Conference of MSc students – Abstracts of the best contributions November 2011

S Conference of MSc students – Abstracts of the best contributions November 2011 Krisztina László (ed.)

ber of microbes extremely rose by means of the added nutrients.On the Vibrio fischeri luminescence inhibition test no toxicity was shown.Alteration of the length of mustard rudiments (Sinapis alba) (Figure 1) showed that in 1 mass percent dosage the material calls forth exhortation, but in 3 and 5 mass percent the unexplained toxicity of the material counteracts.

Cyclodextrin complexation of faradiol-esters extracted from marigold Calendula officinalis L. with supercritical carbon-dioxide
Bálint Baffi e-mail: bafbal@ch.bme.huSupervisor: Béla Simándi Department of Chemical and Environmental Process Engineering, BME e-mail: simandi@mail.bme.huConsultant: Dániel Cossuta Bunge Zrt.e-ail: cossuta@yahoo.co.ukThe marigold is a commonly used medicinal herb that has several positive effects assigned.My aim was to develop a procedure to form complexes of the triterpene anti-inflammatory agents of marigold (faradiol-esters that have a negligible solubility in water which limits their reachable in vivo concentration and efficiency) and cyclodextrins.The fact that the raw material was not a pure substance but a complex herbal extract that contains several hundreds of components differentiates my work from the various procedures for complexation that can be found in the literature.
In addition to the already described procedures experiments were performed with a new method that can be summarized by mixing the extract with a carrier with a high specific surface, pumping carbon-dioxide through it that re-solubilizes the components.Then, the fluid containing the extract is pushed through a cyclodextrin bed where the components form a complex with the cyclodextrin.This procedure enables us to operate it semicontinuously, and the product does not need any post-processing in opposition with the ones described so far.The efficiency of three fundamental technologies, the atmospheric pressure water suspension, the batch and the dynamic reactors, were compared under supercritical carbon-dioxide pressure with a strong emphasis put on differentiating the adsorbed and the really complexed faradiol-esters.HPLC was used to analyse the samples.
After interpreting the results it turned out that the batch supercritical technology can surpass the dynamic method with the applied circumstances.Fortunately, the supercritical fluid technology, that is still spreading and can be considered as a green technology, proved to be more efficient than the conventional suspension procedure.It can be stated that the β-cyclodextrin is capable of forming complexes in the order of magnitude of 1000 ppm while the γ -cyclodextrin is not in the applied circumstances.The aim set, to increase the apparent solubility of the key components in water with several orders, has been achieved by the produced complexes.

