Quantitative proteomics methods for the analysis of histone post-translational modifications

Les histones sont des protéines nucléaires hautement conservées chez les cellules des eucaryotes. Elles permettent d’organiser et de compacter l’ADN sous la forme de nucléosomes, ceux-ci representant les sous unités de base de la chromatine. Les histones peuvent être modifiées par de nombreuses modifications post-traductionnelles (PTMs) telles que l’acétylation, la méthylation et la phosphorylation. Ces modifications jouent un rôle essentiel dans la réplication de l’ADN, la transcription et l’assemblage de la chromatine. L’abondance de ces modifications peut varier de facon significative lors du developpement des maladies incluant plusieurs types de cancer. Par exemple, la perte totale de la triméthylation sur H4K20 ainsi que l’acétylation sur H4K16 sont des marqueurs tumoraux spécifiques a certains types de cancer chez l’humain. Par conséquent, l’étude de ces modifications et des événements determinant la dynamique des leurs changements d’abondance sont des atouts importants pour mieux comprendre les fonctions cellulaires et moléculaires lors du développement de la maladie. De manière générale, les modifications des histones sont étudiées par des approches biochimiques telles que les immuno-buvardage de type Western ou les méthodes d’immunoprécipitation de la chromatine (ChIP). Cependant, ces approches présentent plusieurs inconvénients telles que le manque de spécificité ou la disponibilité des anticorps, leur coût ou encore la difficulté de les produire et de les valider. Au cours des dernières décennies, la spectrométrie de masse (MS) s’est avérée être une méthode performante pour la caractérisation et la quantification des modifications d’histones. La MS offre de nombreux avantages par rapport aux techniques traditionnelles. Entre autre, elle permet d’effectuer des analyses reproductibles, spécifiques et facilite l’etude d’un large spectre de PTMs en une seule analyse. Dans cette thèse, nous présenterons le développement et l’application de nouveaux outils analytiques pour l’identification et à la quantification des PTMs modifiant les histones. Dans un premier temps, une méthode a été développée pour mesurer les changements d’acétylation spécifiques à certains sites des histones. Cette méthode combine l’analyse des histones intactes et les méthodes de séquençage peptidique afin de déterminer les changements d’acétylation suite à la réaction in vitro par l’histone acétyltransférase (HAT) de levure Rtt109 en présence de ses chaperonnes (Asf1 ou Vps75). Dans un second temps, nous avons développé une méthode d’analyse des peptides isomériques des histones. Cette méthode combine la LC-MS/MS à haute résolution et un nouvel outil informatique appelé Iso-PeptidAce qui permet de déconvoluer les spectres mixtes de peptides isomériques. Nous avons évalué Iso-PeptidAce avec un mélange de peptides synthétiques isomériques. Nous avons également validé les performances de cette approche avec des histones isolées de cellules humaines érythroleucémiques (K562) traitées avec des inhibiteurs d’histones désacétylases (HDACi) utilisés en clinique, et des histones de Saccharomyces cerevisiae liées au facteur d’assemblage de la chromatine (CAF-1) purifiées par chromatographie d’affinité. Enfin, en utilisant la méthode présentée précédemment, nous avons fait une analyse approfondie de la spécificité de plusieurs HATs et HDACs chez Schizosaccharomyces pombe. Nous avons donc déterminé les niveaux d’acétylation d’histones purifiées à partir de cellules contrôles ou de souches mutantes auxquelles il manque une HAT ou HDAC. Notre analyse nous a permis de valider plusieurs cibles connues des HATs et HDACs et d’en identifier de nouvelles. Nos données ont également permis de définir le rôle des différentes HATs et HDACs dans le maintien de l’équilibre d’acétylation des histones. Dans l’ensemble, nous anticipons que les méthodes décrites dans cette thèse permettront de résoudre certains défis rencontrés dans l’étude de la chromatine. De plus, ces données apportent de nouvelles connaissances pour l’élaboration d’études génétiques et biochimiques utilisant S. pombe.

