TY - THES
AB - Synthesis of proteins – translation – is a fundamental process of life. Quantitative studies anchor translation into the context of bacterial physiology and reveal several mathematical relationships, called “growth laws,” which capture physiological feedbacks between protein synthesis and cell growth. Growth laws describe the dependency of the ribosome abundance as a function of growth rate, which can change depending on the growth conditions. Perturbations of translation reveal that bacteria employ a compensatory strategy in which the reduced translation capability results in increased expression of the translation machinery.
Perturbations of translation are achieved in various ways; clinically interesting is the application of translation-targeting antibiotics – translation inhibitors. The antibiotic effects on bacterial physiology are often poorly understood. Bacterial responses to two or more simultaneously applied antibiotics are even more puzzling. The combined antibiotic effect determines the type of drug interaction, which ranges from synergy (the effect is stronger than expected) to antagonism (the effect is weaker) and suppression (one of the drugs loses its potency).
In the first part of this work, we systematically measure the pairwise interaction network for translation inhibitors that interfere with different steps in translation. We find that the interactions are surprisingly diverse and tend to be more antagonistic. To explore the underlying mechanisms, we begin with a minimal biophysical model of combined antibiotic action. We base this model on the kinetics of antibiotic uptake and binding together with the physiological response described by the growth laws. The biophysical model explains some drug interactions, but not all; it specifically fails to predict suppression.
In the second part of this work, we hypothesize that elusive suppressive drug interactions result from the interplay between ribosomes halted in different stages of translation. To elucidate this putative mechanism of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using in- ducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks partially causes these interactions.
We extend this approach by varying two translation bottlenecks simultaneously. This approach reveals the suppression of translocation inhibition by inhibited translation. We rationalize this effect by modeling dense traffic of ribosomes that move on transcripts in a translation factor-mediated manner. This model predicts a dissolution of traffic jams caused by inhibited translocation when the density of ribosome traffic is reduced by lowered initiation. We base this model on the growth laws and quantitative relationships between different translation and growth parameters.
In the final part of this work, we describe a set of tools aimed at quantification of physiological and translation parameters. We further develop a simple model that directly connects the abundance of a translation factor with the growth rate, which allows us to extract physiological parameters describing initiation. We demonstrate the development of tools for measuring translation rate.
This thesis showcases how a combination of high-throughput growth rate mea- surements, genetics, and modeling can reveal mechanisms of drug interactions. Furthermore, by a gradual transition from combinations of antibiotics to precise genetic interventions, we demonstrated the equivalency between genetic and chemi- cal perturbations of translation. These findings tile the path for quantitative studies of antibiotic combinations and illustrate future approaches towards the quantitative description of translation.
AU - Kavcic, Bor
ID - 8657
SN - 978-3-99078-011-4
TI - Perturbations of protein synthesis: from antibiotics to genetics and physiology
ER -
TY - THES
AB - Self-organization is a hallmark of plant development manifested e.g. by intricate leaf vein patterns, flexible formation of vasculature during organogenesis or its regeneration following wounding. Spontaneously arising channels transporting the phytohormone auxin, created by coordinated polar localizations of PIN-FORMED 1 (PIN1) auxin exporter, provide positional cues for these as well as other plant patterning processes. To find regulators acting downstream of auxin and the TIR1/AFB auxin signaling pathway essential for PIN1 coordinated polarization during auxin canalization, we performed microarray experiments. Besides the known components of general PIN polarity maintenance, such as PID and PIP5K kinases, we identified and characterized a new regulator of auxin canalization, the transcription factor WRKY DNA-BINDING PROTEIN 23 (WRKY23).
Next, we designed a subsequent microarray experiment to further uncover other molecular players, downstream of auxin-TIR1/AFB-WRKY23 involved in the regulation of auxin-mediated PIN repolarization. We identified a novel and crucial part of the molecular machinery underlying auxin canalization. The auxin-regulated malectin-type receptor-like kinase CAMEL and the associated leucine-rich repeat receptor-like kinase CANAR target and directly phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated repolarization leading to defects in auxin transport, ultimately to leaf venation and vasculature regeneration defects. Our results describe the CAMEL-CANAR receptor complex, which is required for auxin feed-back on its own transport and thus for coordinated tissue polarization during auxin canalization.
