583 research outputs found

    Phosphate sink containing two-component signaling systems as tunable threshold devices.

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    Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tSynthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications.Exeter University Science Strateg

    Exploring and Understanding Signal-response Relationships and Response Dynamics of Microbial Two-Component Signaling Systems

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    Two-component signaling systems are found in bacteria, fungi and plants. They mediate many of the physiological responses of these organisms to their environment and display several conserved biochemical and structural features. This thesis identifies a potential functional role for two commonly found architectures in two-component signaling system, the split kinases and phosphate sink, which suggests that by enabling switch-like behaviors they could underlie physiological decision making. I report that split histidine kinases, where autophosphorylation and phosphotransfer activities are segregated onto distinct proteins capable of complex formation, enable ultrasensitivity and bistability. By employing computer simulations and analytical approaches, I show that the specific biochemical features of split kinases “by design” enable higher nonlinearity in the system response compared to conventional two-component systems and those using bifunctional (but not split) kinases. I experimentally show that one of these requirements, namely segregation of the phosphatase activity only to the free form of one of the proteins making up the split kinase, is met in proteins isolated from Rhodobacter sphaeroides. While the split kinase I study from R. sphaeroides is specifically involved in chemotaxis, other split kinases are involved in diverse responses. Genomics studies suggest 2.3% of all chemotaxis kinases, and 2.8% of all kinases could be functioning as split kinases. Combining theoretical and experimental approaches, I show that the phosphate sink motif found in microbial and plant TCSs allows threshold behaviors. This motif involves a single histidine kinase that can phosphotransfer reversibly to two separate response regulators and examples are found in bacteria, yeast and plants. My results show that one of the response regulators can act as a “sink” or “buffer” that needs to be saturated before the system can generate significant responses. This sink, thereby allows the generation of a signal threshold that needs to be exceeded for there to be significant phosphoryl group flow to the other response regulator. Thus, this system can enable cells to display switch-like behavior to external signals. Using an analytical approach, I identify mathematical conditions on the system parameters that are necessary for threshold dynamics. I find these conditions to be satisfied in both of the natural systems where the system parameters have been measured. Further, by in vitro reconstitution of a sample system, I experimentally demonstrate threshold dynamics for a phosphate-sink containing two-component system. This study provides a link between these architectures of TCSs and signal-response relationship, thereby enabling experimentally testable hypotheses in these diverse two-component systems. These findings indicate split kinases and phosphate as a microbial alternative for enabling ultrasensitivity and bistability - known to be crucial for cellular decision making. By demonstrating ultrasensitivity, threshold dynamics and their mechanistic basis in a common class of two-component system, this study allows a better understanding of cellular signaling in a diverse range of organisms and will open the way to the design of novel threshold systems in synthetic biology. Thus, I believe that this study will have broad implications not only for microbiologists but also systems biologists who aim to decipher conserved dynamical features of cellular networks.University of Exete

    An investigation into dynamic and functional properties of prokaryotic signalling networks

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    In this thesis, I investigate dynamic and computational properties of prokaryotic signalling architectures commonly known as the Two Component Signalling networks and phosphorelays. The aim of this study is to understand the information processing capabilities of different prokaryotic signalling architectures by examining the dynamics they exhibit. I present original investigations into the dynamics of different phosphorelay architectures and identify network architectures that include a commonly found four step phosphorelay architecture with a capacity for tuning its steady state output to implement different signal-response behaviours viz. sigmoidal and hyperbolic response. Biologically, this tuning can be implemented through physiological processes like regulating total protein concentrations (e.g. via transcriptional regulation or feedback), altering reaction rate constants through binding of auxiliary proteins on relay components, or by regulating bi-functional activity in relays which are mediated by bifunctional histidine kinases. This study explores the importance of different biochemical arrangements of signalling networks and their corresponding response dynamics. Following investigations into the significance of various biochemical reactions and topological variants of a four step relay architecture, I explore the effects of having different types of proteins in signalling networks. I show how multi-domain proteins in a phosphorelay architecture with multiple phosphotransfer steps occurring on the same protein can exhibit multistability through a combination of double negative and positive feedback loops. I derive a minimal multistable (core) architecture and show how component sharing amongst networks containing this multistable core can implement computational logic (like AND, OR and ADDER functions) that allows cells to integrate multiple inputs and compute an appropriate response. I examine the genomic distribution of single and multi domain kinases and annotate their partner response regulator proteins across prokaryotic genomes to find the biological significance of dynamics that these networks embed and the processes they regulate in a cell. I extract data from a prokaryotic two component protein database and take a sequence based functional annotation approach to identify the process, function and localisation of different response regulators as signalling partners in these networks. In summary, work presented in this thesis explores the dynamic and computational properties of different prokaryotic signalling networks and uses them to draw an insight into the biological significance of multidomain sensor kinases in living cells. The thesis concludes with a discussion on how this understanding of the dynamic and computational properties of prokaryotic signalling networks can be used to design synthetic circuits involving different proteins comprising two component and phosphorelay architectures.Dorothy Hodgkin Studentship funded by EPSRC and Microsoft Research