Applying scanning electron microscopy for the ultrastructural and clinical analysis of periprosthetic capsules in implant-based breast reconstruction

La reconstruction en deux étapes par expanseur et implant est la technique la plus répandue pour la reconstruction mammmaire post mastectomie. La formation d’une capsule périprothétique est une réponse physiologique universelle à tout corps étranger présent dans le corps humain; par contre, la formation d’une capsule pathologique mène souvent à des complications et par conséquent à des résultats esthétiques sous-optimaux. Le microscope électronique à balayage (MEB) est un outil puissant qui permet d’effectuer une évaluation sans pareille de la topographie ultrastructurelle de spécimens. Le premier objectif de cette thèse est de comparer le MEB conventionnel (Hi-Vac) à une technologie plus récente, soit le MEB environnemental (ESEM), afin de déterminer si cette dernière mène à une évaluation supérieure des tissus capsulaires du sein. Le deuxième objectif est d‘appliquer la modalité de MEB supérieure et d’étudier les modifications ultrastructurelles des capsules périprothétiques chez les femmes subissant différents protocoles d’expansion de tissus dans le contexte de reconstruction mammaire prothétique. Deux études prospectives ont été réalisées afin de répondre à nos objectifs de recherche. Dix patientes ont été incluses dans la première, et 48 dans la seconde. La modalité Hi-Vac s’est avérée supérieure pour l’analyse compréhensive de tissus capsulaires mammaires. En employant le mode Hi-Vac dans notre protocole de recherche établi, un relief 3-D plus prononcé à été observé autour des expanseurs BIOCELL® dans le groupe d’approche d’intervention retardée (6 semaines). Des changements significatifs n’ont pas été observés au niveau des capsules SILTEX® dans les groupes d’approche d’intervention précoce (2 semaines) ni retardée.

Analysis and Design of an Adaptive Polynomial Predistorter with the Loop Delay Estimator

Most adaptive linearization circuits for the nonlinear amplifier have a feedback loop that returns the output signal oj'tne eunplifier to the lineurizer. The loop delay of the linearizer most be controlled precisely so that the convergence of the linearizer should be assured lot this Letter a delay control circuit is presented. It is a delay lock loop (ULL) with it modified early-lute gate and can he easily applied to a DSP implementation. The proposed DLL circuit is applied to an adaptive linearizer with the use of a polynomial predistorter, and the simulalion for a 16-QAM signal is performed. The simulation results show that the proposed DLL eliminates the delay between the reference input signal and the delayed feedback signal of the linearizing circuit perfectly, so that the predistorter polynomial coefficients converge into the optimum value and a high degree of linearization is achieved

Defect analysis of semiconductor thin films for photovoltaic applications using photo-luminescence and photo-conductivity

The present thesis can be divided into three areas:1) the fabrication of a low temperature photo-luminescence and photoconductivity measuring unit 2) photo-luminescence in the chalcopyrite CulnSez and CulnS2 system for defect and composition analysis and 3) photo-luminescence and photo-conductivity of In:JS3. This thesis shows that photo-luminescence is one of most essential semiconductor characterization tool for a scientific group working on photovoltaics. Tools which can be robust, non-destructive, requiring minimal sample preparation for analysis and most informative of the device applications are sought after by industries and this thesis is towards establishing photo-luminescence as "THE" tool for semiconductor characterization. The possible application of photo-luminescence as a tool for compositional and quality analysis of semiconductor thin films has been worked upon by this thesis. Photo-conductivity complement photo-luminescence and together they provide all the information required for the fabrication of an opto-electronic device.

Development and Analysis of a Compact Dual-Band Coplanar Antenna

The coplanar wave guide is an attractive device in microwave integrated circuits due to its uniplanar nature, ease of fabrication and low production cost. Several attempts are already done to explore the radiating modes in coplanar wave guide transmission lines. Usually coplanar wave guides are excited by an SMA connector with its centre conductor connected to the exact middle of the centre strip and the outer ground conductor to the two ground strips. The mode excited on it is purely a bound mode. The E-field distribution in the two slots are out of phase and there for cancels at the far field. This thesis addresses an attempt to excite an in phase E-field distribution in the two slots of the co planar wave guide by employing a feed asymmetry, in order to get radiation from the two large slot discontinuities of the coplanar waveguide. The omni directional distribution of the radiating energy can be achieved by widening the centre strip.The first part of the thesis deals with the investigations on the resonance phenomena of conventional coplanar waveguides at higher frequency bands. Then an offset fed open circuited coplanar waveguide supporting resonance/radiation phenomena is analyzed. Finally, a novel compact co planar antenna geometry with dual band characteristics, suitable for mobile terminal applications is designed and characterized using the inferences from the above study.

