Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

The development of an algorithm to track blood pressure accurately based on the pulse transit time method

Oscillometric blood pressure (BP) monitors are currently used to diagnose hypertension both in home and clinical settings. These monitors take BP measurements once every 15 minutes over a 24 hour period and provide a reliable and accurate system that is minimally invasive. Although intermittent cuff measurements have proven to be a good indicator of BP, a continuous BP monitor is highly desirable for the diagnosis of hypertension and other cardiac diseases. However, no such devices currently exist. A novel algorithm has been developed based on the Pulse Transit Time (PTT) method, which would allow non-invasive and continuous BP measurement. PTT is defined as the time it takes the BP wave to propagate from the heart to a specified point on the body. After an initial BP measurement, PTT algorithms can track BP over short periods of time, known as calibration intervals. After this time has elapsed, a new BP measurement is required to recalibrate the algorithm. Using the PhysioNet database as a basis, the new algorithm was developed and tested using 15 patients, each tested 3 times over a period of 30 minutes. The predicted BP of the algorithm was compared to the arterial BP of each patient. It has been established that this new algorithm is capable of tracking BP over 12 minutes without the need for recalibration, using the BHS standard, a 100% improvement over what has been previously identified. The algorithm was incorporated into a new system based on its requirements and was tested using three volunteers. The results mirrored those previously observed, providing accurate BP measurements when a 12 minute calibration interval was used. This new system provides a significant improvement to the existing method allowing BP to be monitored continuously and non-invasively, on a beat-to-beat basis over 24 hours, adding major clinical and diagnostic value.

Kohärente transiente und effiziente Endzustandskontrolle in Atomen und Molekülen

Es ist ein lang gehegter Traum in der Chemie, den Ablauf einer chemischen Reaktion zu kontrollieren und das Aufbrechen und Bilden chemischer Bindungen zu steuern. Diesem Ziel verschreibt sich auch das Forschungsgebiet der Femtochemie. Hier werden Femtosekunden Laserpulse eingesetzt um auf dem Quantenlevel molekulare Dynamiken auf ihren intrinsischen Zeitskalen zu kontrollieren und das System selektiv und effizient von einem Anfangs- in einen Zielzustand zu überführen. Der Wunsch, mit geformten Femtosekunden Laserpulsen Kontrolle über transiente Dynamiken und finale Populationen auszuüben, zu beobachten und zu verstehen, bildet auch die Motivation für diese Arbeit. Hierzu wurden mit Hilfe der Photoelektronenspektroskopie Untersuchungen zur Wechselwirkung atomarer und molekularer Prototypsysteme mit intensiven, geformten Femtosekunden Laserpulsen durchgeführt. Die Verwendung von Modelsystemen ermöglicht es, grundlegende Mechanismen der kohärenten Kontrolle in intensiven Laserfeldern zu analysieren, ohne dass sie durch komplexe Wechselwirkungen verschleiert werden. Zunächst wurde die Wechselwirkung von Kaliumatomen mit gechirpten Femtosekunden Laserpulsen untersucht. In den Experimenten wurden sowohl transiente Dynamiken als auch die Endbesetzungen der elektronischen Zustände abgebildet. In den folgenden Experimenten wurde das Quantenkontrollszenario SPODS auf die gekoppelte Elektronen-Kern-Dynamik in Molekülen übertragen. Die Kontrolle basiert auf der Erzeugung und Manipulation von Ladungsoszillationen durch Pulssequenzen. Der letzte Teil widmet sich der Entwicklung adiabatischer Kontrollmechanismen in Molekülen. Bei den Experimenten wurden gechirpte Airypulse eingesetzt um robuste Starkfeldanregung in molekularen Systemen zu induzieren. In Zukunft wird die Erforschung immer komplexerer Moleküle im Rahmen der transienten Kontrolle im Fokus stehen. Dabei werden nicht nur die effiziente Besetzung gebundener Zustände von Interesse sein, sondern auch die gezielte Dissoziation in spezifische Fragmente, photoinduzierte Isomerisierungsreaktionen oder die Kontrolle über transiente Dynamiken, die Einfluss auf andere molekulare Eigenschaften haben. Vor dem Hintergrund dieses übergeordneten Wunsches, photochemische Reaktionen immer komplexerer Moleküle, bis hin zu großen, biologisch relevanten Molekülen, zu kontrollieren, ist es umso wichtiger, die zugrundeliegenden Anregungsmechanismen in einfachen Systemen nachzuvollziehen. In den hier präsentierten Experimenten wurde gezeigt, wie die simultane Beobachtung der bekleideten und der stationären Zustände in atomaren Systemen zu einem umfassenden Bild der lichtinduzierte Dynamiken führen kann. Die gewonnenen Erkenntnisse können auf die Steuerung gekoppelter Dynamiken übertragen werden, durch die Kontrolle auch in molekularen Systemen möglich wird.

