The exploration of potato-associated bacteria in the Central Andean Highlands and their application in integrated crop management systems

Potato is the most important food crop after wheat and rice. A changing climate, coupled with a heightened consumer awareness of how food is produced and legislative changes governing the usage of agrochemicals, means that alternative more integrated and sustainable approaches are needed for crop management practices. Bioprospecting in the Central Andean Highlands resulted in the isolation and in vitro screening of 600 bacterial isolates. The best performing isolates, under in vitro conditions, were field trialled in their home countries. Six of the isolates, Pseudomonas sp. R41805 (Bolivia), Pseudomonas palleroniana R43631 (Peru), Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 (Ecuador), showed significant increase in the yield of potato. Using – omic technologies (i.e. volatilomic, transcriptomic, proteomic and metabolomic), the influence of microbial isolates on plant defence responses was determined. Volatile organic compounds of bacterial isolates were identified using GC/MS. RT-qPCR analysis revealed the significant expression of Ethylene Response Factor 3 (ERF3) and the results of this study suggest that the dual inoculation of potato with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 may play a part in the activation of plant defence system via ERF3. The proteomic analysis by 2-DE study has shown that priming by Pseudomonas sp. R41805 can induce the expression of proteins related to photosynthesis and protein folding in in vitro potato plantlets. The metabolomics study has shown that the total glycoalkaloid (TGA) content of greenhouse-grown potato tubers following inoculation with Pseudomonas sp. R41805 did not exceed the acceptable safety limit (200 mg kg-1 FW). As a result of this study, a number of bacteria have been identified with commercial potential that may offer sustainable alternatives in both Andean and European agricultural settings.

Talking about quality: exploring how ‘quality’ is conceptualized in European hospitals and healthcare systems

BACKGROUND: Conceptualization of quality of care - in terms of what individuals, groups and organizations include in their meaning of quality, is an unexplored research area. It is important to understand how quality is conceptualised as a means to successfully implement improvement efforts and bridge potential disconnect in language about quality between system levels, professions, and clinical services. The aim is therefore to explore and compare conceptualization of quality among national bodies (macro level), senior hospital managers (meso level), and professional groups within clinical micro systems (micro level) in a cross-national study. METHODS: This cross-national multi-level case study combines analysis of national policy documents and regulations at the macro level with semi-structured interviews (383) and non-participant observation (803 hours) of key meetings and shadowing of staff at the meso and micro levels in ten purposively sampled European hospitals (England, the Netherlands, Portugal, Sweden, and Norway). Fieldwork at the meso and micro levels was undertaken over a 12-month period (2011-2012) and different types of micro systems were included (maternity, oncology, orthopaedics, elderly care, intensive care, and geriatrics). RESULTS: The three quality dimensions clinical effectiveness, patient safety, and patient experience were incorporated in macro level policies in all countries. Senior hospital managers adopted a similar conceptualization, but also included efficiency and costs in their conceptualization of quality. 'Quality' in the forms of measuring indicators and performance management were dominant among senior hospital managers (with clinical and non-clinical background). The differential emphasis on the three quality dimensions was strongly linked to professional roles, personal ideas, and beliefs at the micro level. Clinical effectiveness was dominant among physicians (evidence-based approach), while patient experience was dominant among nurses (patient-centered care, enough time to talk with patients). Conceptualization varied between micro systems depending on the type of services provided. CONCLUSION: The quality conceptualization differed across system levels (macro-meso-micro), among professional groups (nurses, doctors, managers), and between the studied micro systems in our ten sampled European hospitals. This entails a managerial alignment challenge translating macro level quality definitions into different local contexts.

Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.

FY2010 Annual Report Research, Intelligent Transportation Systems, and Technology Transfer Activities, October 18, 2011

Transportation research makes a difference for Iowans and the nation. Implementation of cost effective research projects contributes to a transportation network that is safer, more efficient, and longer lasting. Working in cooperation with our partners from universities, industry, other states, and FHWA, as well as participation in the Transportation Research Board (TRB), provides benefits for every facet of the DOT. This allows us to serve our communities and the traveling public more effectively. Pooled fund projects allow leveraging of funds for higher returns on investments. In 2010, Iowa led fifteen active pooled fund studies, participated in twenty-two others, and was wrapping-up, reconciling, and closing out an additional 6 Iowa Led pooled fund studies. In addition, non-pooled fund SPR projects included approximately 20 continued, 9 new, and over a dozen reoccurring initiatives such as the technical transfer/training program. Additional research is managed and conducted by the Office of Traffic and Safety and other departments in the Iowa DOT.

