Asynchronous neighborhood task synchronization

Faults are likely to occur in distributed systems. The motivation for designing self-stabilizing system is to be able to automatically recover from a faulty state. As per Dijkstra\u27s definition, a system is self-stabilizing if it converges to a desired state from an arbitrary state in a finite number of steps. The paradigm of self-stabilization is considered to be the most unified approach to designing fault-tolerant systems. Any type of faults, e.g., transient, process crashes and restart, link failures and recoveries, and byzantine faults, can be handled by a self-stabilizing system; Many applications in distributed systems involve multiple phases. Solving these applications require some degree of synchronization of phases. In this thesis research, we introduce a new problem, called asynchronous neighborhood task synchronization ( NTS ). In this problem, processes execute infinite instances of tasks, where a task consists of a set of steps. There are several requirements for this problem. Simultaneous execution of steps by the neighbors is allowed only if the steps are different. Every neighborhood is synchronized in the sense that all neighboring processes execute the same instance of a task. Although the NTS problem is applicable in nonfaulty environments, it is more challenging to solve this problem considering various types of faults. In this research, we will present a self-stabilizing solution to the NTS problem. The proposed solution is space optimal, fault containing, fully localized, and fully distributed. One of the most desirable properties of our algorithm is that it works under any (including unfair) daemon. We will discuss various applications of the NTS problem

Notes on Theory of Distributed Systems

Notes for the Yale course CPSC 465/565 Theory of Distributed Systems

Computational Protein Design for Peptide-Directed Bioconjugation

Proteins enable living organisms to perform many of their critical functions, having been applied over evolutionary time to solve problems of overwhelming diversity and complexity. Protein engineering seeks to deploy these versatile molecules in addressing problems of human concern and would benefit from innovations that improve protein utilization in unnatural environments as well as from increased predictive capability in protein design. Bioconjugation facilitates the use of proteins in unnatural environments by permitting the attachment of molecules, such as polymers, that can modulate protein stability, solubility, and activity and by mediating protein immobilization. We initially explored this propensity of bioconjugation by designing an enzymatic polyurethane-based material in which proteins were covalently immobilized via non-specific, isocyanate reaction chemistries. The resulting material resisted bacterial biofilm formation through the activity of the embedded enzymes, which hydrolyzed signaling molecules involved in quorum sensing.In order to gain better regional and temporal reaction control, we transitioned to working with an enzyme-mediated conjugation system in which lipoic acid ligase functionalizes a specific 13-residue peptide sequence (the LAP sequence) with an azide-bearing lipoic acid derivative. Using GFP as a model protein, we demonstrated that the LAP sequence could be inserted at internal positions within a protein’s structure and successfully ligated and subsequently modified via azide-alkyne click chemistry within that context. Given the ability of the LAP sequence to site-specifically direct conjugation at internal sites within protein structures, we developed a predictive computational approach to facilitate the design of internal LAP insertion sites within diverse protein targets. The kinematic loop modeling application within the Rosetta framework was adapted for rapid scanning and characterization of insertion sites, using Rosetta’s coarse-grained centroid score function for site differentiation. Soluble protein expression of LAP-containing proteins was found to correlate with Rosetta scores, and unintuitive sites were identified relative to B-factor and secondary structure considerations. Our results highlighted the role played by residues in the near-loop environment in determining whether a particular site within a protein accommodates an inserted loop, such as the LAP sequence, and suggest that our computational approach, which is highly user accessible, can usefully predict such sites

Shining light on T6SS mode of action and function within single cells and bacterial communities

