify parasite proteins that usurp shared host cell processes in hepatocytes and erythrocytes, we will examine and characterize exported Plasmodium proteins produced during both stages of infection. Completion of the proposed research will identify host and parasite proteins important for Plasmodium infection of multiple mammalian cell types and help define the minimal requirements necessary to support intracellular parasite replication.

Mechanisms by which organ specific stem cells initiate differentiation (or cell cycle exit) remain largely unknown. In addition it is poorly understood, how cell cycle regulators post-translationally regulate organogenesis. I here propose to explore the link between the cell cycle and retinal development by examining the function and regulation of Id proteins (Inhibitor of differentiation) in retinal stem cells. By utilizing retina of medaka fish (Oryzias latipes), which is amenable to genetic and cell biological analyses, I will first determine the functions of Id in the proliferation of retinal stem cell. In addition, to complement limitations of the medaka retinal system with respect to biochemical analysis, I will be building on my previous expertise and will be using frog (Xenopus laevis) eggs to biochemically address the molecular regulation of Id. For this experiment, I will test two hypotheses concerning the interaction of Id with the APC/C, a ubiquitin ligase specifically degrading cell cycle regulators: (1) The APC/C destroys Id to allow differentiation of retinal stem cell, and (2) Id inhibits the APC/C to maintain stem cells with pluripotency. Finally, on the basis of the results from the Xenopus oocyte system, I will genetically test the models using the medaka fish retina. These results will allow developing the control of cellular differentiation by the cell cycle as new field in developmental biology, which will significantly impact also on other biological processes, such as diseases derived from genetic or developmental abnormalities.

Osteoclasts are the only somatic cells with bone-resorbing capacity and they play a critical role not only in normal bone homeostasis (called "bone remodeling") but also in the pathogenesis of bone destructive disorders such as rheumatoid arthritis and osteoporosis. A major focus of research in the field so far, however, has been on gene regulation by various cytokines, and several key questions about the mode of action of these cells in vivo have been less well studied, including the process of recruitment of osteoclast precursors to their workplace (bone surface) and regulation of terminal maturation. To address these issues, in addition to conventional experimental techniques including biochemistry, molecular biology, and gene-targeting, I am utilizing a new experimental method involving intravital two-photon microscopy for bone tissues that was developed during my postdoctoral research experience. One major subject of the planned work is better defining how the migration and positioning of osteoclast precursors is influenced by a combination of a lipid mediator (e.g. S1P) and chemokines (CXCL12 and CX3CL1). I also plan to analyze osteoclastogenic cell fusion and the role of cell-cell contact in osteoclastogenesis in vivo. Furthermore, recent computational biology advances will be exploited in this proposed study to develop a multiscale mathematical model of osteoclast behavior, helping us to better understand in quantitative terms how bone remodeling is maintained by osteoclast function. Beyond its basic implications, the findings of this multidisciplinary study may lead to insights into human diseases and contribute to new therapeutic strategies.

Heme, consisting of an iron atom contained in the center of a porphyrin ring, is one of the most important and versatile prosthetic groups of proteins in both prokaryotes and eukaryotes. However, when heme is released from proteins, it is toxic to cells. Detoxification of free heme is essential to the survival of malaria parasites. During the growth of malaria parasites within the red blood cell the parasite obtains nutrients by taking up and then digesting hemoglobin, which releases high quantities of free heme. The parasites convert free heme into an insoluble crystalline form called hemozoin that is not toxic to cells. Although several mechanisms have been proposed for the production of hemozoin, the molecular mechanism is highly controversial. Crystal structure of hemozoin is made of a cyclic dimmer of heme in which an iron-oxygen coordinate bond links the central iron of one heme to the oxygen of the propionate group of the adjacent heme. The reciprocal iron-oxygen bonds between two heme molecules are unusual and have not been observed in any other porphyrin dimmer, which could involve a particular kind of enzymatic reaction mechanisms. The objective of the present research project is to reveal the process of heme crystal formation in the parasites. I wish to develop a combined biochemical and biophysical approach to analyze the unusual enzymatic reaction of the hemozoin formation. Since the formation of hemozoin is essential to the survival of the parasites, elucidation of the molecular mechanism of the hemozoin formation will help to develop anti-malarial drugs.

