Study of the bacterial communication in Pseudomonas aeruginosa: a systems and synthetic biology perspective.

openThe major public health concern worldwide is the antibiotic resistance which is making difficult to treat diseases caused by the pathogenic microbiome carrying multiple resistance genes. The increase in frequency of these diseases has led to the new therapeutic approaches as alternative to traditional antibiotics. One of the innovative therapeutic approaches is the examination of bacterial cell communication system, known as Quorum sensing. These systems are laborious to study as they are regulated by a complex network of genes which are multifactorial and interconnected. The genes associated with quorum sensing is also related to toxin production and the formation of extracellular structures i.e., biofilms which is capable to form a physical barrier against antibiotics. Pseudomonas aeruginosa, a Gram-negative bacterium and one of the “superbugs”, is known for evading the effects of multiple drugs due to its innate antibiotic resistance. This serves as a case study in this thesis work.The main objective of this thesis is to study the LasR, RhlI - RhlR systems, of the QS mechanisms present in P.aeruginosa. This is done by creating engineered systems for ease of study. In this we addressed and resolves issues in the Las system by optimizing ribosome binding sites (RBSs). Constructs, including pLasA RFP34, pLasB RFP34, and pLasI RFP34, were designed and transformed into E. coli cells with LasR receptor and ÿD transcription factor. The exploration of pLasR3 promoter and its processing paralleled the study. Co-transformed systems were evaluated with LasR and RhlR receptors in the presence of auto-inductors. Bacterial growth in the presence of 3OC12-HSL revealed nutrient dependence in P. aeruginosa supernatant, impacting E. coli growth. Fluorescence assays indicated suboptimal sensitivity, limiting the system's practicality for screening applications, with pRhlR promoter exhibiting unique behavior. Thorough examination of LasR receptor promoters under varying 3OC12-HSL concentrations revealed distinct expression patterns. In the realm of Synthetic Biology, a groundbreaking approach emerges, aiming to streamline the exploration of intricate systems by untangling the involved gene components. This deliberate reduction of variables serves as a powerful method to dissect individual actors within communication networks. The overarching objective is twofold: firstly, to meticulously identify potential therapeutic targets, and secondly, to engineer systems that facilitate rapid and cost-effective screening of molecules with promising therapeutic effects. This methodology, with its precision and efficiency, becomes especially crucial in the battle against antibiotic resistance, offering a strategic arsenal to confront the challenges posed by resilient bacterial strains.The major public health concern worldwide is the antibiotic resistance which is making difficult to treat diseases caused by the pathogenic microbiome carrying multiple resistance genes. The increase in frequency of these diseases has led to the new therapeutic approaches as alternative to traditional antibiotics. One of the innovative therapeutic approaches is the examination of bacterial cell communication system, known as Quorum sensing. These systems are laborious to study as they are regulated by a complex network of genes which are multifactorial and interconnected. The genes associated with quorum sensing is also related to toxin production and the formation of extracellular structures i.e., biofilms which is capable to form a physical barrier against antibiotics. Pseudomonas aeruginosa, a Gram-negative bacterium and one of the “superbugs”, is known for evading the effects of multiple drugs due to its innate antibiotic resistance. This serves as a case study in this thesis work.The main objective of this thesis is to study the LasR, RhlI - RhlR systems, of the QS mechanisms present in P.aeruginosa. This is done by creating engineered systems for ease of study. In this we addressed and resolves issues in the Las system by optimizing ribosome binding sites (RBSs). Constructs, including pLasA RFP34, pLasB RFP34, and pLasI RFP34, were designed and transformed into E. coli cells with LasR receptor and ÿD transcription factor. The exploration of pLasR3 promoter and its processing paralleled the study. Co-transformed systems were evaluated with LasR and RhlR receptors in the presence of auto-inductors. Bacterial growth in the presence of 3OC12-HSL revealed nutrient dependence in P. aeruginosa supernatant, impacting E. coli growth. Fluorescence assays indicated suboptimal sensitivity, limiting the system's practicality for screening applications, with pRhlR promoter exhibiting unique behavior. Thorough examination of LasR receptor promoters under varying 3OC12-HSL concentrations revealed distinct expression patterns. In the realm of Synthetic Biology, a groundbreaking approach emerges, aiming to streamline the exploration of intricate systems by untangling the involved gene components. This deliberate reduction of variables serves as a powerful method to dissect individual actors within communication networks. The overarching objective is twofold: firstly, to meticulously identify potential therapeutic targets, and secondly, to engineer systems that facilitate rapid and cost-effective screening of molecules with promising therapeutic effects. This methodology, with its precision and efficiency, becomes especially crucial in the battle against antibiotic resistance, offering a strategic arsenal to confront the challenges posed by resilient bacterial strains