Department of Physical Chemistry and Materials Science, BME
There are several methods for film deposition onto solid substrates.Langmuir-Blodgett (LB) method is suitable to prepare nanoparticulate coatings with a planned structure and morphology.The practical applications of nanoparticulate LB films, however, are limited by their poor mechanical stability.Dipcoating technique based on sol-gel processes is a relatively simple method for preparing thin coatings with high durability.The properties of the films can precisely be controlled by changing the conditions of deposition and/or the composition of the precursor materials.
Our aim was to combine the advantageous features of the aforementioned methods for fabricating nanostructural, mechanically stable coatings with desirable morphology.LB films of Stöber silica nanoparticles having diameter of ca.130 nm were coated with silica or titania precursor sols.Microscope glass slides were used as solid substrates.The mechanical stability, the structure and the surface morphology of the combined coatings were investigated.
The combined coatings, demonstrated by the lift-off tests, showed an improved mechanical stability compared to the original LB films.The optical properties were investigated with scanning angle reflectometry and UV-Vis spectroscopy methods and the resulting reflectance and transmittance curves were evaluated in terms of different thin layer optical models.The optical studies and atomic force microscopy investigations gave information about the structure and morphology of coatings.Investigations revealed surfaces with different nanomorphology which was mainly dependent on the thickness of the sol-gel layer deposited onto the nanoparticulate film.For comparison, combined coatings of microparticulate LB films and sol-gel layers were also studied.
Plant based carbon fibres can be used for soil improvement and -in activated form -as adsorbents.These fibres are carbonneutral (the amounts of carbon dioxide developing during their combustion and fixed by the plant through photosynthesis, respectively, are equal), moreover, using of these carbon products ("biochar") for soil amendment is a method for carbon sequestering.
The quality of the carbonized plant material is an important issue in application.According to the literature the properties of carbonized plant depend not only on the ratio of the plant's components, but on the interaction of the components as well.
Carbonization of flax and hemp fibres, advantageously cultivated in Hungary, was investigated.Differences between the two types of cellulose source, the pre-treatment of fibres and carbonization conditions influencing the quality of the product were investigated.Native fibres were modified by alkali/ultrasound treatment used generally for partial disintegration of the fibrils.Parameters of carbonization were selected according to the TG-MS results.Effects of the selected carbonization parameters on the yield and density were determined.The nitrogen adsorption capacities of native, modified, and carbonized fibres were measured, porosity was calculated, SEM pictures of samples were taken.The pre-treatment modified the pore structure of both flax and hemp fibres (Fig. ): the nitrogen adsorption capacity of flax decreased, while that of hemp increased.In both cases, the effect of a combined alkaline/ultrasonic treatment was the highest.
The changes of native fibres were reflected by the properties of the carbonized fibres.In case of hemp both types of pretreatments increased the pore volume of the carbonized fibre; alkali/ultrasound combined technology multiplied the pore volume by almost three.The pore volume of flax was decreased by simple sonication, while combined alkali/ultrasound treatment increased it, although in a smaller degree than in the case of hemp.
The changes of fibre properties due to the pre-treatments are different for flax and hemp.It calls attention on the potential contradictions of ultrasound treatments.

Production of extracts from garlic Allium sativum L. with ethyl alcohol, the scale-up of the procedure and product development based on the extracts
Péter Csépány e-mail: peter.csepany@ch.bme.huSupervisor: Béla Simándi Department of Chemical and Environmental Process Engineering, BME e-mail: simandi@mail.bme.huConsultant: Bence Nagy MOL Nyrt.e-mail: nagybence@yahoo.com Putting the medicinal herbs in order based on their importance in the history of humankind, garlic has always been among the ten most important ones.Nowadays it plays an increasing role in the treatment of the number one illness of the modern age, the heart and circulatory diseases.With a wide spectrum of agents, garlic can reduce the blood pressure, reduce the level of the harmful LDL cholesterol and prevent formation of thrombi.
According to the similar opinions of professionals the most important components of the garlic are the alliin and its metabolites that are formed during the activity of the alliinase enzyme that decomposes the alliin and causes the characteristic and wellknown smell.My aims were to uncover the factors influencing the extraction of alliin, to choose an excellent raw material that is rich in alliin and to map the extraction parameters of the chosen raw material.That work was based on earlier experiments.My recent aims were to develop an economical process for garlic extraction, to scale it up by one order of magnitude and to develop a dietetic supplementary product based on the extract.
Firstly, a multi-stage lab-scale extraction process was developed bearing economic aspects in mind.By further experiments, a method to measure the total alliin content in the garlic raw material was developed.
The second part of the work started with planning the pilotscale equipment.After building it the adaptation of the lab-sized process could begin.I performed test experiments in order to find the adequate settings of the device.In further experiments the desired process in the pilot-scale experiment was reproduced even with better results than expected.In this phase of job several production experiments were performed producing solid extracts of an excellent quality and concentrated solutions of the extracts.
In the third phase, we performed the product development based on the extracts produced in the pilot-scale experiments in a close co-operation with our industrial partners.The product designed has a high alliin potential, moreover we have solved the molecular trapping of the smell produced during the process.

