Elisa Mitri | Elettra- Sincrotrone Trieste (original) (raw)

Papers by Elisa Mitri

Reproductive Toxicology, 2016

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA d... more Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fenton&amp;amp;amp;#39;s reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

The present PhD Thesis is about the development of new fabricative strategies to obtain microflui... more The present PhD Thesis is about the development of new fabricative strategies to obtain microfluidic devices suitable for InfraRed MicroSpectroscopy (IRMS) studies on living cells in physiological environment and the demonstration of the screening and diagnostic capabilities of this technique for bio-medical applications.

Journal of biomedical optics, 2015

Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes i... more Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes in tumoral tissues. The head and neck region is characterized by a great variety of lesions, with different degrees of malignancy, which are often difficult to diagnose. Schneiderian papillomas are sinonasal benign neoplasms arising from the Schneiderian mucosa; they can evolve into malignant tumoral lesions (squamous cell carcinoma). In addition, they can sometimes be confused with the more common inflammatory polyps. Therefore, an early and definitive diagnosis of this pathology is mandatory. Progressing in our research on the study of oral cavity lesions, 15 sections consisting of inflammatory sinonasal polyps, benign Schneiderian papillomas, and sinonasal undifferentiated carcinomas were analyzed using FTIRI. To allow a rigorous description of these pathologies and to gain objective diagnosis, the epithelial layer and the adjacent connective tissue of each section were separately inve...

Biophysical chemistry, Jan 25, 2015

Biophysical Chemistry, 2015

According to the cancer stem cell theory malignant glioma is incurable because of the presence of... more According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample.

Vibrational Spectroscopy, 2014

ABSTRACT

Analytical chemistry, Jan 26, 2015

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeost... more Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian ce...

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellu... more The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemi... more Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemical modifications induced in the resin by exposure to 254 nm (UVC) light. Fourier Transform Infrared microspectroscopy (μ-FTIR) was used to carry out a thorough study on the chemical processes and modifications occurring within the epoxy resin by exposure to 365 nm and 254 nm light. In particular, we established that UVC light promotes the opening of the epoxy rings bypassing the post-exposure bake. The possibility to promote a further activation of the resin, already patterned with standard UV lithography, was exploited to produce closed microfluidic devices. Specifically, we were able to fabricate fluidic chips, characterized by broadband transparency from mid-IR to UV and long term stability in continuous flow conditions. CaF2 was used as substrate, coated by sputtering with a nanometric silicon film, in order to make surface properties of this material more suitable for standard fabrication processes with respect to the original substrate. The fabricated microfluidic chips were used to study by μ-FTIR the biochemical response of live breast cancer MCF-7 cells to osmotic stress and their subsequent lysis induced by the injection of deionized water in the device. μ-FTIR analyses detected fast changes in protein, lipid and nucleic acid content as well as cytosol acidification.

Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal ev... more Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal evolution of multicellular organisms. Its misregulation has been associated with many diseases, making its early and reliable detection a key point for modern cellular biology. In this paper, we propose the use of infrared microspectroscopy (IRMS) as a label-free methodology for the detection of apoptotic-related biochemical processes induced on U937 leukemic monocytes by serum starvation and CCCP-exposure. The spectroscopic results are in agreement with parallel Flow Cytometry (FC) experiments, where plasma membrane integrity and mitochondrial activity were assessed. Spectroscopic outcomes complemented FC data and allowed drawing a more complete picture of the apoptotic pathways. In particular, we established that the two apoptosis-inducing treatments, cell starvation and CCCP treatment, affect the cell cycle in a different way, with the former, cell death is preceded by a cell cycle arrest, whereas the latter causes an increased cell cycle progression. Spectral data demonstrate that for both conditions apoptosis proceeds through the accumulation of lipid droplets within cells. Moreover, we were able to establish a spectral marker for DNA condensation/fragmentation: the enhancement of the PhI band component centred at ∼1206 cm(-1) which is more sensitive than the relative intensity of the PhII band, to which phospholipids and carbohydrates also contribute significantly. In conclusion, we demonstrate that the intrinsic multi-parametric nature of IRMS and its application on cells under physiological conditions can be well exploited for the investigation of apoptotic pathways.