    Engineering the transport of signaling molecules in glycosaminoglycan-based hydrogels

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    Signaling molecules are critically important to regulate cellular processes. Therefore, their incorporation into engineered biomaterials is indispensable for the applications in tissue engineering and regenerative medicine. In particular, the functionalization of highly hydrated polymer networks, so-called hydrogels, with the signaling molecules, has been quite beneficial to provide multiple cell-instructive signals. Following this strategy, the incorporation of sulfated glycosaminoglycans (GAGs) into such polymer networks offers unprecedented options to control the administration of signaling molecules via electrostatic interactions. Moreover, mathematical models can be instrumental in designing materials to tune the transport and adjust the local concentration of the signaling molecules to precisely modulate cell fate decisions. Accordingly, this study aims to systematically investigate the impact of different binary poly(ethylene glycol)-glycosaminoglycan hydrogel networks on the transport of signaling molecules by developing and applying mathematical modeling in combination with experimental approaches. The gained knowledge was then applied to modulate the bioactivities of pro-angiogenic growths factor within the binary hydrogel and rationally design a new class of cytocompatible GAG-based materials for the controlled administration of pro-angiogenic growth factors. Firstly, systematic studies on the mobility of signaling molecules within GAG-based polymer networks revealed differential effects of hydrogel network parameters such as mesh size, GAG content, and the sulfation pattern of the GAG building block on the transport of these signaling molecules. Secondly, the effect of the GAG content of the hydrogel and the sulfation pattern of the GAG building block on the bioactivity of hydrogel administrated vascular endothelial growth factor (VEGF) have been analyzed. Since VEGF is a GAG-affine protein that plays a major role in angiogenesis, its ability to promote vascular morphogenesis has been investigated. The simulation and experimental results demonstrated the determining impact of the availability of free (unbound) VEGF as well as the presence of GAGs with a specific sulfation pattern within the polymer network on the formation of the endothelial capillary network within the hydrogel. Finally, a rational design strategy has been applied to extend a GAG-hydrogel platform to allow for a far-reaching control of its cell instructive properties. The resulting materials are independently tunable over a broad range for their mechanical properties and GAG content. The GAG content of the hydrogel matrices, in particular, was shown to modulate the transport of pro-angiogenic growth factors most. Moreover, the hydrogel also supports endothelial vascular morphogenesis. In conclusion, the in here followed approach of combining experimental results and mathematical modeling for predicting the transport of signaling molecules and the rational design concept for customizing GAG-based hydrogel networks provide the fundamentals to precisely modulate cell fate decisions within GAG-based biohybrid polymer networks rationalizing their application for tissue engineering and regenerative medicin

    Multiple communication mechanisms between sensor kinases are crucial for virulence in Pseudomonas aeruginosa