Utility and safety of invasive (fractional flow reserve) and non-invasive (cardiac magnetic resonance imaging) diagnostic tests in patients with NSTEMI

A prospective randomised controlled clinical trial of treatment decisions informed by invasive functional testing of coronary artery disease severity compared with standard angiography-guided management was implemented in 350 patients with a recent non-ST elevation myocardial infarction (NSTEMI) admitted to 6 hospitals in the National Health Service. The main aims of this study were to examine the utility of both invasive fractional flow reserve (FFR) and non-invasive cardiac magnetic resonance imaging (MRI) amongst patients with a recent diagnosis of NSTEMI. In summary, the findings of this thesis are: (1) the use of FFR combined with intravenous adenosine was feasible and safe amongst patients with NSTEMI and has clinical utility; (2) there was discordance between the visual, angiographic estimation of lesion significance and FFR; (3). The use of FFR led to changes in treatment strategy and an increase in prescription of medical therapy in the short term compared with an angiographically guided strategy; (4) in the incidence of major adverse cardiac events (MACE) at 12 months follow up was similar in the two groups. Cardiac MRI was used in a subset of patients enrolled in two hospitals in the West of Scotland. T1 and T2 mapping methods were used to delineate territories of acute myocardial injury. T1 and T2 mapping were superior when compared with conventional T2-weighted dark blood imaging for estimation of the ischaemic area-at-risk (AAR) with less artifact in NSTEMI. There was poor correlation between the angiographic AAR and MRI methods of AAR estimation in patients with NSTEMI. FFR had a high accuracy at predicting inducible perfusion defects demonstrated on stress perfusion MRI. This thesis describes the largest randomized trial published to date specifically looking at the clinical utility of FFR in the NSTEMI population. We have provided evidence of the diagnostic and clinical utility of FFR in this group of patients and provide evidence to inform larger studies. This thesis also describes the largest ever MRI cohort, including with myocardial stress perfusion assessments, specifically looking at the NSTEMI population. We have demonstrated the diagnostic accuracy of FFR to predict reversible ischaemia as referenced to a non-invasive gold standard with MRI. This thesis has also shown the futility of using dark blood oedema imaging amongst all comer NSTEMI patients when compared to novel T1 and T2 mapping methods.

Study of the Electromagnetic Interference Generated by Wireless Power Transfer Systems

This paper presents a study related with measuring of radio frequency emissions. The purpose is to determine the level of interference generated by wireless power transfer equipment in a specific frequency range, and to compare those levels to the existing standards. The technology of wireless power transfer, especially for electric vehicles batteries charging, is rapidly developing in the recent years. An increasing use of this technology in industrial and consumer electronic products has raised concerns about the possible unfavorable health-effects onto the human being. Another concern is raised from the high intensity fields produced by wireless power transfer systems which will generate highly undesired influence on other electrical and electronic equipment. As a protection against the potential health effects, the governments imposed limits on the occupational and general public exposure to the radio frequencies. These limitations are set out in national and international safety guidelines, standards and regulations. The measurement and evaluation of the human exposure to electromagnetic fields are essential to guarantee occupational and general public safety.

The women role in the agroenvironmental activities of Amazon agroforeststry systems.

This paper presents the results of a research on the way which shows the role of the farmer women to contribute to overcome the life hindrances in the Amazonian agroforest environment, for food safety and familiar income. The sample was the women of the Associação de Produtores Alternativos - APA, Ouro Preto do Oeste, Rondônia, Brazil. Questionnaires were applied to 50 women of this community, in 2004/2005, with additional interviews in 2006, taking into account their role in the farms, and their several invisible activities. The role of APA?s women was compared to that of other Amazonian women, like rubber tappers and riverine people. The APA?s women perform tasks as field manpower - 78.0% of them, making decisions about what to plant - 18.0%, harvest destination - 32.0%, which animals to raise -14.0% and animal products destination -34.0%. Traditionally, in rural zone, the women with their children play a role as non-paid manpower, getting some monetary income with occasional commercialization of on-farm processed products as cheese, pickles, jellies and fruit liqueurs. They are responsible for the cultures practiced around the house. All these Amazonian women edaphoclimatic conditions, greatly contributing to the biodiversity, conservation, and to ecological, social and economic stability.are guardians, perpetuators and disseminators of a rich germplasm adapted to local.