CIRSE Standards of Practice on Conducting Meetings on Morbidity and Mortality

This CIRSE Standards of Practice document is developed by an expert writing group under the guidance of the CIRSE Standards of Practice Committee. It aims to assist Interventional Radiologists in their daily practice by providing best practices for conducting meetings on morbidity and mortality.info:eu-repo/semantics/publishedVersio

Renal Artery Stenting in Consecutive High-Risk Patients With Atherosclerotic Renovascular Disease:A Prospective 2-Center Cohort Study

BACKGROUND: The aim of this study was to prospectively evaluate the effects of renal artery stenting in consecutive patients with severe atherosclerotic renal artery stenosis and high‐risk clinical presentations as defined in a national protocol developed in 2015. METHODS AND RESULTS: Since the protocol was initiated, 102 patients have been referred for revascularization according to the following high‐risk criteria: severe renal artery stenosis (≥70%) with true resistant hypertension, rapidly declining kidney function, or recurrent heart failure/sudden pulmonary edema. At baseline, the mean 24‐hour ambulatory systolic blood pressure was 166.2 mm Hg (95% CI, 162.0–170.4), the defined daily dose of antihypertensive medication was 6.5 (95% CI, 5.8–7.3), and the estimated glomerular filtration rate was 41.1 mL/min per 1.73m(2) (95% CI, 36.6–45.6). In 96 patients with available 3‐month follow‐up data, mean 24‐hour ambulatory systolic blood pressure decreased by 19.6 mm Hg (95% CI, 15.4–23.8; P<0.001), the defined daily dose of antihypertensive medication was reduced by 52% (95% CI, 41%–62%; P<0.001), and estimated glomerular filtration rate increased by 7.8 mL/min per 1.73m(2) (95% CI, 4.5–11.1; P<0.001). All changes persisted after 24 month follow‐up. Among 17 patients with a history of hospitalization for acute decompensated heart failure, 14 patients had no new episodes after successful revascularization. CONCLUSIONS: In this prospective cohort study, we observed a reduction in blood pressure and antihypertensive medication, an increase in estimated glomerular filtration rate, and a decrease in new hospital admissions attributable to heart failure/sudden pulmonary edema after renal artery stenting. REGISTRATION: URL: https://clinicaltrials.gov. Identifier: NCT02770066

Multicomponent polysaccharide alginate-based bioinks

3D-Bioprinting has seen a rapid expansion in the last few years, with an increasing number of reported bioinks. Alginate is a natural biopolymer that forms hydrogels by ionic cross-linking with calcium ions. Due to its biocompatibility and ease of gelation, it is an ideal ingredient for bioinks. This review focuses on recent advances on bioink formulations based on the combination of alginate with other polysaccharides. In particular, the molecular weight of the alginate and its loading level has an impact on materials performance, as well as the loading of the divalent metal salt and its solubility, which affects the cross-linking of the gel. Alginate is often combined with other polysaccharides that can sigificantly modify the properties of the gel, and can optimise alginate for use in different biological applications. It is also possible to combine alginate with sacrificial polymers, which can temporarily reinforce the 3D printed construct, but then be removed at a later stage. Other additives can be formulated into the gels to enhance performance, including nanomaterials that tune rheological properties, peptides to encourage cell adhesion, or growth factors to direct stem cell differentiation. The ease of formulating multiple components into alginate gels gives them considerable potential for further development. In summary, this review will facilitate the identification of different alginate-polysaccharide bioink formulations and their optimal applications, and help inform the design of second generation bioinks, allowing this relatively simple gel system to achieve more sophisticated control over biological processes

Chemická reaktivita z pohledu tradičních a netradičních konceptů

Cílem diplomové práce je pochopení chemické reaktivity ve vybraných oblastech bioanorganické a (bio)organické chemie a to s použitím tradiční teorie tranzitního stavu (TTS), tak i přístupů jdoucích za tuto teorii. Konkrétně v této práci uvádím dvě studie - jednu, která se opírá převážně o TTS při řešení faktorů řídící metanogenezi a jednu ilustrující chemický případ, který nelze řešit v rámci TTS a pro jehož pochopení jsem použila teorerický nástroj, na jehož vývoji jsem se podílela. Cílem mého prvního projektu bylo porozumět katalytické schopnosti koenzymu F430 při tvorbě metanu s důrazem na jeho srovnání se čtyřmi biosyntetickými prekurzory a to za účelem získání věrohodného pohledu na evoluční hnací sílu, jež utváří biokatalytickou způsobilost F430 směrem k tvorbě CH4. V přírodě je produkce metanu usnadněna klíčovým enzymem metyl- koenzym M reduktázou (MCR). MCR katalyzuje reakci mezi koenzymy M a B (H3C-SCoM a CoBS-H) vedoucí na metan a heterodisulfidický produkt. Aktivní místo tohoto enzymu hostí kofaktor F430, v jehož středu se nachází redoxně aktivní ion niklu. Ústřední částí reakčního mechanismu je pak redukční štěpení vazby H3C-S v koenzymu M zprostředkované ionem niklu a uvolňující metylový radikál, jenž v následujícím kroku stěpí vazbu S-H v koenzymu B za vzniku metanu. Navzdory...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemiePřírodovědecká fakultaFaculty of Scienc