AU - Hajny, Jakub
ID - 8822
TI - Identification and characterization of the molecular machinery of auxin-dependent canalization during vasculature formation and regeneration
ER -
TY - THES
AB - In this thesis we study certain mathematical aspects of evolution. The two primary forces that drive an evolutionary process are mutation and selection. Mutation generates new variants in a population. Selection chooses among the variants depending on the reproductive rates of individuals. Evolutionary processes are intrinsically random – a new mutation that is initially present in the population at low frequency can go extinct, even if it confers a reproductive advantage. The overall rate of evolution is largely determined by two quantities: the probability that an invading advantageous mutation spreads through the population (called fixation probability) and the time until it does so (called fixation time). Both those quantities crucially depend not only on the strength of the invading mutation but also on the population structure. In this thesis, we aim to understand how the underlying population structure affects the overall rate of evolution. Specifically, we study population structures that increase the fixation probability of advantageous mutants (called amplifiers of selection). Broadly speaking, our results are of three different types: We present various strong amplifiers, we identify regimes under which only limited amplification is feasible, and we propose population structures that provide different tradeoffs between high fixation probability and short fixation time.
AU - Tkadlec, Josef
ID - 7196
TI - A role of graphs in evolutionary processes
ER -
TY - THES
AB - Many methods for the reconstruction of shapes from sets of points produce ordered simplicial complexes, which are collections of vertices, edges, triangles, and their higher-dimensional analogues, called simplices, in which every simplex gets assigned a real value measuring its size. This thesis studies ordered simplicial complexes, with a focus on their topology, which reflects the connectedness of the represented shapes and the presence of holes. We are interested both in understanding better the structure of these complexes, as well as in developing algorithms for applications.
For the Delaunay triangulation, the most popular measure for a simplex is the radius of the smallest empty circumsphere. Based on it, we revisit Alpha and Wrap complexes and experimentally determine their probabilistic properties for random data. Also, we prove the existence of tri-partitions, propose algorithms to open and close holes, and extend the concepts from Euclidean to Bregman geometries.
AU - Ölsböck, Katharina
ID - 7460
KW - shape reconstruction
KW - hole manipulation
KW - ordered complexes
KW - Alpha complex
KW - Wrap complex
KW - computational topology
KW - Bregman geometry
SN - 2663-337X
TI - The hole system of triangulated shapes
ER -
TY - THES
AB - We study the interacting homogeneous Bose gas in two spatial dimensions in the thermodynamic limit at fixed density. We shall be concerned with some mathematical aspects of this complicated problem in many-body quantum mechanics. More specifically, we consider the dilute limit where the scattering length of the interaction potential, which is a measure for the effective range of the potential, is small compared to the average distance between the particles. We are interested in a setting with positive (i.e., non-zero) temperature. After giving a survey of the relevant literature in the field, we provide some facts and examples to set expectations for the two-dimensional system. The crucial difference to the three-dimensional system is that there is no Bose–Einstein condensate at positive temperature due to the Hohenberg–Mermin–Wagner theorem. However, it turns out that an asymptotic formula for the free energy holds similarly to the three-dimensional case.
We motivate this formula by considering a toy model with δ interaction potential. By restricting this model Hamiltonian to certain trial states with a quasi-condensate we obtain an upper bound for the free energy that still has the quasi-condensate fraction as a free parameter. When minimizing over the quasi-condensate fraction, we obtain the Berezinskii–Kosterlitz–Thouless critical temperature for superfluidity, which plays an important role in our rigorous contribution. The mathematically rigorous result that we prove concerns the specific free energy in the dilute limit. We give upper and lower bounds on the free energy in terms of the free energy of the non-interacting system and a correction term coming from the interaction. Both bounds match and thus we obtain the leading term of an asymptotic approximation in the dilute limit, provided the thermal wavelength of the particles is of the same order (or larger) than the average distance between the particles. The remarkable feature of this result is its generality: the correction term depends on the interaction potential only through its scattering length and it holds for all nonnegative interaction potentials with finite scattering length that are measurable. In particular, this allows to model an interaction of hard disks.
AU - Mayer, Simon
ID - 7514
SN - 2663-337X
TI - The free energy of a dilute two-dimensional Bose gas
ER -
TY - THES
AB - This thesis is based on three main topics: In the first part, we study convergence of discrete gradient flow structures associated with regular finite-volume discretisations of Fokker-Planck equations. We show evolutionary I convergence of the discrete gradient flows to the L2-Wasserstein gradient flow corresponding to the solution of a Fokker-Planck
equation in arbitrary dimension d >= 1. Along the argument, we prove Mosco- and I-convergence results for discrete energy functionals, which are of independent interest for convergence of equivalent gradient flow structures in Hilbert spaces.
The second part investigates L2-Wasserstein flows on metric graph. The starting point is a Benamou-Brenier formula for the L2-Wasserstein distance, which is proved via a regularisation scheme for solutions of the continuity equation, adapted to the peculiar geometric structure of metric graphs. Based on those results, we show that the L2-Wasserstein space over a metric graph admits a gradient flow which may be identified as a solution of a Fokker-Planck equation.