Waste Heat Recovery Systems: Numerical and Experimental Analysis of organic Rankine Cycle Solutions

This thesis aims to present the ORC technology, its advantages and related problems. In particular, it provides an analysis of ORC waste heat recovery system in different and innovative scenarios, focusing on cases from the biggest to the lowest scale. Both industrial and residential ORC applications are considered. In both applications, the installation of a subcritical and recuperated ORC system is examined. Moreover, heat recovery is considered in absence of an intermediate heat transfer circuit. This solution allow to improve the recovery efficiency, but requiring safety precautions. Possible integrations of ORC systems with renewable sources are also presented and investigated to improve the non-programmable source exploitation. In particular, the offshore oil and gas sector has been selected as a promising industrial large-scale ORC application. From the design of ORC systems coupled with Gas Turbines (GTs) as topper systems, the dynamic behavior of the GT+ORC innovative combined cycles has been analyzed by developing a dynamic model of all the considered components. The dynamic behavior is caused by integration with a wind farm. The electric and thermal aspects have been examined to identify the advantages related to the waste heat recovery system installation. Moreover, an experimental test rig has been realized to test the performance of a micro-scale ORC prototype. The prototype recovers heat from a low temperature water stream, available for instance in industrial or residential waste heat. In the test bench, various sensors have been installed, an acquisitions system developed in Labview environment to completely analyze the ORC behavior. Data collected in real time and corresponding to the system dynamic behavior have been used to evaluate the system performance based on selected indexes. Moreover, various operational steady-state conditions are identified and operation maps are realized for a completely characterization of the system and to detect the optimal operating conditions.

Ultra-Low Temperature Partial Oxidation: Detailed Kinetics, Safety, and Environmental Issues

The innovation in several industrial sectors has been recently characterized by the need for reducing the operative temperature either for economic or environmental related aspects. Promising technological solutions require the acquisition of fundamental-based knowledge to produce safe and robust systems. In this sense, reactive systems often represent the bottleneck. For these reasons, this work was focused on the integration of chemical (i.e., detailed kinetic mechanism) and physical (i.e., computational fluid dynamics) models. A theoretical-based kinetic mechanism mimicking the behaviour of oxygenated fuels and their intermediates under oxidative conditions in a wide range of temperature and pressure was developed. Its validity was tested against experimental data collected in this work by using the heat flux burner, as well as measurements retrieved from the current literature. Besides, estimations deriving from existing models considered as the benchmark in the combustion field were compared with the newly generated mechanism. The latter was found to be the most accurate for the investigated conditions and fuels. Most influential species and reactions on the combustion of butyl acetate were identified. The corresponding thermodynamic parameter and rate coefficients were quantified through ab initio calculations. A reduced detailed kinetic mechanism was produced and implemented in an open-source computational fluid dynamics model to characterize pool fires caused by the accidental release of aviation fuel and liquefied natural gas, at first. Eventually, partial oxidation processes involving light alkenes were optimized following the quick, fair, and smoot (QFS) paradigm. The proposed procedure represents a comprehensive and multidisciplinary approach for the construction and validation of accurate models, allowing for the characterization of developing industrial sectors and techniques.

Design of an innovative electric vehicle simulator for Charging Systems' End-of-Line Testing

The trend related to the turnover of internal combustion engine vehicles with EVs goes by the name of electrification. The push electrification experienced in the last decade is linked to the still ongoing evolution in power electronics technology for charging systems. This is the reason why an evolution in testing strategies and testing equipment is crucial too. The project this dissertation is based on concerns the investigation of a new EV simulator design. that optimizes the structure of the testing equipment used by the company who commissioned this work. Project requirements can be summarized in the following two points: space occupation reduction and parallel charging implementation. Some components were completely redesigned, and others were substituted with equivalent ones that could perform the same tasks. In this way it was possible to reduce the space occupation of the simulator, as well as to increase the efficiency of the testing device. Moreover, the possibility of conjugating different charging simulations could be investigated by parallelly launching two testing procedures on a unique machine, properly predisposed for supporting the two charging protocols used. On the back of the results achieved in the body of this dissertation, a new design for the EV simulator was proposed. In this way, space reduction was obtained, and space occupation efficiency was improved with the proposed new design. The testing device thus resulted to be way more compact, enabling to gain in safety and productivity, along with a 25% cost reduction. Furthermore, parallel charging was implemented in the proposed new design since the conducted tests clearly showed the feasibility of parallel charging sessions. The results presented in this work can thus be implemented to build the first prototype of the new EV simulator.

