IST Austria Thesis

One of the most striking hallmarks of the eukaryotic cell is the presence of intracellular vesicles and organelles. Each of these membrane-enclosed compartments has a distinct composition of lipids and proteins, which is essential for accurate membrane traffic and homeostasis. Interestingly, their biochemical identities are achieved with the help of small GTPases of the Rab family, which cycle between GDP- and GTP-bound forms on the selected membrane surface. While this activity switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate decisive signal propagation in space and time is unclear. In my PhD thesis, I present in vitro reconstitution experiments with theoretical modeling to systematically study a minimal Rab5 activation network from bottom-up. We find that positive feedback based on known molecular interactions gives rise to bistable GTPase activity switching on system’s scale. Furthermore, we determine that collective transition near the critical point is intrinsically stochastic and provide evidence that the inactive Rab5 abundance on the membrane can shape the network response. Finally, we demonstrate that collective switching can spread on the lipid bilayer as a traveling activation wave, representing a possible emergent activity pattern in endosomal maturation. Together, our findings reveal new insights into the self-organization properties of signaling networks away from chemical equilibrium. Our work highlights the importance of systematic characterization of biochemical systems in well-defined physiological conditions. This way, we were able to answer long-standing open questions in the field and close the gap between regulatory processes on a molecular scale and emergent responses on system’s level

The Role Of Antibody Subclass In The Pathogenesis Of Pemphigus Vulgaris

A marvel of evolution, the adaptive immune system has the capacity to respond to almost any foreign antigen in a highly specific manner. Antibodies, Y-shaped glycoproteins containing both diverse variable regions responsible for antigen binding and constant regions responsible for effector function, are a key part of this capacity. However, this vast diversity comes with several drawbacks, one of which is the fact that the immune system can deleteriously respond to self-antigens. The focus of this thesis is to characterize the role of class-switching (the changing of antibody constant regions) in the pathogenesis of autoimmune disease, and in particular to trace the lineage of antigen-specific autoreactive B cells by analyzing clonal relationships between antibodies of different constant regions. Analyzing such lineages has the potential to shed light on mechanisms of autoantibody-mediated disease pathogenesis, leading to better understanding of autoimmunity and better therapeutics. The work presented in this thesis focuses on pemphigus vulgaris, or PV, a model antibody-mediated autoimmune disease characterized by a response to the cell adhesion protein desmoglein (Dsg) 3, which holds keratinocytes together in the epidermis. An enigmatic feature of this disease is the predominance of antibodies from the IgG4 subclass during active disease, which ordinarily appears to have few effector functions and may serve as a “brake” on the immune system in the setting of continuous stimulation by antigen. PV patients also display autoantibodies of the IgG1 subclass during disease and remission, but the relationship between IgG1 and IgG4 in the disease in unclear. Because the majority of cases of PV also harbor anti-Dsg antibodies of the IgA1 and IgA2 subclasses, we sought to determine the relationships between autoantibodies belonging to each of these subclasses. First, we address whether the same anti-Dsg variable region, grafted onto either IgG1 or IgG4 constant regions, can show differing affinity or pathogenicity, in order to determine whether antibody subclass is directly modulating pathogenic effect (chapter 2). Finding that the subclass has very little effect on antibody affinity, pathogenicity, or epitope preference, we then sought to determine whether B cells expressing autoantibodies of different subclasses share lineages, indicating common pathways of development (chapter 3). Using a combination of antigen-specific antibody cloning through phage display, and next-generation sequencing of subclass specific repertoires in a panel of PV patients, we managed to trace 80 lineages of anti-Dsg B cells across all four subclasses tested. In particular, we found that anti-Dsg IgG4 B cells, which are believed to be central to disease pathogenesis, tended to not share lineages with other subclasses, and in in general do not appear to share a precursor-product relationship with anti-Dsg IgG1 B cells. We have also found that anti-Dsg IgA1 and IgA2 were tightly related and often arose directly from IgG precursors. These findings are key to understanding the role of class-switching in the pathogenesis of PV, and may shed light on the class-switch mechanisms driving other autoimmune diseases and states of chronic antigen stimulation

Modular analysis of signal transduction networks

Modularity, signaling networks, sytems biologyMagdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2007von Julio Sáez RodríguezZsfassung in dt. Sprach