Bacteria are ubiquitously found in the environment and form the basis for all known ecosystems on our planet. Most bacterial cells reside in complex multi-species bacterial communities, which are often associated with a host, such as the human microbiota. These bacterial communities are shaped by cooperative and competitive interactions amongst their members. Like higher animals, bacteria also compete with their conspecifics for nutrients and space. This evolutionary arms race resulted in a diverse set of strategies for microbial competition. In particular, bacteria residing on solid surfaces can compete with their neighbors through the use of specialized nanomachines, called secretion systems, enabling the direct delivery of toxic effector molecules into by-standing target cells. The most commonly used weapon for contact-dependent antagonism is the bacterial Type VI secretion system (T6SS). The T6SS belongs to the family of contractile injection systems (CISs). All CISs are structurally and functionally related to contractile bacteriophages (e.g. phage T4) and translocate proteins into target cells by means of physical force, which is generated by rapid sheath contraction. This results in the ejection of the inner tube associated with a sharp tip and effector proteins at its end. Effector translocation leads ultimately to target cell death. Importantly, the T6SS is capable translocating effectors across broad ranges of biological membranes making it a powerful weapon in microbial warfare as well as potent virulence mechanism towards eukaryotic host cells. Our current understanding of T6SS mode of action is primarily based on the combination of structural biology and fluorescence live-cell microscopy studies. While in particular cryo-electron microscopy (cryo-EM) revealed the detailed architecture of the T6SS in situ and of isolated subassemblies, fluorescence live-cell microscopy uncovered the remarkable dynamics of T6SS biogenesis. However, a complete understanding of T6SS dynamics is hampered in standard fluorescent microscopy due to: (i) the spatial and temporal resolution limit, (ii) the inability to efficiently label secreted components of the machinery, (iii) the weak signals due to low protein abundance and rapid photobleaching, (iv) the difficulty to perform long-term co-incubation experiments as well as (v) the inability to precisely control spatial and chemical environment. My doctoral thesis aimed to overcome these limitations to allow novel insights into dynamics of the T6SSs of Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. Specifically sheath assembly, initiation of sheath contraction, T6SS mediated protein translocation in to sister cells as well as strategies for prey cell inhibition were studied in this thesis. First, I studied sheath assembly in ampicillin induced V. cholerae spheroplasts. These enlarged cells assemble T6SS sheaths which are up to 10x longer as compared to rod shaped cells. This allowed us to photobleach an assembling sheath structure and demonstrate that new sheath subunits are added to the growing sheath polymer at the distal end opposite the baseplate. Importantly, this was the first direct observation made for any contractile machines studied to date. Moreover, I showed that unlike for all other CISs, T6SS sheath length is not regulated and correlates with cell size. In order to monitor protein translocation into target cells, we developed a T6SS dependent interbacterial protein complementation assay, enabling the indirect detection of translocated T6SS components into the cytosol of recipient cells. This allowed us to demonstrate that secreted T6SS components are exchanged among by-standing sister cells within minutes upon initial cell contact. Importantly, these results were the first experimental indication that T6SS is capable of translocating its components into the cytosol of Gram-negative target cells. Furthermore, we showed that the amount and the composition of the secreted tip influences the number of T6SS assemblies per cell, whereas different concentration of the tube protein influenced sheath length. We also provided evidence that precise aiming of T6SS assemblies through posttranslational regulation in P. aeruginosa increases the efficiency of substrate delivery. In addition, together with two Nanoscience master students we have also been implementing microfluidics in the Basler laboratory. This powerful technology enabled us to control the spatial arrangements of aggressor and prey populations and to follow these populations at single-cell level over time scales of several hours. In collaboration with Prof. Kevin Forster, University of Oxford, we demonstrated that the rate of target cell lysis heavily influences the outcome of contact-dependent T6SS killing and thus drives evolution of lytic effectors. Moreover, microfluidics allows for the dynamic change of the chemical microenvironment during imaging experiments. By following the T6SS dynamics in response to hyperosmotic shocks resulting in a rapid cell volume reduction, we found that physical pressure from the collapsing cell envelope could trigger sheath contraction. This led us to propose a model for sheath contraction under steady-state conditions where continued sheath polymerization against membrane contact site leads to a gradual increase in pressure applied to the assembled sheath. We propose that this could be potentially sensed by the baseplate, which in turn would trigger sheath contraction

Engineering Enhanced Structural Stability to the Streptococcal Bacteriophage Endolysin PlyC