Growth pattern analysis in cell cycle mutants of fission yeast: Linear or bilinear?
yeast cells grow exclusively at their tips almost from birth to division by maintaining a constant diameter, therefore cell length is approximately proportional to cell volume.Length growth patterns may therefore indicate connections between volume changes and cell cycle events.The classical method to study the growth of individual cells is time-lapse microphotography; cells are growing on the surface of an agar pad in a thermostated photomicroscope, and later on, the growth pattern of cell length can be simply studied by a projector.A size control acts in every cell cycle to maintain homeostasis of cell size.With further analyses of growth patterns we are able to estimate the position of this size control.
In different cell types, there is considerable controversy concerning the exact growth profile of size parameters during the cell cycle.Linear, exponential and bilinear (i.e., two linear segments with a Rate Change Point (RCP)) models are commonly considered, and the same model may not apply for all species.The bilinear model is namely a linearised biexponential model, which makes possible a smooth, continuously differentiable transition between two linear segments.Selection of the most adequate model to describe a given data-set requires the use of quantitative model selection criteria, like Akaike Information Criterion (AIC), which are suitable for comparing differently parameterised models.
I have analysed the length growth patterns of 180 fission yeast cells (60 cdc2-3w mutants, 60 cdc2-3w cdc25 double mutants and 60 cdc2-3w cdc25 pyp3 triple mutants).The above mentioned model selection criteria were used for discriminating among linear, exponential and bilinear models and selecting the most adequate one in the case of all these cells' length growth patterns.Although relatively small differences were found in several cases, essentially all the quantitative selection criteria considered here indicated that the bilinear model was generally more adequate than either the exponential or the linear ones.At least the half of the cells had a bilinear growth pattern.In these cases the slopes of the two linear segments were also compared with t-test (Statistica R software, Homogeneity of slopes function)."Average cells" were also constructed from all the individual cells' data for all strains, whose patterns were definitely found to be bilinear by any criterion used.In cdc2-3w cdc25 double and cdc2-3w cdc25 pyp3 triple mutants size control seems to act after RCP, in late G 2 .Furthermore, in cdc2-3w mutant cells, size control probably operates in both G1 and G2 phases.This is in sharp contrast to wild type cells, but confirms our former results.

Acknowledgement
This project is supported by the Hungarian Scientific Research Fund (OTKA K-76229).

The synthesis of phosphinates in an environmentally friendly manner
Nóra Zsuzsa Kiss e-mail: n.zs.kiss@gmail.comSupervisor: György Keglevich Department of Organic Chemistry and Technology, BME e-mail: gkeglevich@mail.bme.hu As phosphinic acids cannot undergo direct esterification with alcohols, phosphinates are usually prepared by the reaction of phosphinic chlorides with alcohols and phenols (Scheme 1).
Scheme 1.A general route for the preparation of phosphinates Although this method has drawbacks from the point of view of "green" chemistry and costs, it is widely applied in industry.
It was found that the otherwise impossible direct esterification of phosphinic acids takes place under MW conditions by irradiating the mixture of the cyclic phosphinic acids selected as model compounds and the alcohol (used in ca 15-fold excess) at around 200 ˚C in a sealed tube (Scheme 2).Scheme 2. Novel method for the preparation of phosphinates Under such conditions, the cyclic phosphinates could be obtained, in most cases, in about 45-60% yields.The only criterion is that the alcohol must not be too volatile to avoid a pressure >15 bar.
The novel reaction is the consequence of the beneficial specific MW effect that was proved by comparative thermal experiments.
Our method, that is an environmentally friendly approach, and which can be accomplished without the use of phosphinic chlorides, is of novelty and of general value.For this, we make efforts for further extensions and applications.
The origin of enzymatic catalysis still remains to be elucidated [1].The goal of our work was to uncover the major driving forces of enzymatic reactions and to design even more efficient enzymes using these principles.As a model system, an artificial enzyme, the Kemp eliminase was used [2].This enzyme is resulted by a rational design process in an effort to find an optimal scaffold for an active site that was obtained by quantum chemical methods.The original, native enzyme showed very good agreement with the computational model, but its activity was far behind the natural enzymes.Therefore it was subjected to an in vitro evolution experiment, which could improve the catalytic rate by 200 fold.We aimed to simulate the reaction mechanism of both the native and the evolved enzyme and compare the major catalytic factors.The activation barriers obtained by the Empirical Valence Bond (EVB) method [3] were in accord with the experimental data.We found that in course of the laboratory evolution the reorganization energy was optimized significantly, meanwhile the activation barrier only modestly decreased.This observation was corroborated by analyzing the individual residue contributions to the catalysis.Almost all mutations in the evolved variant were accompanied by reduction of the reorganization energy, while the electrostatic energies did not exhibit a uniform trend.Based on this observation, we have developed a screening programme that computes reorganization energy contributions of the studied mutations.This code is applicable for high throughput screening and was tested on almost 200 virtual mutations.The computational results were compared to all available mutants that appeared during the in vitro evolution experiments.In all cases, where the mutant enzyme was experimentally available, i.e. selected by evolution, the change in reorganization energy was either negligible or considerably decreased.This verified our conclusion that this quantity is optimized during evolution.Kinetic parameters of the K221A mutant were determined and exhibited excellent agreement with the computational data.Testing of other designed mutants is ongoing.

