CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A hepatite autoimune (HAI) Ã uma doenÃa inflamatÃria crÃnica do fÃgado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruiÃÃo progressiva do parÃnquima hepÃtico e que, sem tratamento imunossupressor, evolui frequentemente para cirrose. Consiste em uma doenÃa rara na infÃncia, com menos de 10% dos pacientes com doenÃa hepÃtica crÃnica, porÃm de alta mortalidade. Caracteriza-se pela presenÃa de hipergamaglobulinemia, autoanticorpos nÃo ÃrgÃos - especÃficos e infiltrado inflamatÃrio portal linfoplasmocitÃrio. O uso da saliva como um mÃtodo de diagnÃstico avanÃou exponencialmente nos Ãltimos anos. DesequilÃbrios na quantidade e qualidade da saliva podem tanto gerar afecÃÃes bucais quanto ser indicativo de alguma alteraÃÃo sistÃmica importante. Este trabalho objetivou estudar parÃmetros de imunoglobulinas salivares e saliva total humana em pacientes portadores de hepatite autoimune. A amostra consistiu em doze indivÃduos com HAI (grupo experimental) e doze indivÃduos sadios (grupo controle), tendo sido avaliado o fluxo salivar, pH, nÃveis de imunoglobulinas e perfil proteico. Saliva total nÃo estimulada foi coletada e centrifugada; o sobrenadante foi retido, liofilizado, armazenado a -80ÂC e analisado para contagem de proteÃnas totais, eletroforese unidimensional e ELISA. Foi possÃvel detectar diferenÃas estatisticamente significativas entre os grupos (p ≤ 0,05) para os parÃmetros salivares analisados. Quando comparados com o grupo controle, portadores de hepatite autoimune apresentaram aumento no nÃmero de imunoglobulinas IgG e bandas proteicas com expressÃo diferenciada. Os resultados deste estudo sugerem haver padrÃes diferenciados na composiÃÃo salivar entre os grupos avaliados. / Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. The use of saliva as a diagnostic tool has advanced exponentially within recent years. Unbalance in the amount and composition of saliva may generate oral diseases and may also indicate important systemic alterations. This study has aimed to investigate the parameters of immunoglobulins and human whole saliva in patients with AIH. Our study sample consisted of 12 individuals with AIH (experimental group) and 12 healthy individuals without AIH (control group). Salivary flow rate, Ph, immunoglobulins and protein profile were evaluated in this population. Non stimulated whole saliva was collected and centrifuged. The supernatant was separated and lyophilized and stored at -80ÂC for posterior total protein, unidimensional electroforetic analysis and ELISA. Statistically significant differences were observed between groups (p ≤ 0, 05) for the analyzed salivary parameters. When compared to the control group, individuals with HAI presented elevated IgG level and protein bands with specific expression. The results of this study suggest the existence of important differences within the pattern of salivary protein composition between groups.

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique.

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.

In this study, we investigated the analytical capabilities of DCHQ5, a new fluorescent chemosensor, belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular magnesium content, and its biological applications. We performed a comparative study of DCHQ5 and DCHQ1, the latter being the mother probe of the series, which showed preliminary encouraging results comparable to atomic absorption spectroscopy. We demonstrated that DCHQ5 is able to accurately quantify the total amount of Mg in a very “small” cellular population, by using a simple spectrofluorimetric assay. Furthermore, DCHQ5 demonstrated to be a versatile tool for different applications: its higher intracellular retentions allow to perform cytofluorimetric assays and two-photon confocal microscopy on whole and viable cells; its photochemical characteristic make it excitable in both UV and visible spectra, and the presence of different lifetimes allow to perform fluorescence life time imaging of intracellular Mg. DCHQ5 was also exploited for studying the involvement of magnesium in the commitment of human adipose-derived mesenchymal stem cells (hASCs) with a mixture of hyaluronic, butyric and retinoic acids (HBR). We found that in normal magnesium availability, hASCs precommitment is associated by an increase of total magnesium content during time and by a block in the G2/M phase of the cell cycle. Moreover, our results demonstrated that magnesium deprivation triggers multilineage enrichments of HBR-induced preconditioning of hASCs. The second part of this research was aimed at comparing single cells elemental analysis performed with synchrotron-based fluorescence and cell population analysis carry out by DCHQ5. We exploited innovative techniques of x-ray fluorescence microscopy by using a multimodal approach in order to achieve within the cells the spatial distribution of the concentration of magnesium and fundamental light elements for life. The combination of classical and innovative analytical techniques can shed new light in the comprehension of magnesium homeostasis.

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.

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 ).