Giancarlo Mascetti

Optical, structural and fluorescence microscopic studies on reduced form of polyaniline : The leucoemeraldine base

Electrochemically deposited thin films of emeraldine salt (ES) on indium-tin-oxide (ITO) glass plate and [111] silicon crystal were treated chemically with a phenyl hydrazine (PNH) solution in order to obtain a leucoemeraldine base (LB) form of polyaniline (PANI). The NMP-treated PANI films were washed using different organic solvents. ES, LB and air-oxidized LB films were examined using UV-visible, optical microscopy, X-ray diffraction and fluorescence microscopic techniques. The obtained results showed a direct correlation in the optical, structural and fluorescence behaviour of the LB form of PANI. An X-ray diffraction study revealed the partial crystallinity in the ES, whereas the LB and the air-oxidized LB showed amorphous structures. The first evidence of high fluorescence microscopic behaviour in the LB form of PANI has been observed. The gradual decrease in fluorescence signal as a function of time for the LB film has been interpreted as being due to the diffusion phenomenon in relation to the contact of the complex of the phenyl ring with O2 molecules.

Effect of Fixatives on Calf Thymocytes Chromatin as Analyzed by 3D High-Resolution Fluorescence Microscopy

Two common fixatives--glutaraldehyde and ethanol/acetic acid mixture--were studied to understand their effects on DNA distribution inside the cell nucleus. Native calf thymocytes were analyzed by using a DNA selective fluorescent dye (DAPI) and computational optical sectioning microscopy on isolated cells before and after fixation. In order to estimate quantitatively the intranuclear DNA distribution, the stained calf thymocytes images were processed by removing the out-of-focus contributions present in each optical section. Although preliminary, the results show that within individual nuclei the frequency distribution of the fluorescence intensity appears significantly and differentially altered by the two fixatives. Namely with respect to the native unfixed preparation, the ethanol/acetic acid causes the complete disappearance of the higher intensity pixels, whereas glutaraldehyde fixation can be associated with the appearance of new ones of an even higher intensity. The quantitative analysis of the processed images allowed us to reconstruct the topological distribution of DNA inside the nucleus and to correlate the data with the results obtained by differential scanning calorimetry on similar samples.

Effects of polyamines on higher-order folding of in situ chromatin.

Modifications of the higher-order chromatin structure induced by polyamines have been quantitatively investigated in situ through a non-invasive biophysical approach using Differential Scanning Calorimetry and Quantitative Fluorescence Microscopy. Calorimetric and intensitometric profiles have been acquired for samples of native thymocytes, alternatively suspended in buffers, with or without natural polyamines (spermine and spermidine). The results here reported show that the structure and distribution of nuclear chromatin in situ considerably change upon the ionic composition of the environment. A quantitative analysis of this data and a comparison with previous results obtained from isolated chromatin fibers was carried out. Finally, an inverse relationship between chromatin condensation and nuclear volume was observed.

High order DNA structure as inferred by optical fluorimetry and scanning calorimetry

New quantitative insights on the native high order chromatin-DNA structure existing within interphase nuclei are obtained by monitoring the effects of two common well-characterized fixatives, glutaraldehyde and ethanol/acetic acid mixture, at the level of the intranuclear DNA distribution and structures. Reproducible distinct levels of DNA fluorescence intensity and their intranuclear distribution are apparent in unfixed and fixed thymocytes by using DAPI and quantitative optical microscopy based on a charge coupled device. The fluorescent histograms correlated with the calorimetric thermograms on the very same thymocytes fixed and unfixed, establish an unequivocal baseline for the different levels of structural organization of the chromatin within the intact nucleus; namely their number, DNA packing ratio and fiber diameter. A systematic comparison among all the numerous models, being so far proposed for the quinternary and quaternary levels of DNA folding, to identifies the rope or ribbon-like and the chromonema as the ones that best fit with the in situ distribution.