Copper(II) complexes of two bis(aminomethyl)phosphinicacid derivatives studied by EPR spectroscopy
Csilla László e-mail: csillalaszlo87@gmail.com Supervisor: Nóra Veronika Nagy, Hungarian Academy of Sciences, Chemical Research Center e-mail: nagyn@chemres.hu There is increasing interest in the research of new chelating ligands for use as molecular imaging agents of metallic radionuclides in medical diagnostic methods (e.g. 64Cu t 1/2 =12,7 h, in positron emission tomography, PET).Up till now, macrocyclic polyaza polycarboxylate and polyphosphonate ligands (with therminal carboxilate and phosphonate groups) were commonly used but nowadays emphasis were made on the preparation and investigation of ligands containing phosphinate groups in a ring or in open chains.Here we report on the complexation study of two new chelating ligands, (1) 2,2'-(3-hydroxi-3-oxo-1,5,3-diazaphosphepan-1,5diil)-diaceticacid (7N2P-2Ac) and (2) (N-methyl-glycino)(Nmethyliminodiacetic)phosphinate (N2P1-3Ac) (Scheme 1).methyliminodiacetic)phosphinate (N2P1-3Ac) (Scheme 1).These chelating ligands must achieve several criteria -the most important is the thermodynamic stability of their complexes.Our goal was to investigate the stabilities and structures of the paramagnetic copper(II) complexes of the above ligands using Electron Paramagnetic Resonance spectroscopic (EPR) methods.A series of EPR spectra were recorded at room temperature in different pHs and ligand to metal concentration ratios and were simulated simultaneously with a "two-dimensional" simulation program [1].By the decomposition of the measured spectra the program can provide the isotropic EPR parameters Per.Pol.Chem.Eng.
as well as the formation constants of the components formed in the overlapping equilibria.EPR spectra at 77 K were also measured at each pH-points, which were simulated by the EPR program [2] to obtain the anizotropic EPR parameters.In order to facilitate the interpretaion of our data geometry optimization of the complexes and calculation of the theoretical EPR parameters were performed by DFT quantum chemical calculations.According to the data of WHO tuberculosis is the most deadly infectious disease, which causes nearly 2 million deaths each year.The dUTPase enzyme that is in focus in our laboratory, is a potential drug target, since it is essential for Mycobacterium tuberculosis.The dUTPase plays a crucial role in DNA integrity, as it catalyses the hydrolysis of dUTP to dUMP and pyrophosphate, thereby it produces the precursor of dTTP biosynthesis and keeps the dUTP:dTTP ratio low.In the absence of dUTPase this ratio will be high, which causes DNA fragmentation and the so-called thymine-less cell death.