Formation, homeostasis and function of tissues and organs depend heavily on a network of cell-to-cell signals that are transmitted through soluble molecules, cell surface bound and transmembrane proteins. Semaphorins and plexins are two protein families with central roles in cell-cell signaling. As such they are found in most tissues where they are often required for viability and involved in a wide range of processes such as guidance of cell migration, neural connectivity, cancer, angiogenesis and regulation of immune responses.

Members of the semaphorin family are secreted or cell surface bound proteins and as such can mediate both long- and short-range signals or act as receptors themselves. Plexins are large (around 1900 residues) single transmembrane spanning molecules and the most important receptors for semaphorins. Although much is known on the cellular function and effects of semaphorin-plexin signaling very little is known on the molecular mechanisms underlying this signaling system.

The goal of the proposal is to elucidate the molecular mechanism underlying semaphorin-plexin signaling. I will use a combined approach of biophysical analysis, cell based fluorescence and functional assays, and structural biology techniques to resolve in detail the structural and oligomeric changes during semaphorin-plexin complex formation and relate these to its functional properties.

Given the importance of semaphorins and plexins in a wide range of functions understanding of the molecular mechanisms and detailed insights into the interactions will be instrumental in the development of new therapies in neural regeneration, immune disorders and cancer.

Bacterial communities in the plant rhizosphere carry out functions that are essential to plant nutrition and disease suppression in terrestrial ecosystems. New cultivation-independent approaches using molecular tools now offer great promise for exploring the genetic diversity of these microorganisms and learning how bacterial communities function to support plant growth and root health. In the research described here, I propose to study the bacterial communities that are associated with the roots of the oldest living plants on Earth. These plants, Creosote bush (Larrea tridentata), are the predominant shrubs in the deserts of the S.W. USA, with some individuals having an age of more than 10,000 years. The long term selection and coevolution of microbial communities with the roots of these plants provides a unique opportunity to examine how certain bacteria may help plants to acquire nutrients and resist disease in poor desert soils, such as we share in the Atacama Desert in Chile and the Mohave Desert in the USA. To carry out this research, I will work with the Crowley research group at the University of California, Riverside, where we will use a metagenomics approach to characterize the bacterial communities and identify genes that encode potentially novel bioactive molecules. This research will provide me with valuable training in metagenomics, and will establish a new collaboration between our research groups. If we are successful, this research will further provide fundamental knowledge on how soil microbial communities function for adapting plants to harsh desert environments.

Many aspects of metabolism and behavior are controlled by circadian (24hs) rhythms. These rhythms rely on intracellular oscillations of clock proteins that have been proposed to be generated by interconnected transcriptional feedback loops. However, and despite the extensive work conducted in the past twenty years, it is still controversial how these clocks keep time. During my post-doc I established the importance of transcriptional oscillations for circadian timekeeping and discovered that miRNAs play a central role in circadian rhythms in Drosophila. miRNAs are small non-coding RNAs that serve as post-transcriptional regulators of gene expression. Through the development of new tools for the study of these small RNAs in fly heads, I was able to determine that the miRNA bantam is a central clock component and regulates the master circadian transcription factor Clk. In addition, I identified miRNAs expressed in the head circadian tissue. The present research plan aims to elucidate the molecular mechanism underlying circadian rhythms with special emphasis on the transcriptional and miRNA regulatory pathways. My specific aims are to: a) study a new transcriptional component of the circadian clock; b) characterize the post-transcriptional control of CLK mRNA; c) characterize temporally and spatially the expression and function of circadian relevant miRNAs. The present project will also deepen the current understanding of miRNA regulation and function in the brain. The HFSP fellowship was essential for the emergence of my current research project, and the CDA will provide the financial support and scientific independence to develop it.

The aim of this proposal is to develop a novel generalisable small molecule biosensor for the in vivo quantification of the phytohormones brassinosteroid and auxin in plant signalling. Although small molecules are universal informational signals, there is currently no available technology for monitoring their abundance in high temporal and spatial resolution. Since hormones are essential for plant growth and development, the model plant Arabidopsis thaliana will be used as a proof of concept system for developing and assessing a novel hormone biosensor, which will be engineered based on molecular evolution and rational design. Once a brassinosteroid and auxin sensor system is developed and validated, it can be utilised for investigating the highly interconnected networks of hormone signalling in plants in response to differential developmental stages and stimuli. Furthermore, the technology of the generalisable hormone biosensor can be applied to other model organisms and small molecules, thus enabling the detailed characterisation of small molecule induced signalling in any biological system.