Amplification d’impulsions brèves de haute énergie par effet Raman stimulé dans les fibres optiques

Le développement au cours des dernières décennies de lasers à fibre à verrouillage de modes permet aujourd’hui d’avoir accès à des sources fiables d’impulsions femtosecondes qui sont utilisées autant dans les laboratoires de recherche que pour des applications commerciales. Grâce à leur large bande passante ainsi qu’à leur excellente dissipation de chaleur, les fibres dopées avec des ions de terres rares ont permis l’amplification et la génération d’impulsions brèves de haute énergie avec une forte cadence. Cependant, les effets non linéaires causés par la faible taille du faisceau dans la fibre ainsi que la saturation de l’inversion de population du milieu compliquent l’utilisation d’amplificateurs fibrés pour l’obtention d’impulsions brèves dont l’énergie dépasse le millijoule. Diverses stratégies comme l’étirement des impulsions à des durées de l’ordre de la nanoseconde, l’utilisation de fibres à cristaux photoniques ayant un coeur plus large et l’amplification en parallèle ont permis de contourner ces limitations pour obtenir des impulsions de quelques millijoules ayant une durée inférieure à la picoseconde. Ce mémoire de maîtrise présente une nouvelle approche pour l’amplification d’impulsions brèves utilisant la diffusion Raman des verres de silice comme milieu de gain. Il est connu que cet effet non linéaire permet l’amplification avec une large bande passante et ce dernier est d’ailleurs couramment utilisé aujourd’hui dans les réseaux de télécommunications par fibre optique. Puisque l’adaptation des schémas d’amplification Raman existants aux impulsions brèves de haute énergie n’est pas directe, on propose plutôt un schéma consistant à transférer l’énergie d’une impulsion pompe quasi monochromatique à une impulsion signal brève étirée avec une dérive en fréquence. Afin d’évaluer le potentiel du gain Raman pour l’amplification d’impulsions brèves, ce mémoire présente un modèle analytique permettant de prédire les caractéristiques de l’impulsion amplifiée selon celles de la pompe et le milieu dans lequel elles se propagent. On trouve alors que la bande passante élevée du gain Raman des verres de silice ainsi que sa saturation inhomogène permettent l’amplification d’impulsions signal à une énergie comparable à celle de la pompe tout en conservant une largeur spectrale élevée supportant la compression à des durées très brèves. Quelques variantes du schéma d’amplification sont proposées, et leur potentiel est évalué par l’utilisation du modèle analytique ou de simulations numériques. On prédit analytiquement et numériquement l’amplification Raman d’impulsions à des énergies de quelques millijoules, dont la durée est inférieure à 150 fs et dont la puissance crête avoisine 20 GW.

HVDC Power Transmission Simulation for Offshore Wind System with Three-Level Converter

An integrated mathematical model for the simulation of an offshore wind system performance is presented in this paper. The mathematical model considers an offshore variable-speed turbine in deep water equipped with a permanent magnet synchronous generator using multiple point full-power clamped three-level converter, converting the energy of a variable frequency source in injected energy into the electric network with constant frequency, through a HVDC transmission submarine cable. The mathematical model for the drive train is a concentrate two mass model which incorporates the dynamic for the blades of the wind turbine, tower and generator due to the need to emulate the effects of the wind and the floating motion. Controller strategy considered is a proportional integral one. Also, pulse width modulation using space vector modulation supplemented with sliding mode is used for trigger the transistors of the converter. Finally, a case study is presented to access the system performance.