On the theory of cell migration: durotaxis and chemotaxis

Cell migration is a fundamental element in a variety of physiological and pathological processes. Alteration of its regulatory mechanisms leads to loss of cellular adhesion and increased motility, which are critical steps in the initial stages of metastasis, before a malignant cell colonizes a distant tissue or organ. Consequently, cell migration has become the focus of intensive experimental and theoretical studies; however the understanding of many of its mechanism remains elusive. Cell migration is the result of a periodic sequence of protrusion, adhesion remodeling and contraction stages that leads to directed movement of cells towards external stimuli. The spatio-temporal coordination of these processes depends on the di erential activation of the signaling networks that regulate them at specific subcellular locations. Particularly, proteins from the family of small RhoGTPases play a central role in establishing cell polarization, setting the direction of migration, regulating the formation of adhesion sites and the generation of the forces that drive motion. Theoretical models based on an independent description of these processes have a limited capacity to predict cellular behavior observed in vitro, since their functionality depends intrinsically on the cross-regulation between their signaling pathways. This thesis presents a model of cell migration that integrates a description of force generation and cell deformation, adhesion site dynamics and RhoGTPases activation. The cell is modeled as a viscoelastic body capable of developing active traction and protrusion forces. The magnitude of stresses is determined by the activation level of the RhoGTPases, whose distribution in the cell body is described by a set of reaction-di usion equations. Adhesion sites are modeled as punctual clusters of transmembrane receptors that dynamically bind and unbind the extracellular matrix depending on the force transmitted to them and the distance with ligands on the substrate. Onthe theoretical level, the major findings concern the relationship between the topology of a crosstalk scheme and the properties, as defined in [1], inherited by the associated reaction network as a gradient sensing and regulatory system: persistent and transient polarization triggered by external gradients, adaptation to uniform stimulus, reversible polarization, multi-stimuli response and amplification. This leads to models that remain functional against the biological diversity associated to di erent cell types and matches the observed cell behaviour in Chemotaxis essays [2, 3, 4, 5]: the capacity of cells to amplify gradients, polarize without featuring Turing patterns of activation, and switch the polarization axis and the direction of migration after the source of the external stimulus is changed. The RhoGTPase model, derived on theoretical premises, challenges a long held view on the mechanisms of RhoGTPase crosstalk and suggests that the role of GDIs, GEFs and GAPs has to be revised. Recent experimental evidence supports this idea[6]. In addition, the model allows to recapitulate a continuous transition between the tear-like shape adopted by neutrophiles and the fan-like shape of keratocytes during migration [7] by varying the relative magnitudes of protrusion and contraction forces or, alternatively, the strength of RhoGTPase Crosstalk. The second mechanism represents a novel explanation of the di erent morphologies observed in migrating cells. Di erences in RhoGTPase crosstalk strength could be mediated by di erences between the activity or concentration of GEFs, GAPs and GDIs in di erent cell types; an idea that can be explored experimentally. On cell mechanosensing, a new hypothesis based on a simple physical principle is proposed as the mechanism that might explain the universal preference of cells (bar neurons) to migrate along sti ness gradients. The theory provides a simple unifying explanation to a number of recent observations on force development and growth in real time at cell Focal adhesions [8, 9, 10, 11]. The apparently conflicting results have been attributed to the di erences in experimental set-ups and cell types used, and have fueled a longstanding controversy on how cells prove the mechanical properties of the extra-cellular matrix. The predictions of the theory recapitulate these experimental observations, and its founding hypothesis can be tested experimentally. This hypothesis directly suggests the mechanism that could explain the preference of cells to migrate along sti ness gradients, and for the first time, a plausible biological function for its existence. This phenomenon is known as Durotaxis, and its abnormal regulation has been associated to the malignant behaviour of cancer cells. &nbsp

Evolution of Ionizing Radiation Research

The industrial and medical applications of radiation have been augmented and scientific insight into mechanisms for radiation action notably progressed. In addition, the public concern about radiation risk has also grown extensively. Today the importance of risk communication among stakeholders involved in radiation-related issues is emphasized much more than any time in the past. Thus, the circumstances of radiation research have drastically changed, and the demand for a novel approach to radiation-related issues is increasing. It is thought that the publication of the book Evolution of Ionizing Radiation Research at this time would have enormous impacts on the society. The editor believes that technical experts would find a variety of new ideas and hints in this book that would be helpful to them to tackle ionizing radiation

Atherogenesis

This monograph will bring out the state-of-the-art advances in the dynamics of cholesterol transport and will address several important issues that pertain to oxidative stress and inflammation. The book is divided into three major sections. The book will offer insights into the roles of specific cytokines, inflammation, and oxidative stress in atherosclerosis and is intended for new researchers who are curious about atherosclerosis as well as for established senior researchers and clinicians who would be interested in novel findings that may link various aspects of the disease

