Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

Abstract Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.

Synthetic biology: a novel approach for the construction of industrial microorganisms

The recent achievement of synthesising a functioning bacterial chromosome marks a coming of age for engineering living organisms. In the future this should allow the construction of novel organisms to help solve the problems facing the human race, including health care, food, energy and environmental protection. In this minireview, the current state of the field is described and the role of synthetic biology in biotechnology in the short and medium term is discussed. It is particularly aimed at the needs of food technologists, nutritionists and other biotechnologists, who might not be aware of the potential significance of synthetic biology to the research and development in their fields. The potential of synthetic biology to produce interesting new polyketide compounds is discussed in detail.

Effect of interaction with micelles on the excited-state optical properties of zinc porphyrins and J-aggregates formation

This work reports on the photophysical properties of zinc porphyrins meso-tetrakis methylpyridiniumyl (Zn2+TMPyP) and meso-tetrakis sulfonatophenyl (Zn2+TPPS) in homogeneous aqueous solutions and in the presence of sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) micelles. The excited-state dynamic was investigated with the Z-scan technique, UV-Vis absorption, and fluorescence spectroscopy. Photophysical parameters were obtained by analyzing the experimental data with a conventional five-energy-level diagram. The interaction of the charged side porphyrin groups with oppositely charged surfactants can reduce the electrostatic repulsion between porphyrin molecules leading to aggregation, which affected the porphyrin characteristics such as absorption cross-sections, lifetimes and quantum yields. The interaction between anionic ZnTPPS with cationic CTAB micelles induced the formation of porphyrin J-aggregates, while this effect was not observed in the interaction of ZnTMPyP with SDS micelles. This difference is, probably, due to the difference in electrostatic repulsion between the porphyrin molecules. The insights obtained by these results are important for the understanding of the photophysical behavior of porphyrins, regarding potential applications in pharmacokinetics as encapsulation of photosensitizer for drug delivery systems and in its interaction with cellular membrane.

Assessment of the initial state of two reserves, Micro áreas Ecoturísticas Litorales (MAEL), in Gran Canaria, Canary Islands

Máster en Oceanografía

Development of musculoskeletal models for the design and the pre-clinical validation of hip resurfacing prosthesis

Background. The surgical treatment of dysfunctional hips is a severe condition for the patient and a costly therapy for the public health. Hip resurfacing techniques seem to hold the promise of various advantages over traditional THR, with particular attention to young and active patients. Although the lesson provided in the past by many branches of engineering is that success in designing competitive products can be achieved only by predicting the possible scenario of failure, to date the understanding of the implant quality is poorly pre-clinically addressed. Thus revision is the only delayed and reliable end point for assessment. The aim of the present work was to model the musculoskeletal system so as to develop a protocol for predicting failure of hip resurfacing prosthesis. Methods. Preliminary studies validated the technique for the generation of subject specific finite element (FE) models of long bones from Computed Thomography data. The proposed protocol consisted in the numerical analysis of the prosthesis biomechanics by deterministic and statistic studies so as to assess the risk of biomechanical failure on the different operative conditions the implant might face in a population of interest during various activities of daily living. Physiological conditions were defined including the variability of the anatomy, bone densitometry, surgery uncertainties and published boundary conditions at the hip. The protocol was tested by analysing a successful design on the market and a new prototype of a resurfacing prosthesis. Results. The intrinsic accuracy of models on bone stress predictions (RMSE < 10%) was aligned to the current state of the art in this field. The accuracy of prediction on the bone-prosthesis contact mechanics was also excellent (< 0.001 mm). The sensitivity of models prediction to uncertainties on modelling parameter was found below 8.4%. The analysis of the successful design resulted in a very good agreement with published retrospective studies. The geometry optimisation of the new prototype lead to a final design with a low risk of failure. The statistical analysis confirmed the minimal risk of the optimised design over the entire population of interest. The performances of the optimised design showed a significant improvement with respect to the first prototype (+35%). Limitations. On the authors opinion the major limitation of this study is on boundary conditions. The muscular forces and the hip joint reaction were derived from the few data available in the literature, which can be considered significant but hardly representative of the entire variability of boundary conditions the implant might face over the patients population. This moved the focus of the research on modelling the musculoskeletal system; the ongoing activity is to develop subject-specific musculoskeletal models of the lower limb from medical images. Conclusions. The developed protocol was able to accurately predict known clinical outcomes when applied to a well-established device and, to support the design optimisation phase providing important information on critical characteristics of the patients when applied to a new prosthesis. The presented approach does have a relevant generality that would allow the extension of the protocol to a large set of orthopaedic scenarios with minor changes. Hence, a failure mode analysis criterion can be considered a suitable tool in developing new orthopaedic devices.