Damage Identification of Structures through Vibration-Based Structural Monitoring Systems

The thesis has been carried out within the “SHAPE Project - Predicting Strength Changes in Bridges from Frequency Data Safety, Hazard, and Poly-harmonic Evaluation” (ERA-NET Plus Infravation Call 2014) which dealt with the structural assessment of existing bridges and laboratory structural reproductions through the use of vibration-based monitoring systems, for detecting changes in their natural frequencies and correlating them with the occurrence of damage. The main purpose of this PhD dissertation has been the detection of the variation of the main natural frequencies as a consequence of a previous-established damage configuration provided on a structure. Firstly, the effect of local damage on the modal feature has been discussed mainly concerning a steel frame and a composite steel-concrete bridge. Concerning the variation of the fundamental frequency of the small bridge, the increasing severity of two local damages has been investigated. Moreover, the comparison with a 3D FE model is even presented establishing a link between the dynamic properties and the damage features. Then, moving towards a diffused damage pattern, four concrete beams and a small concrete deck were loaded achieving the yielding of the steel reinforcement. The stiffness deterioration in terms of frequency shifts has been reconsidered by collecting a large set of dynamic experiments on simply supported R.C. beams discussed in the literature. The comparison of the load-frequency curves suggested a significant agreement among all the experiments. Thus, in the framework of damage mechanics, the “breathing cracks” phenomenon has been discussed leading to an analytical formula able to explain the frequency decay observed experimentally. Lastly, some dynamic investigations of two existing bridges and the corresponding FE Models are presented in Chapter 4. Moreover, concerning the bridge in Bologna, two prototypes of a network of accelerometers were installed and the data of a few months of monitoring have been discussed.

Embedded systems and advanced signal processing for Acousto- Ultrasonic Inspections

Non Destructive Testing (NDT) and Structural Health Monitoring (SHM) are becoming essential in many application contexts, e.g. civil, industrial, aerospace etc., to reduce structures maintenance costs and improve safety. Conventional inspection methods typically exploit bulky and expensive instruments and rely on highly demanding signal processing techniques. The pressing need to overcome these limitations is the common thread that guided the work presented in this Thesis. In the first part, a scalable, low-cost and multi-sensors smart sensor network is introduced. The capability of this technology to carry out accurate modal analysis on structures undergoing flexural vibrations has been validated by means of two experimental campaigns. Then, the suitability of low-cost piezoelectric disks in modal analysis has been demonstrated. To enable the use of this kind of sensing technology in such non conventional applications, ad hoc data merging algorithms have been developed. In the second part, instead, imaging algorithms for Lamb waves inspection (namely DMAS and DS-DMAS) have been implemented and validated. Results show that DMAS outperforms the canonical Delay and Sum (DAS) approach in terms of image resolution and contrast. Similarly, DS-DMAS can achieve better results than both DMAS and DAS by suppressing artefacts and noise. To exploit the full potential of these procedures, accurate group velocity estimations are required. Thus, novel wavefield analysis tools that can address the estimation of the dispersion curves from SLDV acquisitions have been investigated. An image segmentation technique (called DRLSE) was exploited in the k-space to draw out the wavenumber profile. The DRLSE method was compared with compressive sensing methods to extract the group and phase velocity information. The validation, performed on three different carbon fibre plates, showed that the proposed solutions can accurately determine the wavenumber and velocities in polar coordinates at multiple excitation frequencies.