Chemical reactivity through the lens of traditional and non-traditional concepts

Cílem diplomové práce je pochopení chemické reaktivity ve vybraných oblastech bioanorganické a (bio)organické chemie a to s použitím tradiční teorie tranzitního stavu (TTS), tak i přístupů jdoucích za tuto teorii. Konkrétně v této práci uvádím dvě studie - jednu, která se opírá převážně o TTS při řešení faktorů řídící metanogenezi a jednu ilustrující chemický případ, který nelze řešit v rámci TTS a pro jehož pochopení jsem použila teorerický nástroj, na jehož vývoji jsem se podílela. Cílem mého prvního projektu bylo porozumět katalytické schopnosti koenzymu F430 při tvorbě metanu s důrazem na jeho srovnání se čtyřmi biosyntetickými prekurzory a to za účelem získání věrohodného pohledu na evoluční hnací sílu, jež utváří biokatalytickou způsobilost F430 směrem k tvorbě CH4. V přírodě je produkce metanu usnadněna klíčovým enzymem metyl- koenzym M reduktázou (MCR). MCR katalyzuje reakci mezi koenzymy M a B (H3C-SCoM a CoBS-H) vedoucí na metan a heterodisulfidický produkt. Aktivní místo tohoto enzymu hostí kofaktor F430, v jehož středu se nachází redoxně aktivní ion niklu. Ústřední částí reakčního mechanismu je pak redukční štěpení vazby H3C-S v koenzymu M zprostředkované ionem niklu a uvolňující metylový radikál, jenž v následujícím kroku stěpí vazbu S-H v koenzymu B za vzniku metanu. Navzdory..

Identification of heart donors using biochemical probes

Heart transplantation provides a substantial survival benefit for selected patients with advanced heart failure, achieving a 1 year survival rate of ≥80%. Up to two thirds of hearts offered for transplantation are rejected before detailed organ inspection as being likely to fail if transplanted. The decision to discount these organs is based on clinical factors (e.g. blood pressure, electrocardiographic changes, prior cardiopulmonary resuscitation, drug history, history of hypertension and the need for inotropic support). However, none of these factors necessarily preclude successful transplantation. Thus, there is a pool of unused hearts, for which permission for heart donation has been granted, from which additional transplants could be generated if we could be more confident about their current and future function. My research prospectively validated a definition of primary allograft dysfunction following heart transplantation that is suitable for use in multicentre studies. I then investigated the role of biomarkers in the evaluation of potential cardiac donors with objective of increasing the number of donor hearts that will be assessed by direct inspection. This lead to the design of a scoring system to guide donor evaluation

Exact invariant solutions for grooved Couette and channel flows

The dynamical systems approach to turbulence has gained a lot of traction since the turn of the century. A large set of exact invariant solutions for canonical wall-bounded flows such as Couette, channel, and pipe flows has been found by researchers. These solutions, and their connections, are thought to form a skeleton for trajectories of turbulent flow. However, this vision of turbulence has not been extended to rough-walled flows despite the practical signficance of such flows in engineering applications. This thesis describes continuation, by numerical homotopy, of known equilibria from smooth-walled plane shear flows to grooved plane shear flows using a domain transformation method, with the hope that this exploratory work would inform later efforts to extend such solutions to rough-walled flows. As a precursor to computing non-laminar equilibria, laminar solutions are computed for grooved channel flows for transverse, longitudinal, and oblique grooves. In addition to the numerical solutions, analytical solutions are also derived for asymptotically long groove-wavelengths, employing the Stokes-flow approximation for transverse and oblique grooves.Exact invariant solutions can indeed be continued from plane Couette flow (PCoF) with smooth walls to grooved PCoF with longitudinal grooves using a simple domain transformation method. However, smooth PCoF equilibria exist as continuous families of solutions that are identical up to a translational shift; the loss of spanwise homogeneity due to the grooves restricts such continuous families to discrete families due to symmetry-breaking. This phase-based restriction can also be expected to be reflected in turbulent statistics. Continuation of equilibria in grooves of different wavelengths shows a drag increasing tendency for grooves of the same wavelength as the vortex-streak structure, and a drag reducing tendency for grooves of significantly smaller wavelengths. This can relate the optimal spacing of riblets for maximal drag reduction to the spanwise spacing of the vortex-streak structures observed in the self-sustaining near-wall cycle