Validation of an Experimentally Motivated Predictive Model for the Spontaneous Clustering of Receptors on the Cell Membrane

Membrane receptors are specialized membrane-bound proteins that facilitate communication be- tween the intracellular and extracellular membrane. They control signal initiation in many im- portant cellular signaling pathways. Cell signaling (or signal transduction) provides the logical inputs individual cells need in order to perform their role in the context of the organism. Signal- ing molecules such as hormones, neurotransmitters or growth factors, are secreted by cells in the organism as a result of certain conditions; the cells receiving the signal change (or maintain) their state in response to the signaling input. The incoming information is processed and the response is formulated by a complex bio-molecular network.;For many ligand / receptor families, receptor dimerization or cross-linking is a necessary step for activation, making signaling pathways sensitive to the distribution of receptors in the membrane. Microscopic imaging and modern labeling techniques reveal that certain receptor types tend to co-localize in clusters. The origin of these clusters is not well understood; they are likely not the result of chemical binding, but of a pre-existing micro-domain structure of the membrane. In this work, we analyze a set of micrographs resulting from a study of vascular endothelial growth factor (VEGF) receptor. VEGF is a protein that is involved in the process of the growth and maintenance of blood vessels. The micrographs represent static snapshots of VEGF receptors. They are obtained by fixing the cells from a cell culture, separating their cell membrane, and then labeling the receptors with nano-gold particles. The samples are then imaged by high-resolution transmission electron microscopy (TEM).;The first part of the work presented here consists of characterizing the two dimensional point di- stirbutions obtained by identifying the location of the labelled receptor particles. We first applied a number of statistical tests used to establish whether the distributions are consistent with random placement, and whether clustering was present. After establishing the presence of clustering in vir- tually all images, we proceeded to separate the points in each image into clusters using hierarchic distance based clustering. This method relies on a characteristic length scale that is not a priori identified. Building on previous work, we developed a more refined approach to the identification of an optimal length parameter. We implemented this approach to cluster identification as well as a procedure that assigns a geometric shape to each cluster, in computer script that performs all of these analyses for a set of files. Using the analysis pipeline, we processed approximately 80 images that were available and summarized a number of image parameters, measures of clustering, as well as distributions of cluster sizes.;The second part of the dissertation aims to develop and validate a stochastic model of clustering, based on the hypothesis of pre-existing domains that have a high affinity for receptors. The proxi- mate objective is to clarify the mechanism behind cluster formation, and in the longer perspective, to estimate the effect on signaling. We showed that the observed particle distribution results were consistent with the random placement of receptors within the clusters and, to a lesser extent, the random placement of the clusters on the cell membrane. We then defined a simple statistical model, based on the pre-existing domain hypothesis, to predict the probability distribution of cluster sizes.;The model parameters can be identified by fitting to the experimentally derived cluster size distri- butions. Using a Metropolis-Hastings algorithm, we found that the majority of the images (close to 75%) could be fit individually. The remaining images exhibited large scale features that were not meant to be captured in the model. The global fit of the 60 images with a single model pa- rameter set was less successful. We obtained better results by separating the images into groups using k-means clustering, and then performing global fits to each group taken separately. The bi- ological significance of these emerging groups is not clear at the moment; however, the process yielded sets of parameter values that can readily be used in dynamical calculations as estimates of the quantitative characteristics of the clustering domains

Cellular interactions in the tumor microenvironment: the role of secretome

Over the past years, it has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells, fibroblasts, endothelial cells, adipocytes, and extracellular matrix. These components of the tumor microenvironment and the neoplastic cells interact with each other providing pro and antitumor signals. The tumor-stroma communication occurs directly between cells or via a variety of molecules secreted, such as growth factors, cytokines, chemokines and microRNAs. This secretome, which derives not only from tumor cells but also from cancer-associated stromal cells, is an important source of key regulators of the tumorigenic process. Their screening and characterization could provide useful biomarkers to improve cancer diagnosis, prognosis, and monitoring of treatment responses.Agência financiadora Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) FAPESP 10/51168-0 12/06048-2 13/03839-1 National Council for Scientific and Technological Development (CNPq) CNPq 306216/2010-8 Fundacao para a Ciencia e a Tecnologia (FCT) UID/BIM/04773/2013 CBMR 1334info:eu-repo/semantics/publishedVersio