With the aim of expanding the structure-activity relationship investigation, the series of Ru(II)... more With the aim of expanding the structure-activity relationship investigation, the series of Ru(II) half sandwich coordination compounds of the type [Ru([9]aneS3)(chel)(L)](n+) previously described by us (where [9]aneS3 is the neutral face-capping ligand 1,4,7-trithiacyclononane, chel is a neutral or anonic chelating ligand, L = Cl(-) or dmso-S, n = 0-2) was extended to 1,4,7-triazacyclononane ([9]aneN3). In addition, new neutral N-N, and anionic N-O and O-O chelating ligands, i.e. dach (trans-1,2-diaminocyclohexane), pic(-) (picolinate), and acac(-) (acetylacetonate), were investigated in combination with both [9]aneS3 and [9]aneN3. Overall, ten new half-sandwich complexes were prepared and fully characterized and their chemical behaviour in aqueous solution was established. The single-crystal X-ray structures of eight of them, including the versatile precursor [Ru([9]aneN3)(dmso-S)(2)Cl]Cl (9), were also determined. The results of in vitro antiproliferative tests performed on selected compounds against MDA-MB-231 human mammary carcinoma cells confirmed that, in this series, only compounds that hydrolyse the monodentate ligand at a reasonable rate show moderate activity, provided that the chelate ligand is a hydrogen bond donor.

In this contribution we present the first example of a microfluidic chip based on BaF2 for Infrar... more In this contribution we present the first example of a microfluidic chip based on BaF2 for Infrared Microspectroscopy (IRMS) of living cells. The advantage in using barium fluoride as platform relies on its high IR transparency, especially in the spectral region below 1300 cm−1, where the absorption bands of nucleic acids and carbohydrates are located.Barium fluoride is slightly soluble in water (0.12 g/100 g water) and it is potentially harmful for living cells. To overcome these problems, here we exploit an approach whose feasibility has been demonstrated previously on CaF2: the surface modification obtained by sputtering a thin Si layer on the surface. The Surface Modified Microfluidic Devices (SM-MD) hence obtained not only solve the BaF2 drawbacks, but also provide a silicon-like substrate fully compatible with standard micro-fabrication processes. These potentialities are here further explored in the direction of chemical or topographical nano-patterning of the silicon-like surface. The silicon thin layer was structured in the shape of 300 nm wide grooves (500 nm pitch) with a thickness of 35 nm by using standard NIL and etching processes; chemical patterning was achieved by exploiting silane chemistry.Finally, we tested the performances of these devices at SISSI beamline@Elettra, collecting IR spectra of single MDA-MB-231 living cells maintained either in physiological solution or complete medium. A comparison of the spectra of a single cell obtained in BaF2 and CaF2 MDs is reported.

Infrared Microspectroscopy (IRMS) has been proposed as a powerful diagnostic tool in biology, due... more Infrared Microspectroscopy (IRMS) has been proposed as a powerful diagnostic tool in biology, due to the rich molecular, structural and conformational information contained in IR spectra of cells and tissues. In particular, IRMS of live cells in microfluidic devices has to cope with the strong water absorption in the medium infrared spectral region and the scarce knowledge about fabrication protocols suitable for micro-structuring infrared-transparent materials. Based on these motivations we are developing and testing a class of microfluidic devices consisting of a patterned photoresist sandwiched between two CaF 2 optical windows. In this paper we propose solutions to a few specific issues, namely, (i) the poor resist adhesion during micro-fabrication processes due to the low surface energy of CaF 2 , (ii) the potentially harmful effects of CaF 2 dissolution on interesting cellular lines (such as neurons or stem cells), (iii) the sealing of the devices. Specifically, we modified the surface properties of CaF 2 substrates by sputtering a thin layer of Si, as to obtain the following advantages: (a) all lithographic steps can be performed as if they were carried out on silicon wafers; (b) the chemical functionalization and nanostructuring of the surface in contact with cells can be obtained by usual protocols used for Si; (c) the deposited silicon separates living cells and their environment from CaF 2 . A device sealing process is discussed, based on a polymer bonding protocol, in order to tune the content of residual solvent. Finally, we present IR hyperspectral images acquired on MCF-7 living cells, cultured inside our devices for 48 h. Ó 2012 Elsevier B.V. All rights reserved.

Reproductive Toxicology, 2016

Journal of biomedical optics, 2015

Biophysical chemistry, Jan 25, 2015

Biophysical Chemistry, 2015

Vibrational Spectroscopy, 2014

ABSTRACT

Analytical chemistry, Jan 26, 2015