In the third part, we focus again on the discrete gradient flows, already encountered in the first part. We propose a variational structure which extends the gradient flow structure to Markov chains violating the detailed-balance conditions. Using this structure, we characterise contraction estimates for the discrete heat flow in terms of convexity of
corresponding path-dependent energy functionals. In addition, we use this approach to derive several functional inequalities for said functionals.
AU - Forkert, Dominik L
ID - 7629
SN - 2663-337X
TI - Gradient flows in spaces of probability measures for finite-volume schemes, metric graphs and non-reversible Markov chains
ER -
TY - THES
AB - Proteins and their complex dynamic interactions regulate cellular mechanisms from sensing and transducing extracellular signals, to mediating genetic responses, and sustaining or changing cell morphology. To manipulate these protein-protein interactions (PPIs) that govern the behavior and fate of cells, synthetically constructed, genetically encoded tools provide the means to precisely target proteins of interest (POIs), and control their subcellular localization and activity in vitro and in vivo. Ideal synthetic tools react to an orthogonal cue, i.e. a trigger that does not activate any other endogenous process, thereby allowing manipulation of the POI alone.
In optogenetics, naturally occurring photosensory domain from plants, algae and bacteria are re-purposed and genetically fused to POIs. Illumination with light of a specific wavelength triggers a conformational change that can mediate PPIs, such as dimerization or oligomerization. By using light as a trigger, these tools can be activated with high spatial and temporal precision, on subcellular and millisecond scales. Chemogenetic tools consist of protein domains that recognize and bind small molecules. By genetic fusion to POIs, these domains can mediate PPIs upon addition of their specific ligands, which are often synthetically designed to provide highly specific interactions and exhibit good bioavailability.
Most optogenetic tools to mediate PPIs are based on well-studied photoreceptors responding to red, blue or near-UV light, leaving a striking gap in the green band of the visible light spectrum. Among both optogenetic and chemogenetic tools, there is an abundance of methods to induce PPIs, but tools to disrupt them require UV illumination, rely on covalent linkage and subsequent enzymatic cleavage or initially result in protein clustering of unknown stoichiometry.
This work describes how the recently structurally and photochemically characterized green-light responsive cobalamin-binding domains (CBDs) from bacterial transcription factors were re-purposed to function as a green-light responsive optogenetic tool. In contrast to previously engineered optogenetic tools, CBDs do not induce PPI, but rather confer a PPI already upon expression, which can be rapidly disrupted by illumination. This was employed to mimic inhibition of constitutive activity of a growth factor receptor, and successfully implement for cell signalling in mammalian cells and in vivo to rescue development in zebrafish. This work further describes the development and application of a chemically induced de-dimerizer (CDD) based on a recently identified and structurally described bacterial oxyreductase. CDD forms a dimer upon expression in absence of its cofactor, the flavin derivative F420. Safety and of domain expression and ligand exposure are demonstrated in vitro and in vivo in zebrafish. The system is further applied to inhibit cell signalling output from a chimeric receptor upon F420 treatment.
CBDs and CDD expand the repertoire of synthetic tools by providing novel mechanisms of mediating PPIs, and by recognizing previously not utilized cues. In the future, they can readily be combined with existing synthetic tools to functionally manipulate PPIs in vitro and in vivo.
AU - Kainrath, Stephanie
ID - 7680
TI - Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals
ER -
TY - THES
AB - A search problem lies in the complexity class FNP if a solution to the given instance of the problem can be verified efficiently. The complexity class TFNP consists of all search problems in FNP that are total in the sense that a solution is guaranteed to exist. TFNP contains a host of interesting problems from fields such as algorithmic game theory, computational topology, number theory and combinatorics. Since TFNP is a semantic class, it is unlikely to have a complete problem. Instead, one studies its syntactic subclasses which are defined based on the combinatorial principle used to argue totality. Of particular interest is the subclass PPAD, which contains important problems
like computing Nash equilibrium for bimatrix games and computational counterparts of several fixed-point theorems as complete. In the thesis, we undertake the study of averagecase hardness of TFNP, and in particular its subclass PPAD.
Almost nothing was known about average-case hardness of PPAD before a series of recent results showed how to achieve it using a cryptographic primitive called program obfuscation.