From Graph Coloring to Receptor Clustering

1. Hued colorings for planar graphs, graphs of higher genus and K4-minor free graphs.;For integers k, r \u3e 0, a (k,r) -coloring of a graph G is a proper coloring of the vertices of G with k colors such that every vertex v of degree d(v) is adjacent to vertices with at least min{lcub}d(v) ,r{rcub} different colors. The r-hued chromatic number, denoted by Xr (G), is the smallest integer k for which a graph G has a ( k,r)-coloring. A list assignment L of G is a function that assigns to every vertex v of G a set L(v) of positive integers. For a given list assignment L of G, an ( L,r)-coloring of G is a proper coloring c of the vertices such that every vertex v of degree d(v) is adjacent to vertices with at least min{lcub} d(v),r{rcub} different colors and c(v) epsilon L(v). The r-hued choice number of G, XL,r(G), is the least integer k such that every list assignment L with | L(v)| = k, ∀ v epsilon V(G), permits an (L,r)-coloring. It is known that for any graph G, Xr(G) ≤ XL,r( G). Using Euler distributions, we proved the following results, where (ii) and (iii) are best possible. (i) If G is planar, then XL,2(G) ≤ 6. Moreover, XL,2( (G) ≤ 5 when Delta (G) ≤ 4. (ii) If G is planar, then X2( G) ≤ 5. (iii) If G is a graph with genus g(G) ≥ 1, then XL,2 (G) ≤ ½ 7+1+48gG .;Let K(r) = r + 3 if 2 ≤ r ≤ 3, and K(r) = 3r/2+1 if r≥ 4. We proved that if G is a K4-minor free graph, then (i) Xr(G) ≤ K(r), and the bound can be attained; (ii) XL,r(G) ≤ K( r)+1. This extends a previous result in [Discrete Math. 269 (2003) 303--309].;2. Quantitative description and impact of VEGF receptor clustering .;Cell membrane-bound receptors control signal initiation in many important cellular signaling pathways. Microscopic imaging and modern labeling techniques reveal that certain receptor types tend to co-localize in clusters, ranging from a few to hundreds of members. Here, we further develop a method of defining receptor clusters in the membrane based on their mutual distance, and apply it to a set of transmission microscopy (TEM) images of vascular endothelial growth factor (VEGF) receptors. We clarify the difference between the observed distributions and random placement. Moreover, we outline a model of clustering based on the hypothesis of pre-existing domains that have a high affinity for receptors. The observed results are consistent with the combination of two distributions, one corresponding to the placement of clusters, and the other to that of random placement of individual receptors within the clusters. Further, we use the preexisting domain model to calculate the probability distribution of cluster sizes. By comparing to the experimental result, we estimate the likely area and attractiveness of the clustering domains.;Furthermore, as VEGF signaling is involved in the process of blood vessel development and maintenance, it is of our interest to investigate the impact of VEGF receptors (VEGFR) clustering. VEGF signaling is initiated by binding of the bivalent VEGF ligand to the membrane-bound receptors (VEGFR), which in turn stimulates receptor dimerization. To address these questions, we have formulated the simplest possible model. We have postulated the existence of a single high affinity region in the cell membrane, which acts as a transient trap for receptors. We have defined an ODE model by introducing high- and low-density receptor variables and introduce the corresponding reactions from a realistic model of VEGF signal initiation. Finally, we use the model to investigate the relation between the degree of VEGFR concentration, ligand availability, and signaling. In conclusion, our simulation results provide a deeper understanding of the role of receptor clustering in cell signaling

Regulation of Tie2 Extracellular Complex Formation in Angiogenesis

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α5β1 and αVβ3, providing a convenient mechanism for the integration of extracellular stimuli. Our data confirm the interaction between Integrins and Tie2 and additionally indicate an interaction with the orphan co-receptor Tie1. To elucidate the biological role of these macromolecular complexes, biochemical and biophysical methods including co-immunoprecipitation, FRET microscopy, and cellular based assays were used to follow receptor/Integrin association in response to the Tie2 ligands Angiopoietin-1 and -2 as well as the Integrin ligand fibronectin. Furthermore, structural analysis by small angle x-ray scattering of Tie2-ligand complexes and specific Integrin and Tie complexes are being used to identify the basis for growth factor receptor and Integrin signal transduction

Single cell decisions in endothelial population in the context of inflammatory angiogenesis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 161-171).Normalizing angiogenesis is a promising strategy for treatments of cancer and several disorders plagued by misregulated blood supplies. To address the daunting complexity of angiogenesis arising from multiple phenotypic behaviors governed by multiple stimuli, computational approaches have been developed to predict sprouting angiogenic outcomes. In recent years, the agent based model, in which individual cells are modeled as autonomous decision making entities, has become an important tool for simulating complex phenomena including angiogenesis. The reliability of these models depends on model validation by quantitative experimental characterization of the cellular (agent) behaviors which so far has been lacking. To this end, I develop an experimental and computational method to semi-automatically estimate parameters describing the single-cell decision in the agent based model based on flow cytometry aggregate headcount data and single cell microscopy which yields full panel single cell trajectories of individual endothelial cells. Applying thees method to the single cell decision data, I propose two conceptual models to account for the different state transition patterns and how they are modulated in the presence of opposing inflammatory cytokines. The observed unique state transition patterns in the angiogenic endothelial cell population are consistent with one of these descriptions, the diverse population model (DPM). The DPM interpretation offers an alternative view from the traditional paradigm of cell population heterogeneity. This understanding is important in designing appropriate therapeutic agents that take effect at the cellular level to meet a tissue level therapeutic goal.by Tharathorn Rimchala.Ph.D

Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Development of Targeted Pro-Angiogenic Therapies for Ischemic Diabetic Foot Ulcer Repair

Between 15-25% of diabetic patients develop chronic foot ulcers within their lifetime. These ulcers are characterized by delayed wound healing, are highly susceptible to infection, and can lead to lower-limb/foot amputation. Impaired ulcer healing is most often due to ischemia and insufficient formation of new vessels in the wound bed, as well as rapid turnover of healing tissue by excessive matrix metalloproteinase (MMP) activity. To improve healing of chronic ischemic wounds, researchers have sought to increase angiogenesis at the wound site using growth factors such as vascular endothelial growth factor (VEGF). VEGF is a key angiogenic mediator and uniquely participates in multiple aspects of wound healing including revascularization, reepithelialization, and collagen deposition. However, the clinical success of growth factor therapies such as VEGF has been limited largely due to the overexpression MMPs in the wound environment that degrade or inactivate the growth factors. Furthermore, non-targeted angiogenic growth factor therapies raise significant concerns because they are not restricted to the wound site, but potentially diffuse into systemic circulation and cause malignancies. This thesis reviews current advances in understanding the pathogenesis and pathophysiology of the compromised wound healing environment leading to DFUs with particular emphasis on the roles of neovascularization and matrix remodeling. It also addresses recent progress in VEGF therapy for DFUs and the current limitations in clinical translation imposed by co-existing pathophysiological defects of diabetic wound healing. Given the current understanding of the impaired healing components suggesting a need to correct multiple derangements while maintaining efficacy in the complex and highly proteolytic environment, we propose multiple collagen-targeted, degradation resistant, proangiogenic therapies to activate and potentiate VEGF pathways and simultaneously protect existing collagen matrices. Specifically, we discuss the development of VEGF-loaded collagen-binding nanoparticles and two variants of engineered decorin mimetics functionalized with pro-angiogenic VEGF-mimicking or αvβ3 integrin-binding peptides to increase vascularization of the wound bed. Both the nanoparticles and the angiogenic proteoglycan mimics can be targeted to endogenous collagen or exogenous collagen dressings by a collagen-binding peptide and could be used in combination to exploit VEGF activation and potentiation. This project (i) develops a thermosensitive nanoparticle VEGF-delivery system, (ii) develops and characterizes pro-angiogenic peptide-functionalized decorin mimics, and (iii) assesses the in vitro and in vivo angiogenic potential of these pro-angiogenic decorin mimics as proof-of-concept supporting their potential to accelerate ischemic dermal wound healing in animal models

Characterization of endothelial cells of lymphatic vessels

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice only tumours of the blood vessel endothelium (haemangiomas) have been thus far reported. In the first part of this thesis is described a highly reproducible method for the induction of benign tumours of the lymphatic endothelial cells (lymphangioma) in mice, by intraperitoneal injection of incomplete Freund's adjuvant. Different criteria have been used in order to establish the nature of the induced lesion. Morphological and histophatological studies of the tumour developed in the peritoneal cavity revealed the presence of cells at various levels of vascular development. Expression of the endothelial markers PECAM/CD31, ICAM-l/CD54, ICAM 2/CDI02 as well as the vascular endothelial growth factor (VEGF) receptor Flk-I, the endothelial cell specific receptors Tie-I, Tie-2, and the lymphatic endothelial specific Flt-4 was identified. When the lesion was induced in ~- galactosidase knock-in Flt-4+/- mice, the tumour endothelia could be stained blue in a number of tumour cells. Tumour-derived cells were propagated in vitro where they spontaneously differentiated, forming vessel-like structures. This evidence leads to the conclusion that this is the first experimental protocol for the induction of a lymphatic endothelium hyperplasia in mice peritoneum. The second part of this thesis describes the use of this model system to investigate the profile of chemokine expression in murine lymphangiomas and in lymphangioma-derived lymphatic endothelial primary cultures. Chemokines are a superfamily of small, secreted chemoattractant molecules that plays a key role in the immune cell trafficking. Although production of chemokines by vascular endothelial cells has been extensively documented, there is much less information regarding the lymphatic endothelium. The reported results are the first detailed analysis of chemokine production by lymphatic endothelial cells. Chemokines belonging to all three subfamilies (CXC, CC and C), were found to be expressed in lymphangioma. Among these molecules is remarkable the identification of CIO, a molecule previously identified only in the bone marrow. The molecular as well as functional assays performed provide an indication of the signals that mediate the recruitment of leukocytes into lymphatic vessels