Antibiotic misuse and overuse has prompted bacteria to rapidly develop resistance, thereby hindering the efficacy of these chemotherapeutics. Due to antibiotic resistant strains expeditiously disseminating, antimicrobial resistance has been labeled as one of the greatest threats to human health globally. An emerging alternative antimicrobial strategy involves using bacteriophage-derived enzymes, termed endolysins. Endolysins are peptidoglycan hydrolases that liberate lytic bacteriophage virions late in the infection cycle by cleaving critical covalent bonds in the bacterial cell wall. As a result, the high intracellular osmotic pressure induces cell lysis. Antimicrobial strategies have been devised involving the extrinsic application of recombinant endolysins to susceptible Gram-positive pathogens. The efficacy of these enzymes has been validated in vitro and in vivo, with no resistance observed to date. One such example is the streptococcal-specific endolysin PlyC. This endolysin is currently the most bacteriolytically-active and possesses the ability to lyse human and animal pathogens known to cause serious health complications. Unfortunately, like numerous other endolysins, PlyC is relatively unstable and accordingly has short shelf life expectancy. With a long-term goal of using endolysins for industrial applications, furthering the development of a thermolabile translational antimicrobial with a short shelf life is ambitious. The main objective of this dissertation is to develop and validate bioengineering strategies for thermostabilizing bacteriolytic enzymes. Using PlyC as the model enzyme, we first used a rationale-based computational screening methodology to identify stabilizing mutations to a thermosusceptible region of the catalytic subunit, PlyCA. One mutation, T406R, caused a 2.27°C increase in thermodynamic stability and a 16 fold improvement in kinetic stability. Next, we developed a substantiated novel directed evolution protocol that involves randomly incorporating mutations into a bacteriolytic enzyme followed by a screening process that effectively identifies mutations that are stabilizing. Finally, applying multiple rounds of directed evolution to PlyC allowed for the identification of a thermostabilizing mutation, N211H, which increased the thermodynamic stability by 4.10°C and kinetic stability 18.8 fold. Combining the N211H and T406R mutations was additive in terms of thermal stability, with thermodynamic and kinetic stability enhancements of 7.46°C and 28.72 kcal/mol activation energy (EA) of PlyCA unfolding, respectively

Modelling the spread of plasmid-encoded antibiotic resistance in aquatic environments considering evolutionary modifications, individual heterogeneity and complex biotic interactions

Plasmids providing antibiotic resistance to their host bacteria pose a major threat to society, as antibiotics are often the only way to treat infectious diseases. Here the existence conditions of plasmids are investigated in an ecological framework with mathematical methods such as ordinary differential equations and individual-based models. It is shown how (i) the arise of different kinds of compensatory mutation, (ii) intra- and intercellular interactions of plasmids representing opposing plasmid lifestyles as well as (iii) a diverse plasmid community affect plasmid dynamics, community composition and persistence. The results indicate that evolutionary modifications and interactions between plasmids broaden the existence conditions of plasmids in a way that has not been recognized before, but explains their occurrence in nature. This includes that biotic interactions could maintain costly plasmid-encoded antibiotic resistance despite the absence of abiotic selection. These findings open a way to study remaining research questions related to the complexity of natural environments.:1. Introduction 2. Article I (published) – Mobile compensatory mutations promote plasmid survival 3. Article II (published) – Conjugative plasmids enable the maintenance of low cost non-transmissible plasmids 4. Article III (submitted) – The autopoiesis of plasmid diversity 5. Supervised Master thesis I – The propagation of antibiotic resistances considering migration between microhabitats 6. Supervised Master thesis II – Estimation of the pB10 conjugation rate in Escherichia coli combining laboratory experiments and modelling 7. Supervised research internship – Plasmid population dynamics considering individual plasmid copy numbers 8. DiscussionPlasmide, die Antibiotikaresistenzen an ihre Wirtsbakterien vermitteln, stellen eine große Bedrohung füur die Gesellschaft dar, weil Antibiotika oft die einzige Möglichkeit sind Infektionskrankheiten zu behandeln. In dieser Arbeit werden die Existenzbedingungen von Plasmiden aus einer ökologischen Perspektive mit mathematischen Methoden wie gewöhnlichen Differentialgleichungen und Individuen-basierten Modellen untersucht. Es wird gezeigt, wie (i) das Aufkommen verschiedener Kosten-kompensierender Mutationen, (ii) intra- und interzelluläre Wechselwirkungen von Plasmiden, die gegensätzliche Plasmidlebensstile repräsentieren, sowie (iii) eine vielfältige Plasmidgemeinschaft einen Einfluss auf die Dynamik, Gemeinschaftszusammensetzung und Persistenz von Plasmiden ausüben. Die Ergebnisse deuten darauf hin, dass evolutionäre Modifikationen und Wechselwirkungen zwischen Plasmiden die Existenzbedingungen von Plasmiden in einer Weise erweitern, die bisher nicht erkannt wurde, aber ihr Auftreten in der Natur erklärt. Dazu gehört auch, dass biotische Wechselwirkungen trotz fehlender abiotischer Selektion eine kostspielige Plasmid-vermittelte Antibiotikaresistenz aufrechterhalten könnten. Die Erkentnisse dieser Arbeit können dazu genutzt werden verbleibende Forschungsfragen anzugehen, die im Zusammenhang mit der Komplexität der natürlichen Umwelt stehen.:1. Introduction 2. Article I (published) – Mobile compensatory mutations promote plasmid survival 3. Article II (published) – Conjugative plasmids enable the maintenance of low cost non-transmissible plasmids 4. Article III (submitted) – The autopoiesis of plasmid diversity 5. Supervised Master thesis I – The propagation of antibiotic resistances considering migration between microhabitats 6. Supervised Master thesis II – Estimation of the pB10 conjugation rate in Escherichia coli combining laboratory experiments and modelling 7. Supervised research internship – Plasmid population dynamics considering individual plasmid copy numbers 8. Discussio