However, it is currently not known how to construct program obfuscation from standard cryptographic assumptions. Therefore, it is desirable to relax the assumption under which average-case hardness of PPAD can be shown. In the thesis we take a step in this direction. First, we show that assuming the (average-case) hardness of a numbertheoretic
problem related to factoring of integers, which we call Iterated-Squaring, PPAD is hard-on-average in the random-oracle model. Then we strengthen this result to show that the average-case hardness of PPAD reduces to the (adaptive) soundness of the Fiat-Shamir Transform, a well-known technique used to compile a public-coin interactive protocol into a non-interactive one. As a corollary, we obtain average-case hardness for PPAD in the random-oracle model assuming the worst-case hardness of #SAT. Moreover, the above results can all be strengthened to obtain average-case hardness for the class CLS ⊆ PPAD.
Our main technical contribution is constructing incrementally-verifiable procedures for computing Iterated-Squaring and #SAT. By incrementally-verifiable, we mean that every intermediate state of the computation includes a proof of its correctness, and the proof can be updated and verified in polynomial time. Previous constructions of such procedures relied on strong, non-standard assumptions. Instead, we introduce a technique called recursive proof-merging to obtain the same from weaker assumptions.
AU - Kamath Hosdurg, Chethan
ID - 7896
TI - On the average-case hardness of total search problems
ER -
TY - THES
AB - Mosaic genetic analysis has been widely used in different model organisms such as the fruit fly to study gene-function in a cell-autonomous or tissue-specific fashion. More recently, and less easily conducted, mosaic genetic analysis in mice has also been enabled with the ambition to shed light on human gene function and disease. These genetic tools are of particular interest, but not restricted to, the study of the brain. Notably, the MADM technology offers a genetic approach in mice to visualize and concomitantly manipulate small subsets of genetically defined cells at a clonal level and single cell resolution. MADM-based analysis has already advanced the study of genetic mechanisms regulating brain development and is expected that further MADM-based analysis of genetic alterations will continue to reveal important insights on the fundamental principles of development and disease to potentially assist in the development of new therapies or treatments.
In summary, this work completed and characterized the necessary genome-wide genetic tools to perform MADM-based analysis at single cell level of the vast majority of mouse genes in virtually any cell type and provided a protocol to perform lineage tracing using the novel MADM resource. Importantly, this work also explored and revealed novel aspects of biologically relevant events in an in vivo context, such as the chromosome-specific bias of chromatid sister segregation pattern, the generation of cell-type diversity in the cerebral cortex and in the cerebellum and finally, the relevance of the interplay between the cell-autonomous gene function and cell-non-autonomous (community) effects in radial glial progenitor lineage progression.
This work provides a foundation and opens the door to further elucidating the molecular mechanisms underlying neuronal diversity and astrocyte generation.
AU - Contreras, Ximena
ID - 7902
TI - Genetic dissection of neural development in health and disease at single cell resolution
ER -
TY - THES
AB - This thesis considers two examples of reconfiguration problems: flipping edges in edge-labelled triangulations of planar point sets and swapping labelled tokens placed on vertices of a graph. In both cases the studied structures – all the triangulations of a given point set or all token placements on a given graph – can be thought of as vertices of the so-called reconfiguration graph, in which two vertices are adjacent if the corresponding structures differ by a single elementary operation – by a flip of a diagonal in a triangulation or by a swap of tokens on adjacent vertices, respectively. We study the reconfiguration of one instance of a structure into another via (shortest) paths in the reconfiguration graph.
For triangulations of point sets in which each edge has a unique label and a flip transfers the label from the removed edge to the new edge, we prove a polynomial-time testable condition, called the Orbit Theorem, that characterizes when two triangulations of the same point set lie in the same connected component of the reconfiguration graph. The condition was first conjectured by Bose, Lubiw, Pathak and Verdonschot. We additionally provide a polynomial time algorithm that computes a reconfiguring flip sequence, if it exists. Our proof of the Orbit Theorem uses topological properties of a certain high-dimensional cell complex that has the usual reconfiguration graph as its 1-skeleton.
In the context of token swapping on a tree graph, we make partial progress on the problem of finding shortest reconfiguration sequences. We disprove the so-called Happy Leaf Conjecture and demonstrate the importance of swapping tokens that are already placed at the correct vertices. We also prove that a generalization of the problem to weighted coloured token swapping is NP-hard on trees but solvable in polynomial time on paths and stars.
AU - Masárová, Zuzana
ID - 7944
KW - reconfiguration
KW - reconfiguration graph
KW - triangulations
KW - flip
KW - constrained triangulations
KW - shellability
KW - piecewise-linear balls
KW - token swapping
KW - trees
KW - coloured weighted token swapping
SN - 978-3-99078-005-3
TI - Reconfiguration problems
ER -