Network analysis and machine learning assist drug repurposing and safety assessment in neurological diseases

In recent decades, two prominent trends have influenced the data modeling field, namely network analysis and machine learning. This thesis explores the practical applications of these techniques within the domain of drug research, unveiling their multifaceted potential for advancing our comprehension of complex biological systems. The research undertaken during this PhD program is situated at the intersection of network theory, computational methods, and drug research. Across six projects presented herein, there is a gradual increase in model complexity. These projects traverse a diverse range of topics, with a specific emphasis on drug repurposing and safety in the context of neurological diseases. The aim of these projects is to leverage existing biomedical knowledge to develop innovative approaches that bolster drug research. The investigations have produced practical solutions, not only providing insights into the intricacies of biological systems, but also allowing the creation of valuable tools for their analysis. In short, the achievements are: • A novel computational algorithm to identify adverse events specific to fixed-dose drug combinations. • A web application that tracks the clinical drug research response to SARS-CoV-2. • A Python package for differential gene expression analysis and the identification of key regulatory "switch genes". • The identification of pivotal events causing drug-induced impulse control disorders linked to specific medications. • An automated pipeline for discovering potential drug repurposing opportunities. • The creation of a comprehensive knowledge graph and development of a graph machine learning model for predictions. Collectively, these projects illustrate diverse applications of data science and network-based methodologies, highlighting the profound impact they can have in supporting drug research activities.

Industrial Demilitarized Zone and Zero Trust cybersecurity models for Industrial Control Systems

Today more than ever, with the recent war in Ukraine and the increasing number of attacks that affect systems of nations and companies every day, the world realizes that cybersecurity can no longer be considered just as a “cost”. It must become a pillar for our infrastructures that involve the security of our nations and the safety of people. Critical infrastructure, like energy, financial services, and healthcare, have become targets of many cyberattacks from several criminal groups, with an increasing number of resources and competencies, putting at risk the security and safety of companies and entire nations. This thesis aims to investigate the state-of-the-art regarding the best practice for securing Industrial control systems. We study the differences between two security frameworks. The first is Industrial Demilitarized Zone (I-DMZ), a perimeter-based security solution. The second one is the Zero Trust Architecture (ZTA) which removes the concept of perimeter to offer an entirely new approach to cybersecurity based on the slogan ‘Never Trust, always verify’. Starting from this premise, the Zero Trust model embeds strict Authentication, Authorization, and monitoring controls for any access to any resource. We have defined two architectures according to the State-of-the-art and the cybersecurity experts’ guidelines to compare I-DMZ, and Zero Trust approaches to ICS security. The goal is to demonstrate how a Zero Trust approach dramatically reduces the possibility of an attacker penetrating the network or moving laterally to compromise the entire infrastructure. A third architecture has been defined based on Cloud and fog/edge computing technology. It shows how Cloud solutions can improve the security and reliability of infrastructure and production processes that can benefit from a range of new functionalities, that the Cloud could offer as-a-Service.We have implemented and tested our Zero Trust solution and its ability to block intrusion or attempted attacks.

A Microleakage Study Of Gutta-percha/ah Plus And Resilon/real Self-etch Systems After Different Irrigation Protocols.

The development and maintenance of the sealing of the root canal system is the key to the success of root canal treatment. The resin-based adhesive material has the potential to reduce the microleakage of the root canal because of its adhesive properties and penetration into dentinal walls. Moreover, the irrigation protocols may have an influence on the adhesiveness of resin-based sealers to root dentin. The objective of the present study was to evaluate the effect of different irrigant protocols on coronal bacterial microleakage of gutta-percha/AH Plus and Resilon/Real Seal Self-etch systems. One hundred ninety pre-molars were used. The teeth were divided into 18 experimental groups according to the irrigation protocols and filling materials used. The protocols used were: distilled water; sodium hypochlorite (NaOCl)+eDTA; NaOCl+H3PO4; NaOCl+eDTA+chlorhexidine (CHX); NaOCl+H3PO4+CHX; CHX+eDTA; CHX+ H3PO4; CHX+eDTA+CHX and CHX+H3PO4+CHX. Gutta-percha/AH Plus or Resilon/Real Seal Se were used as root-filling materials. The coronal microleakage was evaluated for 90 days against Enterococcus faecalis. Data were statistically analyzed using Kaplan-Meier survival test, Kruskal-Wallis and Mann-Whitney tests. No significant difference was verified in the groups using chlorhexidine or sodium hypochlorite during the chemo-mechanical preparation followed by eDTA or phosphoric acid for smear layer removal. The same results were found for filling materials. However, the statistical analyses revealed that a final flush with 2% chlorhexidine reduced significantly the coronal microleakage. A final flush with 2% chlorhexidine after smear layer removal reduces coronal microleakage of teeth filled with gutta-percha/AH Plus or Resilon/Real Seal SE.