LINEAR AND NON-LINEAR PHOTOACTIVATION A TOOL FOR NEUROSCIENCE AND CANCER RESEARCH.

Photoactivation is a physical process that allows using light-matter interaction as a precursor to induce a specific Physico-chemical reaction. This reaction is induced after the absorption of a photon, having a suitable wavelength, by specific photoactivable molecules. Such molecules act as a molecular machine that performs functions useful both for therapeutic purposes but also for the study of biophysical processes. The ability to have high temporal and spatial control is one of the main advantages of photoactivation. Therefore combining the photoactivation with appropriate optical methods, manipulation of living specimens at molecular precision is possible. Nevertheless, a set of photoactivable molecules is the key player in realizing super-resolution microscopy, allowing the quantitative study of biological challenges at the subcellular scale. This work of thesis focuses the attention on two classes of photoactivable molecules, i.e., caged compounds and photosensitizers, with the attention at applications in the neuroscience and photodynamic therapy fields, respectively. Caged compounds are realized via covalent appendage of a light-sensitive protecting group \u201cthe cage\u201d to a signaling molecule. In particular, it was used as a GABA caged molecule (RuBi-GABA). One photon (UV-VIS light) and two-photon (700-900nm) absorption were used to break the \u201ccage\u201d binding. The uncaged molecule (GABA) becomes active and can bind the GABAA receptor site generating a Cl- current across the neuronal membrane that can be recorded using the Patch Clamp Technique. This approach allows controlling the neurotransmitter release in time, space, and relative concentration. In particular, the uncaging method and fluorescence microscopy coupled to the patch-clamp technique provides a useful approach to detect a selected biological target in a temporally and spatially confined way. It was analyzed how the change of physical parameters such as uncaging distance, exposure time, laser power, linear and non-linear photoactivation influence the measurements, and it was determined how these parameters change concentration and volume of GABA release and consequently the GABAA response. Specifically, localization precision can be improved using advanced fluorescent optical methods such as super-resolved and non-linear fluorescence microscopy. This allows exploring the release of caged GABA topically applied in situ at defined concentration and in a specific region of neuronal cells for mapping the localization and the functional distribution of GABAA receptors in cerebellar granule cells in vitro. Finally, it was possible to explore the responses generated by specifics drugs in different regions of neurons. Photosensitizers are photoactivable molecules that, after absorption of light, can produce reactive species of oxygen, which induce cell damage and death. When those molecules are targeted to cancer cells the process could lead to the death of tumor cells. Such molecules are typically exploited in photodynamic therapy (PDT). This treatment modality is not invasive and explicates its function by the simultaneous presence of a PS, visible light, and tissue oxygen. Indeed, these specific molecules can target and localize in the neoplastic tissue and, upon photoactivation with visible light, they generate reactive oxygen species (ROS), causing confined damage. The reactive chemical species, and in particular singlet oxygen (1O\uac2), are characterized by an active region of the order of 0.02 \u3bcm, where a series of reactions can produce oxidative damages and consequently cellular death for apoptosis or necrosis. The cellular death mechanism,such as apoptosis or necrosis,depends on(i)the cell line used,(ii)thequantity of light usedand(iii)and the cellarea in which the PSs accumulate. Besides, these cellular damages could produce an immune response, improving therapy efficiency. A particular class of PSs, such as hypericin, phthalocyanine, porphyrin, and curcumin, are an object of study for PDT. However, a general characteristic and drawback of PSs is the low solubility in water and the aggregate formation, which impair their photophysical properties, such as the quenching of productive, excited states. To overcome this limit, PSs carriers were exploited. Among them, proteins offer several potential advantages. It has been demonstrated that PS molecules spontaneously bind internal hydrophobic cavities of particular proteins, preserving their monomeric, photoactive (both photosensitizing and fluorescent) state. Proteins such as ApoMb and BSA were used as a carrier (ApoMb and BSA) for hypericin, phthalocyanine, porphyrin, and curcumin. These complexes were studied through advanced microscopical methods (spinning disk confocal, confocal, and STED microscopy) analyzing the difference in the use of PS or PS-protein complex, demonstrating how a protein can increase the PS efficacy. Therefore, were performed accumulation measurements, biocompatibility and bioavailability tests, colocalization measurements, and were studied the interactions of PSs with tumoral spheroids. In summary, the main goals of this work are: (i) the study of cellular tumor damages induced by photosensitizing molecules and its localization in cells and the increase of its efficacy using protein carriers exploit advanced microscopy technique (ii) the study of GABAA receptor with the use of GABA caged molecule developing a set-up that combines confocal and two-photon excitation fluorescence microscope with patch-clamp technique