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    This is the author accepted manuscript. The final version is available as an open access article from the publisher via the DOI in this recordBacteria and many non-metazoan Eukaryotes respond to stresses and threats using two-component systems (TCSs) comprising sensor kinases (SKs) and response regulators (RRs). Multikinase networks, where multiple SKs work together, detect and integrate different signals to control important lifestyle decisions such as sporulation and virulence. Here, we study interactions between two SKs from Pseudomonas aeruginosa, GacS and RetS, which control the switch between acute and chronic virulence. We demonstrate three mechanisms by which RetS attenuates GacS signalling: RetS takes phosphoryl groups from GacS-P; RetS has transmitter phosphatase activity against the receiver domain of GacS-P; and RetS inhibits GacS autophosphorylation. These mechanisms play important roles in vivo and during infection, and exemplify an unprecedented degree of signal processing by SKs that may be exploited in other multikinase networks.This work was supported by the Medical Research Council (MRC) (grant number MR/ M020045/1), the Leverhulme Trust (grant number RPG-2014-228), the RoseTrees Trust (grant number M328) and a NERC PhD studentship (grant number 1076449)

    BIOERODIBLE CALCIUM SULFATE BONE GRAFTING SUBSTITUTES WITH TAILORED DRUG DELIVERY CAPABILITIES

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    Bone regeneration or augmentation is often required prior to or concomitant with implant placement. With the limitations of many existing technologies, a biologically compatible synthetic bone grafting substitute that is osteogenic, bioerodible, and provides spacing-making functionality while acting as a drug delivery vehicle for bioactive molecules could provide an alternative to ‘gold standard’ techniques. In the first part of this work, calcium sulfate (CS) space-making synthetic bone grafts with uniformly embedded poly(β-amino ester) (PBAE) biodegradable hydrogel particles was developed to allow controlled release of bioactive agents. The embedded gel particles’ influence on the physical and chemical characteristics of CS was tested. Namely, the compressive strength and modulus, dissolution, and morphology, were studied. All CS samples dissolved via zero-order surface erosion consistent to one another. Compression testing concluded that the amount, but not size, of embedded gel particles significantly decreased (up to 75%) the overall mechanical strength of the composite. Release studies were conducted to explore this system’s ability to deliver a broad range of drug types and sizes. Lysozyme (model protein for larger growth factors like bone morphogenic protein [BMP]) was loaded into PBAE particles embedded in CS matrix. The release of simvastatin, a small molecule drug capable of up regulating BMP production, was also examined. The release of both lysozyme and simvastatin was governed by dissolution of CS. The second part of this work proposed a bilayered CS implant. The physical and chemical properties were characterized similarly to the CS composites above. Release kinetics of directly loaded simvastatin in either the shell, core, or both were investigated. A sequential release of simvastatin was witnessed giving foresight of the composite’s tunability. The sequential release of an antibacterial, metronidazole, loaded into poly(lactic-co-glycolic acid) (PLGA) particles embedded into the shell along with directly loaded simvastatin either in the shell, core, or both layers was also observed. Through controlled release of bioactive agents, as well as a tunable layered geometry, CS-based implants have the potential to be optimized in order to help streamline the steps required for the healing and regeneration of compromised bone tissue

    BIOENGINEERED SCAFFOLDS TO INDUCE ALIGNMENT AND PROMOTE AXON REGENERATION FOLLOWING SPINAL CORD INJURY

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    Scaffolds delivered to injured spinal cords to stimulate axon connectivity often act as a bridge to stimulate regeneration at the injured area, but current approaches lack the permissiveness, topology and mechanics to mimic host tissue properties. This dissertation focuses on bioengineering scaffolds through the means of altering topology in injectables and tuning mechanics in 3D-printed constructs as potential therapies for spinal cord injury repair. A self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunohistochemistry assays verify that vascularized peptide scaffolds promote axon infiltration, attenuate inflammation and reduce astrogliosis. Furthermore, magnetically-responsive (MR) RADA-16I injections are patterned along the rostral-caudal direction in both in-vitro and in-vivo conditions. ELISA and histochemical assays validate the efficacy of MR hydrogels to promote and align axon infiltration at the site of injury. In addition to injectable scaffolds, this thesis uses digital light processing (DLP) to mimic the mechanical heterogeneity of the spinal cord caused by white and gray matter, and demonstrate that doing so improves axon infiltration into the scaffold compared to controls exhibiting homogeneous mechanical properties. Taken together, this work contributes to advancing the field of tissue engineering and regenerative medicine by demonstrating the potential of bioengineered scaffolds to repair the damaged spinal cord