Ethidium bromide intercalation and chromatin structure: A thermal analysis

Abstract Differential Scanning Calorimetry has been performed in the temperature range 310 K-410 K on intact thymocytes and physiologically isolated chromatin following Ethidium bromide intercalation. Native thymocytes exhibited four main thermal transitions (at 339 K, 347 K, 362 K and 375 K) that were assigned to the melting of different cellular components. At increasing dye concentrations an enthalpy redistribution became evident between the thermal transition at 362 K related to the melting of nucleosome organized in the 10 nm filament, and the transition at 375 K related to the melting of nucleosome organized in the 30 nm (or more) fiber. In correlation with increasing concentrations of Ethidium bromide, the disappearance and the subsequent reappearance of the highest temperature transition seem to be related to the unwrapping and subsequent wrapping of the chromatin fiber. Under similar condition, free DNA and digested chromatin do not show any enthalpy redistribution in their calorimetric profiles following Ethidium bromide intercalation. On the contrary, physiologically isolated chromatin displayed similar enthalpy redistribution between transitions assigned to chromatin DNA melting. An interesting difference appeared in the calorimetric profile of isolated chromatin with respect to the in situ material after chromatin extraction. In fact, a transition at 354 K, probably related to the melting of linker DNA became apparent (the transition at 362 K was assigned to the melting of DNA around the core particle). Selective digestions with different enzymes (micrococcal nuclease, proteinase K and DNase I) were carried out on thymocytes to verify the assignment of the main thermal transitions. In order to clarify the nature of the high temperature transitions native thymocytes were treated with topoisomerase I that removes superhelical turns from topologically closed DNA molecules. A comparison of calorimetric data with thermal denaturation profiles obtained by spectropolarimetric measurements on physiologically isolated chromatin gave further confirmation to the peak assignment by distinguishing the thermal transitions related to protein denaturation from the ones assigned to chromatin-DNA.

Chromatin of Trypanosoma cruzi: In situ analysis revealed its unusual structure and nuclear organization

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life‐cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis. J. Cell. Biochem. 85: 798–808, 2002.

Changes of nuclear structure induced by increasing temperatures.

Abstract Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.

Effects of fixatives on high order chromatin structure: a calorimetric study

Abstract Differential scanning calorimetry has been carried out on native calf thymus cells in order to investigate, by a non-invasive tool, the effects of the most common fixatives at the level of higher order chromatin structure. Therefore the effects of 2% glutaraldehyde in saline buffer and ethanol acetic acid 3:1 v/v mixture have been analysed: the first one is known to induce cross-linking between the various cell constituents, and the second one induces dehydration. Looking at the thermal profiles the disappearance of all four transitions and the appearance of a new single broad transition at 390 K are the most remarkable effects following the glutaraldehyde fixation of thymocytes. In contrast ethanol/acetic acid causes a reduction of the protein melting at 345 K, a loss of the 375 K transition and a shift of the 360 K transition. These data are explained in terms of opposite dramatic chromatin structure changes.

X-ray small angle scattering study of chromatin as a function of fiber length.

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.

A Neural Network Based Method Selecting Gabor Wavelet Filters for the Detection of Microcalcifications

Texture analysis methods are largely used in order to extract features both for enhancement and segmentation of mammographical images. In particular, multires-olution wavelet analysis may describe structures in images as microcalcifications by pointing out spatially localized features at different resolutions. Wavelet transforms may be chosen as mathematical models for this texture analysis due to their intrinsical adaptability in resolution both in frequency and space domains according with a scale parameter [1]. Within this conceptual frame, Gabor functions may be implemented as mother functions of the Wavelet transform because they assure the lowest limit of the indetermination principle in the space-frequency domain. On these bases, a set of 2-D bandpass filters, named as a set of Wavelet/Gabor Transform (WGT) filters, may be easily defined through contractions and dilations of non isotropic 2-D Gabor functions described by their central frequency and their orientation. On the other hand, the outputs of such WGT filters reflect the character of the image within the space-frequency regions where the wavelet functions are localized. As a consequence, the straightforward selection of a subset of such filters, describing the character of regions containing microcalcifications, may be an efficient way to enhance the visibility of low contrast structures. A computational intelligence approach has been chosen in order to simulate the sequence of analysis peculiar to an human expert.