Pulse shape studies of neutral particles with a liquid scintillator

The hadrontherapy exploits beams of charged particles against deep cancers. These ions have a depth-dose profile in which there is a little release of energy at the beginning of their path, whereas there is a sharp maximum, the Bragg Peak, near its end path. However, if heavy ions are used, the fragmentation of the projectile can happen and the fragments can release some dose outside the treatment volume beyond the Bragg peak. The fragmentation process takes place also when the Galactic Cosmic Rays at high energy hit the spaceship during space missions. In both cases some neutrons can be produced and if they interact with the absorbing materials nuclei some secondary particles are generated which can release energy. For this reason, studies about the cross section measurements of the fragments generated during the collisions of heavy ions against the tissues nuclei are very important. In this context, the FragmentatiOn Of Target (FOOT) experiment was born, and aims at measuring the differential and double differential fragmentation cross sections for different kinetic energies relevant to hadrontherapy and space radioprotection with high accuracy. Since during fragmentation processes also neutrons are produced, tests of a neutron detection system are ongoing. In particular, recently a neutron detector made up of a liquid organic scintillator, BC-501A with neutrons/gammas discrimination capability was studied, and it represents the core of this thesis. More in details, an analysis of the data collected at the GSI laboratory, in Darmstadt, Germany, is effectuated which consists in discriminating neutral and charged particles and then to separate neutrons from gammas. From this analysis, a preliminary energy-differential reaction cross-section for the production of neutrons in the 16O + (C_2H_4)_(n) and 16O + C reactions was estimated.

Obesity And Inflammation And The Effect On The Hematopoietic System.

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

The Value Of The 2005 International Society Of Urological Pathology (isup) Modified Gleason Grading System As A Predictor Of Biochemical Recurrence After Radical Prostatectomy.

To compare time and risk to biochemical recurrence (BR) after radical prostatectomy of two chronologically different groups of patients using the standard and the modified Gleason system (MGS). Cohort 1 comprised biopsies of 197 patients graded according to the standard Gleason system (SGS) in the period 1997/2004, and cohort 2, 176 biopsies graded according to the modified system in the period 2005/2011. Time to BR was analyzed with the Kaplan-Meier product-limit analysis and prediction of shorter time to recurrence using univariate and multivariate Cox proportional hazards model. Patients in cohort 2 reflected time-related changes: striking increase in clinical stage T1c, systematic use of extended biopsies, and lower percentage of total length of cancer in millimeter in all cores. The MGS used in cohort 2 showed fewer biopsies with Gleason score ≤ 6 and more biopsies of the intermediate Gleason score 7. Time to BR using the Kaplan-Meier curves showed statistical significance using the MGS in cohort 2, but not the SGS in cohort 1. Only the MGS predicted shorter time to BR on univariate analysis and on multivariate analysis was an independent predictor. The results favor that the 2005 International Society of Urological Pathology modified system is a refinement of the Gleason grading and valuable for contemporary clinical practice.

The Applicability Of Ordered Mesoporous Sba-15 And Its Hydrophobic Glutaraldehyde-bridge Derivative To Improve Ibuprofen-loading In Releasing System.

The mesoporous SBA-15 silica with uniform hexagonal pore, narrow pore size distribution and tuneable pore diameter was organofunctionalized with glutaraldehyde-bridged silylating agent. The precursor and its derivative silicas were ibuprofen-loaded for controlled delivery in simulated biological fluids. The synthesized silicas were characterized by elemental analysis, infrared spectroscopy, (13)C and (29)Si solid state NMR spectroscopy, nitrogen adsorption, X-ray diffractometry, thermogravimetry and scanning electron microscopy. Surface functionalization with amine containing bridged hydrophobic structure resulted in significantly decreased surface area from 802.4 to 63.0 m(2) g(-1) and pore diameter 8.0-6.0 nm, which ultimately increased the drug-loading capacity from 18.0% up to 28.3% and a very slow release rate of ibuprofen over the period of 72.5h. The in vitro drug release demonstrated that SBA-15 presented the fastest release from 25% to 27% and SBA-15GA gave near 10% of drug release in all fluids during 72.5 h. The Korsmeyer-Peppas model better fits the release data with the Fickian diffusion mechanism and zero order kinetics for synthesized mesoporous silicas. Both pore sizes and hydrophobicity influenced the rate of the release process, indicating that the chemically modified silica can be suggested to design formulation of slow and constant release over a defined period, to avoid repeated administration.

Electronic And Structural Study Of Pt-modified Au Vicinal Surfaces: A Model System For Pt-au Catalysts.