Elucidating gene expression adaptation of phylogenetically divergent coral holobionts under heat stress

As coral reefs struggle to survive under climate change, it is crucial to know whether they have the capacity to withstand changing conditions, particularly increasing seawater temperatures. Thermal tolerance requires the integrative response of the different components of the coral holobiont (coral host, algal photosymbiont, and associated microbiome). Here, using a controlled thermal stress experiment across three divergent Caribbean coral species, we attempt to dissect holobiont member metatranscriptome responses from coral taxa with different sensitivities to heat stress and use phylogenetic ANOVA to study the evolution of gene expression adaptation. We show that coral response to heat stress is a complex trait derived from multiple interactions among holobiont members. We identify host and photosymbiont genes that exhibit lineage-specific expression level adaptation and uncover potential roles for bacterial associates in supplementing the metabolic needs of the coral-photosymbiont duo during heat stress. Our results stress the importance of integrative and comparative approaches across a wide range of species to better understand coral survival under the predicted rise in sea surface temperatures

Biomedical applications of Surface Enhanced Raman Spectroscopy - a step forward to clinical practice

Lo scopo di questo progetto di dottorato \ue8 quello di utilizzare delle superfici metalliche nanostrutturate come substrati per la spettroscopia Raman amplificata da superfici (SERS) per l\u2019analisi di biofluidi. Questa tecnica analitica restituisce l\u2019impronta digitale vibrazionale del campione grazie alla presenza della nanostruttura metallica. Queste caratteristiche anticipano le potenzialit\ue0 della spettroscopia SERS in campo bioanalitico che ha visto un aumento esponenziale delle sue applicazioni nell\u2019ultimo decennio. In particolare, la SERS richiede la fabbricazione di substrati metallici nanostrutturati che possano funzionare da sensori. Questo progetto si basa sullo sviluppo di un approccio privo di marcatura (label-free:): nessuna funzionalizzazione \ue8 presente sulla superficie metallica al fine di rilevare in modo aspecifico gli analiti presenti della matrice di interesse biologico. Il risultato del segnale SERS sar\ue0 un\u2019istantanea della soluzione in analisi depositata sulla superficie metallica, cio\ue8 l\u2019impronta specifica del campione. Per esempio, l\u2019analisi label-free dei biofluidi riflette il suo contenuto metabolico. Nell\u2019era \u201comica\u201d, il SERS pu\uf2 essere integrato nella metabolomica non funzionalizzata in quanto fornisce il profilo metabolico del soggetto in esame e di conseguenza distinguere campioni diversi basandosi sulle differenze di ogni profilo analizzato. I colloidi stabilizzati elettrostaticamente sono stati scelti per la loro nota compatibilit\ue0 con i biofluidi. Verranno usati sia in forma colloidale in sospensione acquosa, sia fissati su un supporto di carta, definiti supporti solidi e sviluppati grazie a un protocollo validato nel nostro laboratorio. Il vantaggio portato dai supporti in carta risiede nella stabilit\ue0 della risposta spettroscopica: sono di lunga durata, facili da fabbricare e da maneggiare, economici e veloci, potenzialmente fabbricabili su ampia scala. Queste sono le caratteristiche che nell\u2019ambito delle applicazioni del SERS possono promuovere la costruzione di un dispositivo Point of Care. Basandosi sulle competenze acquisite dal nostro gruppo di ricerca, lo scopo di questa tesi di dottorato \ue8 duplice: aumentare le nostre conoscenze sull\u2019interazione biofluidi-nanostrutture e utilizzare il metodo SERS per lo studio di specifici problemi clinici. Al fine di soddisfare tali richieste questo lavoro \ue8 diviso in tre parti: 1. Sviluppare protocolli per l\u2019analisi label-free delle frazioni di sangue (siero, plasma, eritrociti, cellule mononucleate del sangue periferico, e sangue intero) con il SERS, sfruttando le loro caratteristiche in base alla diversa preparazione dei campioni e ai substrati SERS utilizzati; 2. Caratterizzare il comportamento delle biomolecole sulla superficie di nanoparticelle metalliche su sistemi modello, cio\ue8 capire il ruolo delle corone di proteine e non proteine nell\u2019interazione metabolita-nanoparticelle. Il sistema modello usato si basa su un insieme di albumina di siero umano (la pi\uf9 abbondante proteina del siero) e molecole che sono comunemente osservate nei biofluidi: adenina, ipoxantina e acido urico; 