    BIOACTIVE POLY(BETA-AMINO ESTER) BIOMATERIALS FOR TREATMENT OF INFECTION AND OXIDATIVE STRESS

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    Polymers have deep roots as drug delivery tools, and are widely used in clinical to private settings. Currently, however, numerous traditional therapies exist which may be improved through use of polymeric biomaterials. Through our work with infectious and oxidative stress disease prevention and treatment, we aimed to develop application driven, enhanced therapies utilizing new classes of polymers synthesized in-house. Applying biodegradable poly(β-amino ester) (PBAE) polymers, covalent-addition of bioactive substrates to these PBAEs avoided certain pitfalls of free-loaded and non-degradable drug delivery systems. Further, through variation of polymer ingredients and conditions, we were able to tune degradation rates, release profiles, cellular toxicity, and material morphology. Using these fundamentals of covalent drug-addition into biodegradable polymers, we addressed two problems that exist with the treatment of patients with high-risk wound-sites, namely non-biodegradability that require second-surgeries, and free-loaded antibiotic systems where partially degraded materials fall below the minimum inhibitory concentration, allowing biofilm proliferation. Our in situ polymerizable, covalently-bound vancomycin hydrogel provided active antibiotic degradation products and drug release which closely followed the degradation rate over tunable periods. With applications of antioxidant delivery, we continued with this concept of covalent drug addition and modified a PBAE, utilizing a disulfide moiety to mimic redox processes which glutathione/glutathione disulfide performs. This material was found to not only be hydrolytically biodegradable, but tunable in reducibility through cleavage of the disulfide crosslinker, forming antioxidant groups of bound-thiols, similar to drugs currently used in radioprotective therapies. The differential cellular viability of degradation products containing disulfide or antioxidant thiol forms was profound, and the antioxidant form significantly aided cellular resistance to a superoxide attack, similar to that of a radiation injury. Pathophysiological oxidation in the form of radiation injury or oxidative stress based diseases are often region specific to the body and thus require specific targeting, and nanomaterials are widely researched to perform this. Utilizing a tertiary-amine base-catalyst, we were able to synthesize a high drug content (20-26 wt%) version of the disulfide PBAE previously unattainable. The reduced version of this material created a linear-chain polymer capable of single-emulsion nanoparticle formulation for use with intravenous antioxidant delivery applications instead of local

    Microengineering The Neural Tube

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    Early embryonic development is a complex and highly regulated orchestra of instructive cues that collectively guide naïve stem cells towards progressively more specialized fates. In the neural tube, the precursor structure to the brain and spinal cord, these signals emanate from ‘organizing centers’ surrounding the neural tube. These organizing centers send out soluble cues or morphogens that diffuse tens to hundreds of microns to recipient cells residing in the neural tube. Re-creating this dynamic landscape of cues in vitro is impossible using standard cell culture tools and techniques. However, microfluidics is perfectly suited to fill this gap, allowing precise control over the microenvironment on the same length scale as the developing embryo. A microfluidic device is presented that is able to re-create some of the spatial patterning events that occur during the early development of the neural tube. This platform enables developmental biologists to reverse engineer development from the ground up, enabling researchers to pose radically new experiments to help answer some of the most relevant questions regarding fate specification in the developing neural tube. Here the device is used to guide mouse embryonic stem cells into motor neurons. Importantly, these motor neurons are able to be directed to differentiate in a defined region of the microdevice, a spatial patterning event that is the hallmark of the developing neural tube. For the first time it is now possible to study the effect of development cues on live populations of stem cells. The characterization of these fundamental developmental processes will prove invaluable in understanding how humans acquire both form and function. One day, it may allow researchers to harness these developmental techniques, which have been refined over thousands of years of evolution, to guide patient derived cells into any user defined cell fate
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