Farm Programmes of Electronic Media: A Comparative Study of Audience Perception in Kerala

Farm communication and extension programs are vital part of the farm development attempts. Electronic media plays a major role in farm extension activities. Kerala, the consumer state, which was a complete agricultural state in pre-independence period, is the sprouting land of agricultural extension and publication activities in print media. Later AIR (All India Radio) farm programs and farm broadcasting of Doordarshan enriched the role of electronic media in farm extension activities. The media saturated southern state of India received this new electronic media farm communication revolution whole heartedly. However, after 1990, Kerala witnessed a flood of private T V channels and currently there are 24 channels in this regional language, named Malayalam. All major news and entertainment channels are broadcasting farm programs. Farm programs of AIR and Doordarshan, broadcasted in Malayalam language, have been well accepted to the farmers‘ in Kerala. However, post-independence period, witnessed the formation of Kerala state in Indian Union and the first ballot-elected communist Government started its administration. After the land reform bills, the state witnessed a gradual decrease in agricultural production. Even if it is not reflected much in the attitude and practices of farm community and farm broadcast of traditional electronic broadcasting, a change is observable after the post-liberalization era of India. Private Television channels, which were focused on entertainment value of programs, started broadcasting farm programs and the parameters of program production went through certain changes. In this situation, there is ample relevance for a study about the farm programs of electronic media in terms of a comparative study of audience perception. The study is limited in the state of Kerala as it is the most media saturated state in India. The study analyzes the rate, nature and scope of adoption of farming methods transmitted through electronic media (T.V. and Radio) in Malayalam language.All kinds of Farm programs including comprehensive program serials, success stories, seasonal cropping methods, experts opinion, been analyzed on the basis of the following objectives.  To find whether propagating new farm methods through farm programs in electronic media or the availability of adequate infrastructure and economic factors make a farmer to adopt a new farming method.  To find which electronic media has more influence on farmers to adopt agricultural programs.  To find which form of electronic media gets better feedback from farmers  To find out whether the programs of T.V. or Radio is more acceptable to farmers than the print media.  To find whether farmers gets the message through their preferred medium for the message. The researcher recorded opinions from a panel of agricultural officers, farm Information officers, agro extension researchers and experts. According to their opinions and guidelines, a pilot study is designed and conducted in Kanjikuzhy Panchayath, in Alappuzha district, Kerala. The Panchayath is selected by considering its ideal nature of being the sample for a social Science research. Besides, the nature of farming in the Panchayath, which devoid of the cultivation of cash crops also supported its sample value. As per the observations from the pilot study, researcher confirmed the Triangulation method as the methodology of research. The questionnaire survey, being the primary part contained 42 Questions with 6 independent and 32 dependent variables. The survey is conducted among 400 respondents in Idukki, Alappuzha and Pathanamthitta districts considering geographical differences and distribution of different types of crops. The response from a total of 360 respondents, 120 from each district, finally selected for tabulation and data analysis.The data analysis, based on percentage analysis, along with the results from focus group discussion among a selected group of 20 farmers, together produced the results as follows. Farmers, who are the audience of farm programs, have a very serious approach towards the medium. They are maintaining a critical point of view towards the content of the programs. Farmers are reasonably aware about the financial side of the programs and the monitory aspirations of both private and Government owned Television channels. Even though, the farmers are not aware on the technical terminology and jargons, they have ideas about success stories, program serials and they are even informed about channels are not maintaining an audience research section like AIR. Though the farmers accept Doordarshan as the credential source of farm information and methods, they are inclined to the entertainment value of programs too. They prefer to have more entertainment value for the programs of Doordarshan. Surprisingly, they have very solid suggestions on even about the shots which add entertainment value to the farm broadcasting methods of Doordarshan. Farmers are very much aware about the fact that media is just an instrument for inspiration and persuasion. They strongly believe that the source of information and new methods is agricultural research and an effective change happens only when there are adequate infrastructure and marketing facilities, along with the proper support from Government agricultural guideline and support systems like Krishi Bhavans. They strongly believe that media alone cannot create any magic in increasing agricultural production. Farmers are pointing out the lack of response to the feedback and queries of farmers on farming methods, as an evidence for the difference in levels of commitment of Government and private owned Television channels.Farmers are still perceiving AIR farm programs are far more committed to farmers and farming than any other electronic medium. However, they are seriously lacking Radio receivers with medium wave reception facility. Farmers perceive that the farming methods on new crops are more adoptable than the farming methods of traditional crops in both private and Government owned Television channels. There are multiple factors behind this observation from farmers. Farmers changed in terms of viewing habits and they prefer success stories, which are totally irrelevant and they even think that such stories encourage people to go for farming and they opined that such stories are good sources of inspiration. However, they are all very much sure about the importance and particular about the presence of entertainment factor even in farm programs. Farmers expect direct interaction of any expert of the new farming method to implement the method in their agriculture practices. Though introduction of a new idea in the T.V. is acceptable, farmers need the direct instruction of expert on field to start implementing the new farming practices Farmers still have an affinity towards print media reports and agricultural pages and they have complaints to print media on the removal of agricultural information pages from news papers. They prefer the reports in print media as it facilitates them to collect and refer articles when they need it. Farmers are having an eye of doubt about the credibility of farm programs by private T.V. channels. Even if they prefer private Television channels for listening and adopting new farming methods and other farm information, they scrutinize programs to know whether they are sponsored programs by agrochemical or agro-fertilizer manufacturer.