3. Applicare le nozioni di cui sopra per la diagnosi precoce di diverse malattie (tumore al seno, fegato grasso non alcolico, cirrosi e carcinoma epatocellulare) tramite campioni di sangue e plasma e l\u2019uso di analisi dati multivariata per spettri SERS. Lo scopo dell\u2019utilizzo del SERS in ambito medico \ue8 di proporre nuovi approcci diagnostici complementari alle tecniche gi\ue0 in uso in clinica come ad esempio i metodi di immunochimica e istopatologia. Il vantaggio del SERS risiede nella rapida risposta e in un approccio non invasivo tramite l\u2019utilizzo di biopsia liquida. Lo scopo futuro \ue8 lo sviluppo di una piattaforma SERS label-free come dispositivo point of care integrato allo strumento RamanThis PhD project aims to apply nanostructured metal surfaces as substrates for Surface Enhanced Raman Spectroscopy for the study of biofluids. This analytical technique provides the vibrational fingerprint of a sample assisted by nanostructured metal surfaces, which can enhance the scattering signal of analytes adsorbed on them: this allows detection of analytes in very low concentrations. These features tell a lot about the potential of SERS in the bioanalytics, and indeed, in this field, the use of SERS has increased over the past decade taking advantage of both sensitive detection and fingerprinting features. Above all, SERS requires the manufacturing of metal nanostructured substrates as sensors. In particular, this project is based on the development of a label-free approach: no functionalization is present on the nanoparticles surface, and, hence, no preferential affinity for a given analyte in the biological matrix is sought. Briefly, once nanoparticles are in contact with the specimen, the analytes may adsorb on them without any specific interaction other than their affinity for the metal. The outcoming SERS signal will be a snapshot of what actually reached the metal surface, namely a fingerprint of the sample. For instance, the label-free analysis of biofluids reflect the metabolic content of the fluid itself. In the \u201comic\u201d era, SERS can integrate with untargeted metabolomics and provide the metabolic profile of a specimen and distinguish different samples accordingly, based on differences in such profiles. Silver colloids have been chosen, given that their performances with biofluids are known. They have been used both as colloidal suspension in water, and fixed on a paper support, according to an in-house developed protocol for the fabrication of solid substrates. The coupling of metal nanostructures substrates with SERS acts as actual sensors, able to interact with aqueous environment and detect dissolved analytes. The real advantage of the paper supports lay in the stability of the spectroscopic response: they are long lasting, easy to fabricate and to handle, cost and time effective, prone to scale up. These reasons make them potential Point of Care tool in the frame of SERS applications. The aim of this PhD thesis is twofold: to push forward our fundamental knowledge of the nanostructure-biofluid interaction and to test the feasibility of the application of SERS for specific clinical problems. These goals were pursued in three steps: 1. to develop protocols for the label-free analysis of blood fractions (serum, plasma, erythrocytes, periphereal blood mononuclear cells, and whole blood) with SERS, exploiting their features according to several treatments and SERS substrates; 2. to characterize the behaviour of biomolecules at the interface with metal nanoparticles on model systems, namely to understand the role of the protein and non-protein corona in the metabolites-nanoparticle interaction. The model system used is based on mixture of human serum albumin (i.e. the most abundant serum protein) and molecules which are commonly detected in SERS of biofluids: adenine, hypoxanthine and uric acid; 3. to apply the aforementioned knowledge to the early diagnosis of several diseases (breast cancer, non-alcoholic fatty liver diseases, cirrhosis and hepatocellular carcinoma) through serum and plasma samples by means of multivariate data analysis of SERS spectra. Considering the latter application of SERS in the field of disease diagnosis, the aim is to provide new diagnostic methods complementary to the accepted gold standards such as immunochemistry and histopathology methods. The advantages of SERS lay on the rapid response and on the non-invasiveness of the liquid biopsy approach. As a future goal, the development of SERS platforms as label-free point of care tools integrated to portable Raman instruments could bring the diagnosi