Evaluation of a multi variated analysis for the selection of t-tbar multijet events in the current CMS implemented environment at LHC

In the present study we are using multi variate analysis techniques to discriminate signal from background in the fully hadronic decay channel of ttbar events. We give a brief introduction to the role of the Top quark in the standard model and a general description of the CMS Experiment at LHC. We have used the CMS experiment computing and software infrastructure to generate and prepare the data samples used in this analysis. We tested the performance of three different classifiers applied to our data samples and used the selection obtained with the Multi Layer Perceptron classifier to give an estimation of the statistical and systematical uncertainty on the cross section measurement.

Physical and mineralogical changes of Hungarian monumental stones exposed to different conditions: stone-testing in-situ and under laboratory conditions

The Székesfehérvár Ruin Garden is a unique assemblage of monuments belonging to the cultural heritage of Hungary due to its important role in the Middle Ages as the coronation and burial church of the Kings of the Hungarian Christian Kingdom. It has been nominated for “National Monument” and as a consequence, its protection in the present and future is required. Moreover, it was reconstructed and expanded several times throughout Hungarian history. By a quick overview of the current state of the monument, the presence of several lithotypes can be found among the remained building and decorative stones. Therefore, the research related to the materials is crucial not only for the conservation of that specific monument but also for other historic structures in Central Europe. The current research is divided in three main parts: i) description of lithologies and their provenance, ii) physical properties testing of historic material and iii) durability tests of analogous stones obtained from active quarries. The survey of the National Monument of Székesfehérvár, focuses on the historical importance and the architecture of the monument, the different construction periods, the identification of the different building stones and their distribution in the remaining parts of the monument and it also included provenance analyses. The second one was the in situ and laboratory testing of physical properties of historic material. As a final phase samples were taken from local quarries with similar physical and mineralogical characteristics to the ones used in the monument. The three studied lithologies are: fine oolitic limestone, a coarse oolitic limestone and a red compact limestone. These stones were used for rock mechanical and durability tests under laboratory conditions. The following techniques were used: a) in-situ: Schmidt Hammer Values, moisture content measurements, DRMS, mapping (construction ages, lithotypes, weathering forms) b) laboratory: petrographic analysis, XRD, determination of real density by means of helium pycnometer and bulk density by means of mercury pycnometer, pore size distribution by mercury intrusion porosimetry and by nitrogen adsorption, water absorption, determination of open porosity, DRMS, frost resistance, ultrasonic pulse velocity test, uniaxial compressive strength test and dynamic modulus of elasticity. The results show that initial uniaxial compressive strength is not necessarily a clear indicator of the stone durability. Bedding and other lithological heterogeneities can influence the strength and durability of individual specimens. In addition, long-term behaviour is influenced by exposure conditions, fabric and, especially, the pore size distribution of each sample. Therefore, a statistic evaluation of the results is highly recommended and they should be evaluated in combination with other investigations on internal structure and micro-scale heterogeneities of the material, such as petrographic observation, ultrasound pulse velocity and porosimetry. Laboratory tests used to estimate the durability of natural stone may give a good guidance to its short-term performance but they should not be taken as an ultimate indication of the long-term behaviour of the stone. The interdisciplinary study of the results confirms that stones in the monument show deterioration in terms of mineralogy, fabric and physical properties in comparison with quarried stones. Moreover stone-testing proves compatibility between quarried and historical stones. Good correlation is observed between the non-destructive-techniques and laboratory tests results which allow us to minimize sampling and assessing the condition of the materials. Concluding, this research can contribute to the diagnostic knowledge for further studies that are needed in order to evaluate the effect of recent and future protective measures.