Performance Analysis of Reversible Fast Decimal Adders

This paper presents a performance analysis of reversible, fault tolerant VLSI implementations of carry select and hybrid decimal adders suitable for multi-digit BCD addition. The designs enable partial parallel processing of all digits that perform high-speed addition in decimal domain. When the number of digits is more than 25 the hybrid decimal adder can operate 5 times faster than conventional decimal adder using classical logic gates. The speed up factor of hybrid adder increases above 10 when the number of decimal digits is more than 25 for reversible logic implementation. Such highspeed decimal adders find applications in real time processors and internet-based applications. The implementations use only reversible conservative Fredkin gates, which make it suitable for VLSI circuits.

Role of chlorine on the opto-electronic properties of β-In2S3 thin films

Effect of chlorine doping on the opto-electronic properties of β-In2S3 thin film, deposited by spray pyrolysis technique is studied for the first time. Chlorine was incorporated in the spray solution, using HCl. Pristine sample prepared using In(NO3)3 and thiourea as the precursors showed very low photosensitivity. But upon adding optimum quantity of chlorine, the photosensitivity increased by 3 orders. X-ray analysis revealed that crystallinity was also increasing up to this optimum level of Cl concentration. It was also observed that samples with high photosensitivity were having higher band gap. The present study proved that doping with chlorine was beneficial as this could result in forming crystalline and photosensitive films of indium sulfide.

State dependent volume isotope shift analysis of the low lying states of Ba I and Ba II

Relativistic multi-configuration Dirac-Fock wavefunctions, coupled to good angular momentum J, have been calculated for low lying states of Ba I and Ba II. The resulting electronic factors show good agreement with data derived from recent high-resolution laser spectroscopy experiments and results from a comparison of muonic and optical data.

The electronic structure of anionic halide complexes of element 105 in aqueous solutions and their extraction by aliphatic amines

To study the complex formation of group 5 elements (Nb, Ta, Ha, and pseudoanalog Pa) in aqueous HCI solutions of medium and high concentrations the electronic structures of anionic complexes of these elements [MCl_6]^-, [MOCl_4]^-, [M(OH)-2 Cl_4]^-, and [MOCl_5]^2- have been calculated using the relativistic Dirac-Slater Discrete-Variational Method. The charge density distribution analysis has shown that tantalum occupies a specific position in the group and has the highest tendency to form the pure halide complex, [TaCl_6-. This fact along with a high covalency of this complex explains its good extractability into aliphatic amines. Niobium has equal trends to form pure halide [NbCl_6]^- and oxyhalide [NbOCl_5]^2- species at medium and high acid concentrations. Protactinium has a slight preference for the [PaOCl_5]^2- form or for the pure halide complexes with coordination number higher than 6 under these conditions. Element 105 at high HCl concentrations will have a preference to form oxyhalide anionic complex [HaOCl_5]^2- rather than [HaCl_6]^-. For the same sort of anionic oxychloride complexes an estimate has been done of their partition between the organic and aqueous phases in the extraction by aliphatic amines, which shows the following succession of the partition coefficients: P_Nb < P_Ha < P_Pa.