More than Clearing the Clutter: The Imperative Role of Efferocytosis in Repair and Immune Reprogramming in the Damaged Nervous System

Evolutionarily, the nervous system and immune system have been intertwined for hundreds of millions of years. In healthy conditions, these systems work diligently to maintain homeostasis and proper functioning. In summation, they keep our bodies moving, our organs operating, our minds thinking, and our bodies safe from foreign pathogenic invaders. However, in the event of a challenge to homeostasis, like a traumatic injury, both systems engage complex signaling cascades to degenerate parts of cells that can’t be saved, protect those that can, remove harmful debris, and regenerate and repair to again obtain homeostasis. A common system to study these complex response mechanisms is that of a peripheral nerve injury. My research over the past several years has been focused around fully understanding the complex immune-nerve communication and consequences that occur following peripheral nerve injury. The work herein keenly elaborates on the time course and content of the immune response after peripheral nerve crush injury. We show that granulocytes are the first to respond with infiltrating monocytes entering a few days later and finally dendritic cells about a week after injury. We however show little evidence of significant immune infiltration into dorsal root ganglia of the sciatic nerve and rather DRG-resident immune cell morphological changes. It is also demonstrated that mesenchymal progenitor cells are key in shaping the inflammatory milieu after injury. The requirement of Csf2 for conditioning-lesion-induced dorsal column axon regeneration is evidenced as well as its role in skewing the inflammatory response. The dynamicity of the immune non-immune responses to nerve injury in wild-type an SARM1 knockout animals at multiple timepoints is compared and contrasted. Finally, we are the first group to show the occurrence of efferocytosis (the phagocytosis of apoptotic cells) in the injured nerve, identify a specific transcriptomic identity for macrophages engaged in this action, and investigate the anti-inflammatory signaling this process propagates.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/170010/1/lucashu_1.pd

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Modelling forward-programmed megakaryocyte culture for manufacturing process development

Advanced therapy medicinal products (ATMPs), which include cell and gene therapies, have the potential to revolutionise the healthcare industry through curing conditions which have previously been manageable at best. ATMP developers face challenges due to the complexity of their products since cells dynamically change, and are changed by, their environment through complex interactions. Defining the relationships between cells and their environment will allow developers to better characterise and control their processes, leading to more consistent and lower risk processes, which in turn would reduce COGs.Here, it was hypothesised that modelling approaches could be used to define complex cell-environment interactions and produce meaningful process improvements for cell-based therapy manufacture. To test this hypothesis, two modelling approaches were applied to a clinically relevant, cell type that would face the same generic challenges a commercial, allogenic cell-based therapy (system productivity and maintaining or producing the required cell quality) – forward programmed platelet precursor cells (megakaryocytes) FOPMKs.The first modelling approach selected was Quality by Design (QbD) due to its acceptance and promotion by regulatory bodies for the manufacture of small molecules. A Quality Target Product Profile (QTPP) was compiled for an in vitro platelet product and Critical Quality Attributes (CQAs) – extracellular marker positive expression of CD41a, CD42a and CD42b and negative expression of CD235a - were identified. In order to reproducibly measure CQAs, substantial analytical development was undertaken to develop a novel flow cytometry assay that measured extracellular marker expression and characterise the differentiation of FOPMKs including on-target and off-target expression.Following assay development statistical Design of Experiments (DOE), was used to link control variables to CQAs. This work showed that concentrations of medium consumables doxycycline (Dox) and thrombopoietin (TPO) correlated with increased yield and CD41a expression. Whereas seeding density and removal of Dox from culture correlated with lower cell yields and lower CD41a expression.The second modelling approach applied was a novel, dynamic, mechanistic modelling approach which showed that cell growth was inhibited, and viable cells were converted non-viable cells as a function of cell.time mediated medium exhaustion. Firstly, the system productivity was found to be approximately 1.48 ± 0.28 × 106 viable cells.mL-1 and the system limitation was a product of the number of cells present and time. Dynamic modelling confirmed this hypothesis and indicated that growth inhibition as a function of medium exhaustion was also present.Dynamic models were also applied to pluripotent cell culture where growth inhibition was shown to be a function of cell density, and the density threshold at which the cells became growth inhibited could be increased through the addition of Rhok Inhibitor in the first 24 hours of culture.Future work for FOPMKs should focus on identifying the root cause of the medium limitation (initial screening showed it was unlikely to be glucose, lactate or ammonium concentrations). Modelling frameworks for phenotypically unstable populations require the ability to handle higher numbers of parameters, or more efficient methods to screen and reduce the number parameters required.</div