NMR studies on supramolecular aggregates in solution and the solid state

The aim of this work presented here is the characterization of structure and dynamics of different types of supramolecular systems by advanced NMR spectroscopy. One of the characteristic features of NMR spectroscopy is based on its high selectivity. Thus, it is desirable to exploit this technique for studying structure and dynamics of large supramolecular systems without isotopic enrichment. The observed resonance frequencies are not only isotope specific but also influenced by local fields, in particular by the distribution of electron density around the investigated nucleus. Barbituric acid are well known for forming strongly hydrogen-bonded complexes with variety of adenine derivatives. The prototropic tautomerism of this material facilitates an adjustment to complementary bases containing a DDA(A = hydrogen bond acceptor site, D = hydrogen bond donor site) or ADA sequences, thereby yielding strongly hydrogen-bonded complexes. In this contribution solid-state structures of the enolizable chromophor "1-n-butyl-5-(4-nitrophenyl)-barbituric acid" that features adjustable hydrogen-bonding properties and the molecular assemblies with three different strength of bases (Proton sponge, adenine mimetic 2,6-diaminopyridine (DAP) and 2,6-diacetamidopyridine (DAC)) are studied. Diffusion NMR spectroscopy gives information over such interactions and has become the method of choice for measuring the diffusion coefficient, thereby reflecting the effective size and shape of a molecular species. In this work the investigation of supramolecular aggregates in solution state by means of DOSY NMR techniques are performed. The underlying principles of DOSY NMR experiment are discussed briefly and more importantly two applications demonstrating the potential of this method are focused on. Calix[n]arenes have gained a rather prominent position, both as host materials and as platforms to design specific receptors. In this respect, several different capsular contents of tetra urea calix[4]arenes (benzene, benzene-d6, 1-fluorobenzene, 1-fluorobenzene-d5, 1,4-difluorobenzene, and cobaltocenium) are studied by solid state NMR spectroscopy. In the solid state, the study of the interaction between tetra urea calix[4]arenes and guest is simplified by the fact that the guests molecule remains complexed and positioned within the cavity, thus allowing a more direct investigation of the host-guest interactions.

Mapping the elastic response of epithelial apical cell membranes suspended across porous array

As the elastic response of cell membranes to mechanical stimuli plays a key role in various cellular processes, novel biophysical strategies to quantify the elasticity of native membranes under physiological conditions at a nanometer scale are gaining interest. In order to investigate the elastic response of apical membranes, elasticity maps of native membrane sheets, isolated from MDCK II (Madine Darby Canine kidney strain II) epithelial cells, were recorded by local indentation with an Atomic Force Microscope (AFM). To exclude the underlying substrate effect on membrane indentation, a highly ordered gold coated porous array with a pore diameter of 1.2 μm was used to support apical membranes. Overlays of fluorescence and AFM images show that intact apical membrane sheets are attached to poly-D-lysine coated porous substrate. Force indentation measurements reveal an extremely soft elastic membrane response if it is indented at the center of the pore in comparison to a hard repulsion on the adjacent rim used to define the exact contact point. A linear dependency of force versus indentation (-dF/dh) up to 100 nm penetration depth enabled us to define an apparent membrane spring constant (kapp) as the slope of a linear fit with a stiffness value of for native apical membrane in PBS. A correlation between fluorescence intensity and kapp is also reported. Time dependent hysteresis observed with native membranes is explained by a viscoelastic solid model of a spring connected to a Kelvin-Voight solid with a time constant of 0.04 s. No hysteresis was reported with chemically fixated membranes. A combined linear and non linear elastic response is suggested to relate the experimental data of force indentation curves to the elastic modulus and the membrane thickness. Membrane bending is the dominant contributor to linear elastic indentation at low loads, whereas stretching is the dominant contributor for non linear elastic response at higher loads. The membrane elastic response was controlled either by stiffening with chemical fixatives or by softening with F-actin disrupters. Overall, the presented setup is ideally suitable to study the interactions of the apical membrane with the underlying cytoskeleton by means of force indentation elasticity maps combined with fluorescence imaging.