Utilizzo del compost nel processo di biorecupero di suoli contaminati da idrocarburi: valutazione dei parametri chimici e biologici

L'aggiunta di compost a suoli contaminatipuò favorire il loro biorecupero. Nel presente lavoro è stato simulato un possibile inquinamento da derivati petroliferi aggiungendo ad un suolo (tendenzialmente sabbioso e povero di sostanza organica) 10000 ppm di gasolio per autotrazione. Per favorire il loro biorecupero sono state aggiunte diverse quantità di ammendante compostato verde (1%, 2% e 4% sul peso secco del terreno) mantenendo costanti condizioni di temperatura e umidità. Oltre alla quantità di idrocarburi totali (TPH) presenti sono stati monitorati per una durata complessiva di 107 giorni alcuni parametri chimici (pH,TOC), biochimici (lipasi, deidrogenasi) e biologici (attività respiratoria del terreno, ATP), con l'obiettivo di valutare se le loro variazioni temporali fossero eventualmente correlabili alla degradazione degli idrocarburi

An epistatic mini-circuitry between the transcription factors Snail and HNF4a controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs

Preservation of the epithelial state involves the stable repression of EMT program while maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes, may provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4, directly represses the expression of the epithelial microRNAs-200c and -34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4, previously identified as a transcriptional repressor of Snail, induces the microRNAs-34a and -200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4 and microRNAs -200a, b, c and -34a as epistatic elements controlling hepatic stem cell maintenance/differentiation

Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29

Background and aims: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. Methods: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. Results: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR- 29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. Conclusions: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels

YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation

Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases

SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation

International audienceDespite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation. We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation. Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation

Experimental generalized quantum suppression law in Sylvester interferometers

Photonic interference is a key quantum resource for optical quantum computation, and in particular for so-called boson sampling machines. In interferometers with certain symmetries, genuine multiphoton quantum interference effectively suppresses certain sets of events, as in the original Hong-Ou-Mandel effect. Recently, it was shown that some classical and semi-classical models could be ruled out by identifying such suppressions in Fourier interferometers. Here we propose a suppression law suitable for random-input experiments in multimode Sylvester interferometers, and verify it experimentally using 4- and 8-mode integrated interferometers. The observed suppression is stronger than what is observed in Fourier interferometers of the same size, and could be relevant to certification of boson sampling machines and other experiments relying on bosonic interference.Comment: 5 pages, 3 figures + 11 pages, 3 figures Supplementary Informatio

Correlative imaging of cystic lymphangiomas: ultrasound, CT and MRI comparison

Cystic lymphangioma is a rare benign lesion derived from the detachment of the lymph sacs from venous drainage systems; the treatment of choice is a surgical excision and the final diagnosis is of histological type

Is diabetes in Cushing's syndrome only a consequence of hypercortisolism?

OBJECTIVE: Diabetes mellitus (DM) is one of the most frequent complications of Cushing's syndrome (CS). The aim of this study was to define the changes in insulin sensitivity and/or secretion in relation to glucose tolerance categories in newly diagnosed CS patients. DESIGN: Cross-sectional study on 140 patients with CS. METHODS: A total of 113 women (80 with pituitary disease and 33 with adrenal disease, aged 41.7±15.7 years) and 27 men (19 with pituitary disease and eight with adrenal disease, aged 38.1±20.01 years) at diagnosis were divided according to glucose tolerance into normal glucose tolerance (CS/NGT), impaired fasting glucose and/or impaired glucose tolerance (CS/prediabetes), and diabetes (CS/DM) groups. RESULTS: Seventy-one patients had CS/NGT (49.3%), 26 (18.5%) had CS/prediabetes and 43 (30.8%) had CS/DM. Significant increasing trends in the prevalence of family history of diabetes (P<0.001), metabolic syndrome (P<0.001), age (P<0.001) and waist circumference (P=0.043) and decreasing trends in HOMA-β (P<0.001) and oral disposition index (DIo) (P<0.002) were observed among the groups. No significant trends in fasting insulin levels, area under the curve for insulin (AUCINS), Matsuda index of insulin sensitivity (ISI-Matsuda) and visceral adiposity index were detected. CONCLUSIONS: Impairment of glucose tolerance is characterized by the inability of β-cells to adequately compensate for insulin resistance through increased insulin secretion. Age, genetic predisposition and lifestyle, in combination with the duration and degree of hypercortisolism, strongly contribute to the impairment of glucose tolerance in patients with a natural history of CS. A careful phenotypic evaluation of glucose tolerance defects in patients with CS proves useful for the identification of those at a high risk of metabolic complications