Two single crystalline surfaces of Au vicinal to the (111) plane were modified with Pt and studied using scanning tunneling microscopy (STM) and X-ray photoemission spectroscopy (XPS) in ultra-high vacuum environment. The vicinal surfaces studied are Au(332) and Au(887) and different Pt coverage (θPt) were deposited on each surface. From STM images we determine that Pt deposits on both surfaces as nanoislands with heights ranging from 1 ML to 3 ML depending on θPt. On both surfaces the early growth of Pt ad-islands occurs at the lower part of the step edge, with Pt ad-atoms being incorporated into the steps in some cases. XPS results indicate that partial alloying of Pt occurs at the interface at room temperature and at all coverage, as suggested by the negative chemical shift of Pt 4f core line, indicating an upward shift of the d-band center of the alloyed Pt. Also, the existence of a segregated Pt phase especially at higher coverage is detected by XPS. Sample annealing indicates that the temperature rise promotes a further incorporation of Pt atoms into the Au substrate as supported by STM and XPS results. Additionally, the catalytic activity of different PtAu systems reported in the literature for some electrochemical reactions is discussed considering our findings.

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Blockade Of Renin-angiotensin System Prevents Micturition Dysfunction In Renovascular Hypertensive Rats.

Association between hypertension and bladder symptoms has been described. We hypothesized that micturition dysfunction may be associated with renin-angiotensin system (RAS) acting in urethra. The effects of the anti-hypertensive drugs losartan (AT1 antagonist) and captopril (angiotensin-converting enzyme inhibitor) in comparison with atenolol (β1-adrenoceptor antagonist independently of RAS blockade) have been investigated in bladder and urethral dysfunctions during renovascular hypertension in rats. Two kidney-1 clip (2K-1C) rats were treated with losartan (30 mg/kg/day), captopril (50mg/kg/day) or atenolol (90 mg/kg/day) for eight weeks. Cystometric study, bladder and urethra smooth muscle reactivities, measurement of cAMP levels and p38 MAPK phosphorylation in urinary tract were determined. Losartan and captopril markedly reduced blood pressure in 2K-1C rats. The increases in non-voiding contractions, voiding frequency and bladder capacity in 2K-1C rats were prevented by treatments with both drugs. Likewise, losartan and captopril prevented the enhanced bladder contractions to electrical-field stimulation (EFS) and carbachol, along with the impaired relaxations to β-adrenergic-cAMP stimulation. Enhanced neurogenic contractions and impaired nitrergic relaxations were observed in urethra from 2K-1C rats. Angiotensin II also produced greater urethral contractions that were accompanied by higher phosphorylation of p38 MAPK in urethral tissues of 2K-1C rats. Losartan and captopril normalized the urethral dysfunctions in 2K-1C rats. In contrast, atenolol treatment largely reduced the blood pressure in 2K-1C rats but failed to affect the urinary tract smooth muscle dysfunction. The urinary tract smooth muscle dysfunction in 2K-1C rats takes place by local RAS activation irrespective of levels of arterial blood pressure.

Transmission Of Intra-cellular Genetic Information: A System Proposal.

One of the great challenges of the scientific community on theories of genetic information, genetic communication and genetic coding is to determine a mathematical structure related to DNA sequences. In this paper we propose a model of an intra-cellular transmission system of genetic information similar to a model of a power and bandwidth efficient digital communication system in order to identify a mathematical structure in DNA sequences where such sequences are biologically relevant. The model of a transmission system of genetic information is concerned with the identification, reproduction and mathematical classification of the nucleotide sequence of single stranded DNA by the genetic encoder. Hence, a genetic encoder is devised where labelings and cyclic codes are established. The establishment of the algebraic structure of the corresponding codes alphabets, mappings, labelings, primitive polynomials (p(x)) and code generator polynomials (g(x)) are quite important in characterizing error-correcting codes subclasses of G-linear codes. These latter codes are useful for the identification, reproduction and mathematical classification of DNA sequences. The characterization of this model may contribute to the development of a methodology that can be applied in mutational analysis and polymorphisms, production of new drugs and genetic improvement, among other things, resulting in the reduction of time and laboratory costs.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

Hypothalamic Inflammation And The Central Nervous System Control Of Energy Homeostasis.

The control of energy homeostasis relies on robust neuronal circuits that regulate food intake and energy expenditure. Although the physiology of these circuits is well understood, the molecular and cellular response of this program to chronic diseases is still largely unclear. Hypothalamic inflammation has emerged as a major driver of energy homeostasis dysfunction in both obesity and anorexia. Importantly, this inflammation disrupts the action of metabolic signals promoting anabolism or supporting catabolism. In this review, we address the evidence that favors hypothalamic inflammation as a factor that resets energy homeostasis in pathological states.