The gas mass fraction and the dynamical state in x-ray luminous clusters of galaxies at low redshift

Gli ammassi di galassie sono le strutture gravitazionalmente legate con le più profonde buche di potenziale, pertanto è previsto che questi contengano una frazione di barioni non molto diversa da quella cosmologica. Con l’introduzione di modelli sempre più accurati di fisica barionica all’interno di simulazioni idrodinamiche è stato possibile predire la percentuale cosmica di barioni presente negli ammassi di galassie. Unendo questi modelli previsionali con misure della frazione di gas in ammassi e informazioni sulla densità di barioni dell’Universo si può ottenere una stima della densità di materia cosmica Ωm. L'obiettivo di questo lavoro di Tesi è la stima di Ωm a partire dalla frazione di gas osservata in questi sistemi. Questo lavoro era stato già fatto in precedenza, ma tenendo in considerazione solo gli ammassi più massivi e dinamicamente rilassati. Usando parametri che caratterizzano la morfologia della distribuzione di brillanza superficiale nei raggi X, abbiamo classificato i nostri oggetti come rilassati o disturbati, laddove presentassero evidenze di recenti attività di interazione. Abbiamo dunque valutato l’impatto degli oggetti disturbati sulla stima del parametro cosmologico Ωm, computando il Chi2 tra la frazione di massa barionica nell’Universo e quella da noi ricavata. Infine abbiamo investigato una relazione tra il valore della frazione di gas degli ammassi rilassati e quello dei disturbati, in modo da correggere quindi questi ultimi, riportandoli nei dintorni del valore medio per i rilassati e usarli per ampliare il campione e porre un vincolo più stringente su Ωm. Anche con il limitato campione a nostra disposizione, è stato possibile porre un vincolo più stretto su Ωm, utilizzando un maggior numero di oggetti e riducendo così l’errore statistico.

Simulated switching of the resting state functional connectivity in mouse brain using a real mesoscale connectome

Capire come modellare l'attività del cervello a riposo, resting state, è il primo passo necessario per avvicinarsi a una reale comprensione della dinamica cerebrale. Sperimentalmente si osserva che, quando il cervello non è soggetto a stimoli esterni, particolari reti di regioni cerebrali presentano un'attività neuronale superiore alla media. Nonostante gli sforzi dei ricercatori, non è ancora chiara la relazione che sussiste tra le connessioni strutturali e le connessioni funzionali del sistema cerebrale a riposo, organizzate nella matrice di connettività funzionale. Recenti studi sperimentali mostrano la natura non stazionaria della connettività funzionale in disaccordo con i modelli in letteratura. Il modello implementato nella presente tesi per simulare l'evoluzione temporale del network permette di riprodurre il comportamento dinamico della connettività funzionale. Per la prima volta in questa tesi, secondo i lavori a noi noti, un modello di resting state è implementato nel cervello di un topo. Poco è noto, infatti, riguardo all'architettura funzionale su larga scala del cervello dei topi, nonostante il largo utilizzo di tale sistema nella modellizzazione dei disturbi neurologici. Le connessioni strutturali utilizzate per definire la topologia della rete neurale sono quelle ottenute dall'Allen Institute for Brain Science. Tale strumento fornisce una straordinaria opportunità per riprodurre simulazioni realistiche, poiché, come affermato nell'articolo che presenta tale lavoro, questo connettoma è il più esauriente disponibile, ad oggi, in ogni specie vertebrata. I parametri liberi del modello sono stati scelti in modo da inizializzare il sistema nel range dinamico ottimale per riprodurre il comportamento dinamico della connettività funzionale. Diverse considerazioni e misure sono state effettuate sul segnale BOLD simulato per meglio comprenderne la natura. L'accordo soddisfacente fra i centri funzionali calcolati nel network cerebrale simulato e quelli ottenuti tramite l'indagine sperimentale di Mechling et al., 2014 comprovano la bontà del modello e dei metodi utilizzati per analizzare il segnale simulato.