References
To understand the enzymatic mechanism mutant enzymes were previously generated, which differ from the wild type at the substrate binding C-terminal arm.The structures of two mutants -one with alanine instead of histidin, which stacks over the uracile ring and one, which lacks the full C-terminal armcould be resolved and have been deposited to the PDB database with ID of 3LOJ and 3I93.To compare the substrate hydrolysis mechanism of the wild type and the mutants, in silico analysis have been performed at the NIH (National Institutes of Health, Bethesda, Maryland, USA).
Using a previously defined [1] reaction coordinate, the geometries have been minimized starting with the initial enzymesubstrate structures and following the reaction through the tran-sition state to the product state.The substrate and the whole active site of the wild type and mutant enzymes were included in the quantum region, which was calculated using density functional theory, while the remaining part of the enzyme and the solvent within 20 Å from the substrate-bound Mg 2+ ion was treated with classical molecular mechanics and was coupled to the quantum region using full electrostatic embedding.
The calculated activation energies are in good agreement with the experimentally observed catalytic rate constants.The moieties important to the efficient catalytic activity of the dUTPase enzyme can be identified according to the charge distribution of the active site within the obtained structures, and to the changes in relevant bond lengths and angles.
establishing an electric field gradient in case of species with high electrophoretic mobility or inducing electroosmotic flow to capture uncharged species.To determine the relative extent of various transport mechanism and their efficiency to capture the targeted species and direct them into the nanopore sensing zone we solved the coupled Poisson, Nernst-Planck and Navier-Stokes differential equations using COMSOL Multiphysics finite element package.The results confirm the importance of considering the entrance resistance into the nanopores when correlating the transpore current and pore geometry as well as that both electrophoresis and electroosmosis can increase the translocation efficiency of the target species through the nanopore.The present study was successful in providing the theoretical predictive basis for the development of single-nanopore electrochemical sensors for virus and nanoparticle counting.
In the past two decades more and more evidence has accumulated supporting the view that intrinsically disordered proteins comprise a large fraction of eukaryotic proteomes.Although these proteins do not have a well-defined tertiary structure, they play key roles in essential processes such as cell-cycle control, regulation, signaling and apoptosis [1].It was also observed that disordered proteins with disordered regions are often involved in cancer and other disease associated proteins [2].Thus, it has been suggested that protein disorder entails a biological cost.To address this statement it is necessary to understand the associations between the structural properties of proteins and the mutations which are responsible for the development of disease.
The aim of my research was the detailed analysis of the frequency of cancer-associated point mutations and of polymorphisms in ordered and disordered protein segments and disordered binding regions by means of bioinformatics and statistical methods.The experimentally verified mutational data of cancerassociated proteins were taken from the Swiss-Prot database.In the statistical analysis of the distribution of mutations, both the random distribution and the distribution weighted by the observed polymorphisms were used as a reference.
The analysis of the polymorphisms showed that they are not evenly distributed in proteins and can be found more frequently in disordered regions.This points to the higher tolerance of disordered regions to mutations.The connection between cancerassociated mutations and the structural properties of proteins indicated a more complex picture.Contrary to the generally accepted view, in general, the cancer-associated mutations were more likely to fall into the ordered parts of the sequence than expected based on the random distribution, and this effect was even more significant when the uneven distribution of the polymorphisms was used as a reference.In addition, I presented a few examples that illustrated the connection between protein function and mutations in various ordered and disordered regions.
To summarize my findings, the presumption that protein disorder imposes a biological cost per se in the context of point mutations can be refuted.The results may contribute to the understanding of the role of protein disorder in cancer, which can be applied in a wide range of disciplines from basic research to rational drug design.Steric arrangements of the ring substituents were confirmed in three cases (R = Me, Et, Bn) by two dimensional NMR measurements.In addition, N -Boc protected azetidine was prepared using the same superbase and an N -allyl azetidine derivative

Scheme 1 :
Scheme 1: Structures of the ligands studied