La biochimica clinica dei rettili e dei cheloni in particolare, non ha ottenuto al momento lo stesso livello di attenzione rivolto ai mammiferi. Con il presente lavoro viene proposta la valutazione dei più importanti parametri ematologici e biochimici in campioni di sangue di Testudo hermanni al fine di facilitare l'interpretazione dei dati di laboratorio e la diagnosi di eventuali patologie. A questo scopo, sono stati calcolati gli intervalli di riferimento di questi parametri in individui clinicamente sani e sono state analizzate le influenze di fattori ambientali e fisiologici. Sono state inoltre determinate le concentrazioni di alcuni importanti elementi chimici sia essenziali che non essenziali. Su campioni di chirottero del genere Tadarida teniotis sono state determinate le concentrazioni di PCB DL, PCB NDL, PCDD/F, PFAS e di elementi chimici essenziali e non, al fine di valutare: il possibile livello di contaminazione in relazione alla specie e all’habitat, la prevalenza e/o il rapporto delle classi di composti nel caso di una possibile contaminazione, la eventuale fonte di contaminazione. A quanto ci risulta, il nostro lavoro rappresenta il primo tentativo di analizzare su vasta scala le concentrazioni di questi inquinanti in una popolazione di pipistrelli residenti in un’area urbana. Tra i vari contaminanti esaminanti in questo studio, il Pb e le diossine possono costituire un serio problema per Tadarida teniotis. / Hermann’s tortoise, Testudo hermanni, is currently on the IUCN red list of endangered species. Reptile medicine uses laboratory analyses to evaluate health status, but data on hematology, blood biochemistry and protein electrophoresis are limited in tortoises.The purposes of this study were to establish the most important hematological and biochemical parameters for health assessment in Hermann’s tortoises, calculate the reference intervals and evaluate the impact of environmental and physiological factors.The reference intervals defined in the present study can be considered a useful tool for clinical pathologists and researchers working in tortoise medicine and conservation. In Europe bat populations are gradually declining due to environmental pollution; in this thesis we have studied the concentrations of many contaminants including essential and non essential trace elements, dioxins, PCB DL and NDL and PFAS in tissues of juveniles Tadarida teniotis. The high concentrations of Pb and dioxin determined are indicative of acute evnironmental exposure.

An acceptable agreement permits interchangeability of the instruments. For this purpose, we have investigated the agreement of several clinical instruments frequently used in clinical practice with their laboratory counterpart. We have estimated the agreement between a point-of-care blood gas analyzer (i-Stat, Abaxis) and a bench-top blood gas analyzer (Nova, Biomedical) in venous samples from Hermann’s tortoises. We have estimated the agreement between a point-of-care chemistry analyzer (VetScan VS2, Abaxis) and a laboratory analyzer (Olympus AU400, Olympus Co.) in venous samples from Hermann’s tortoises. We have estimated the agreement between portable blood glucose meters (Accu-Chek, Aviva; AlphaTrak 2, Abbott) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. We have estimated the agreement between point-of-care bench-top glucose measurement (VetScan VS2, Abaxis) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. Beyond method comparison and validation, reference interval determination for common laboratory testing is required to allow the clinician to discriminate individuals that are different from the remaining population for a certain parameter. We have calculated reference intervals for blood gas in Hermann’s tortoises. We have calculated reference intervals for protein electrophoresis in Hermann’s tortoises. We have described normal hematology in Hermann’s tortoises. We have calculated reference intervals for clinical chemistry in Hermann’s tortoises. We have calculated reference intervals for aldosterone in ferrets. Based on our results, animal species requires individual validation of laboratory methods and reference intervals. Lack of consideration of these findings may result in clinical misdiagnosis and improper treatment of animals.

No description available.