Biologically Plausible Neural Circuits for Realization of Maximum Operations

Object recognition in the visual cortex is based on a hierarchical architecture, in which specialized brain regions along the ventral pathway extract object features of increasing levels of complexity, accompanied by greater invariance in stimulus size, position, and orientation. Recent theoretical studies postulate a non-linear pooling function, such as the maximum (MAX) operation could be fundamental in achieving such invariance. In this paper, we are concerned with neurally plausible mechanisms that may be involved in realizing the MAX operation. Four canonical circuits are proposed, each based on neural mechanisms that have been previously discussed in the context of cortical processing. Through simulations and mathematical analysis, we examine the relative performance and robustness of these mechanisms. We derive experimentally verifiable predictions for each circuit and discuss their respective physiological considerations.

A User-Centric Approach to eCertificate for Electronic Identities (eIDs) Management in Mobile Environment

The use of electronic documents is constantly growing and the necessity to implement an ad-hoc eCertificate which manages access to private information is not only required but also necessary. This paper presents a protocol for the management of electronic identities (eIDs), meant as a substitute for the paper-based IDs, in a mobile environment with a user-centric approach. Mobile devices have been chosen because they provide mobility, personal use and high computational complexity. The inherent user-centricity also allows the user to personally manage the ID information and to display only what is required. The chosen path to develop the protocol is to migrate the existing eCert technologies implemented by the Learning Societies Laboratory in Southampton. By comparing this protocol with the analysis of the eID problem domain, a new solution has been derived which is compatible with both systems without loss of features.

Critical analysis and extension of the Hirshfeld atoms in molecules

The computational approach to the Hirshfeld [Theor. Chim. Acta 44, 129 (1977)] atom in a molecule is critically investigated, and several difficulties are highlighted. It is shown that these difficulties are mitigated by an alternative, iterative version, of the Hirshfeld partitioning procedure. The iterative scheme ensures that the Hirshfeld definition represents a mathematically proper information entropy, allows the Hirshfeld approach to be used for charged molecules, eliminates arbitrariness in the choice of the promolecule, and increases the magnitudes of the charges. The resulting "Hirshfeld-I charges" correlate well with electrostatic potential derived atomic charges

Estimation of electronic coupling in π -stacked donor-bridge-acceptor systems: correction of the two-state model

Comparison of donor-acceptor electronic couplings calculated within two-state and three-state models suggests that the two-state treatment can provide unreliable estimates of Vda because of neglecting the multistate effects. We show that in most cases accurate values of the electronic coupling in a π stack, where donor and acceptor are separated by a bridging unit, can be obtained as Ṽ da = (E2 - E1) μ12 Rda + (2 E3 - E1 - E2) 2 μ13 μ23 Rda2, where E1, E2, and E3 are adiabatic energies of the ground, charge-transfer, and bridge states, respectively, μij is the transition dipole moments between the states i and j, and Rda is the distance between the planes of donor and acceptor. In this expression based on the generalized Mulliken-Hush approach, the first term corresponds to the coupling derived within a two-state model, whereas the second term is the superexchange correction accounting for the bridge effect. The formula is extended to bridges consisting of several subunits. The influence of the donor-acceptor energy mismatch on the excess charge distribution, adiabatic dipole and transition moments, and electronic couplings is examined. A diagnostic is developed to determine whether the two-state approach can be applied. Based on numerical results, we showed that the superexchange correction considerably improves estimates of the donor-acceptor coupling derived within a two-state approach. In most cases when the two-state scheme fails, the formula gives reliable results which are in good agreement (within 5%) with the data of the three-state generalized Mulliken-Hush model