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Il Tumore a Cellule Giganti dell’osso (TCG) è una rara neoplasia che rappresenta il 5% dei tumori di natura ossea; sebbene venga considerato un tumore a decorso benigno può manifestare caratteri di aggressività locale dando origine a recidive locali nel 10-25% dei casi, e nel 2-4% dei casi metastatizza a livello polmonare. In questo studio è stata valutata l’espressione dei miRNA mediante miRNA microarray in 10 pazienti affetti da TCG, 5 con metastasi e 5 liberi da malattia; sono stati riscontrati miRNA differenzialmente espressi tra i 2 gruppi di pazienti e la successiva validazione mediante Real Time PCR ha confermato una differenza significativa per il miR-136 (p=0.04). Mediante analisi bioinformatica con il software TargetScan abbiamo identificato RANK e NF1B come target del miR-136 e ne abbiamo studiato l’espressione mediante Real Time PCR su una più ampia casistica di pazienti affetti da TCG, metastatico e non, evidenziando una maggior espressione di NF1B nel gruppo di pazienti metastatici, mentre RANK non ha dimostrato una differenza significativa. L’analisi di Western Blot ha rilevato una maggiore espressione di entrambe le proteine nei pazienti metastatici rispetto ai non metastatici. Successivamente è stato condotto uno studio di immunoistochimica su TMA di 163 campioni di pazienti affetti da TCG a diverso decorso clinico che ha dimostrato una maggiore e significativa espressione di entrambe i target nei pazienti con metastasi rispetto ai non metastatici; le analisi di popolazione mediante Kaplan-Meier hanno confermato la correlazione tra over-espressione di RANK, NF1B e ricaduta con metastasi (p=0.001 e p<0.0005 rispettivamente). Lo studio di immunoistochimica è stato ampliato alle proteine maggiormente coinvolte nell’osteolisi che risultano avere un significato prognostico; tuttavia mediante analisi di ROC, la co-over-espressione di RANK, RANKL e NF1B rappresenta il migliore modello per predire la comparsa di metastasi (AUC=0.782, p<0.0005). / Giant Cell Tumor of bone (GCTb) is a rare locally aggressive tumour representing 5% of all bone tumors. Local recurrences occur in 10-25% of cases, and it may occasionally give lung metastases in 2-5% of cases. In this study, the expression of miRNAs was evaluated using miRNA microarray analysis in 10 patients with GCTb, 5 with lung metastases and 5 disease-free; several miRNAs were differentially expressed between the 2 groups of patients and the subsequent validation by Real Time PCR confirmed a significant difference for miR-136 (p=0.04. Using bioinformatic analysis with the TargetScan software, we identified RANK and NF1B as miR-136 targets and their expression was analysed by Real Time PCR on a larger series of GCTb, showing higher levels of NF1B in metastatic than in disease-free patients. RANK didn’t show any difference. Western Blot analysis revealed an increased expression of both NF1B and RANK in the first group and an immunohistochemistry study performed on TMA sections of 163 GCTb samples with different clinical course included other proteins involved in osteolysis. A significant higher expression of both targets NF1B and RANK was seen in metastatic compared to disease-free patients; the Kaplan-Meier analysis confirmed the correlation between over-expression of RANK and NF1B and probability of metastasis occurrence (Log Rank p=0.001 and p<0.0005 respectively). Although other proteins involved in osteolysis presented a prognostic impact, ROC curve revealed that the simultaneous over-expression of RANK, RANKL and NF1B was the best model to predict the risk of metastases (AUC=0.782, p<0.0005 ).

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

Cell-free expression systems are widely used to synthesize proteins for subsequent further characterization, to manufacture potentially useful commercial end products, and to construct cellular mimics in the laboratory. The first part of the thesis explores the feasibility of preparing two of the commercially available and widely used E. coli-based cell-free expression systems: the PURE System and the S30 Bacterial Extract. The second part focuses on the characterization of in vitro transcription and translation. The third part of the thesis features an example of an application of S30 Bacterial Extract cell-free expression systems i.e. the building of cell-like structures that can work together with engineered bacteria to achieve a predetermined task. Finally, the construction of a microfluidic dialysis device compatible with cell-free synthetic biology projects is presented.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.