Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.

Vegetables biofortified with iodine: chemical, agronomic and qualitative aspects of raw and fried potatoes

Iodine is an essential microelement for human health because it is a constituent of the thyroid hormones that regulate growth and development of the organism. Iodine Deficiency Disorders (IDDs) are believed to be one of the commonest preventable human health problems in the world today, according to the World Health Organization: that diseases include endemic goiter, cretinism and fetal abnormalities, among others, and they are caused by lack of iodine in the diet, that is the main source of iodine. Since iodine intake from food is not enough respect to human needs, this can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentration and/or bioavailability of mineral elements in the edible portions of crops through agricultural intervention or genetic selection (biofortification). The introduction of iodized salt is a strategy widely used and accepted to eradicate iodine deficiency, because it is an inexpensive source of stable iodine. Since the intake of salt, though iodized, must still be limited according to the risk of cardiovascular disease, so the increase of iodine content in plants for the production of functional foods is representing a field of study of particular interest and a potential market. In Italy potatoes enriched with iodine are produced by a patented procedure of agronomic biofortification for the fresh market since several years, furthermore they are recently accepted and recommended by Italian Thyroid Association, as an alternative source of iodine. Researches performed during the PhD course intended to characterize this innovative vegetables products, focusing the attention on different aspects, such as chemistry, agriculture, and quality of fresh and fried potatoes. For this purpose, lipid fraction of raw material was firstly investigated, in order to assess whether the presence of iodine in plant metabolism can affect fatty acid or sterol biosynthesis, according to the hypothesis that iodine can be bounded to polyunsaturated fatty acids of cell membranes, protecting them from peroxydation; phytosterols of plant sterol are also studied because their importance in reducing serum cholesterol, especially in potato plant sterols are also involved in synthesis of glycoalkaloid, a family of steroidal toxic secondary metabolites present in plants of the Solanaceae family. To achieve this goal chromatographic analytical techniques were employed to identify and quantify fatty acids and sterols profile of common and iodine enriched row potatoes. Another aim of the project was to evaluate the effects of frying on the quality of iodine-enriched and common potatoes. Since iodine-enriched potatoes are nowadays produced only for the fresh market, preliminary trials of cultivation under controlled environment were carried out to verify if potato varieties suitable for processing were able to absorb and accumulate iodine in the tuber. In a successive phase, these varieties were grown in the field, to evaluate their potential productivity and quality at harvest and after storage. The best potato variety to be destined for processing purposes, was finally subjected to repeated frying cycles; the effects of lipid oxidation on the composition and quality of both potatoes and frying oil bath were evaluated by chromatographic and spectrophotometric analytical techniques. Special attention were paid on volatile compounds of fried potatoes.

Diet Effects on Activation and Maturation of Feed Control over the Gastrointestinal Defence Barrier in Piglets

Weaning is an important and complex step involving many stresses that interfere deeply with feed intake, gastro-intestinal tract (GIT) development and adaptation to the weaning diet in young pigs. The health of the pig at weaning, its nutrition in the immediate post-weaning period, and the physical, microbiological and psychological environment are all factors that interact to determine food intake and subsequent growth. GIT disorders, infections and diarrhoea increase at the time of weaning, in fact pathogens such as enterotoxigenic Escherichia coli (ETEC) are major causes of mucosal damage in post-weaning disease contributing to diarrhoea in suckling and post-weaned pigs. The European ban in 2006 put on antibiotic growth promoters (AGP) has stimulated research on the mechanisms of GIT disorders and on nutritional approaches for preventing or reducing such disturbances avoiding AGPs. Concerning these aspects here are presented five studies based on the interplay among nutrition, genomic, immunity and physiology with the aim to clarify some of these problematic issues around weaning period in piglets. The first three evaluate the effects of diets threonine or tryptophan enriched on gut defence and health as possible alternatives to AGP in the gut. The fourth is focused on the possible immunological function related with the development of the stomach. The fifth is a pilot study on the gastric sensing and orexygenic signal given by fasting or re-feeding conditions. Although some results are controversial, it appears that both tryptophan and threonine supplementation in weaning diets have a preventive role in E.coli PWD and favorable effects in the gut especially in relation to ETEC susceptible genotype. While the stomach is believed as almost aseptic organ, it shows an immune activity related with the mucosal maturation. Moreover it shows an orexygenic role of both oxyntic mucosa and pyloric mucosa, and its possible relation with nutrient sensing stimuli.

Motor control system in Parkinson’s disease: a modeling approach

Parkinson’s disease is a neurodegenerative disorder due to the death of the dopaminergic neurons of the substantia nigra of the basal ganglia. The process that leads to these neural alterations is still unknown. Parkinson’s disease affects most of all the motor sphere, with a wide array of impairment such as bradykinesia, akinesia, tremor, postural instability and singular phenomena such as freezing of gait. Moreover, in the last few years the fact that the degeneration in the basal ganglia circuitry induces not only motor but also cognitive alterations, not necessarily implicating dementia, and that dopamine loss induces also further implications due to dopamine-driven synaptic plasticity got more attention. At the present moment, no neuroprotective treatment is available, and even if dopamine-replacement therapies as well as electrical deep brain stimulation are able to improve the life conditions of the patients, they often present side effects on the long term, and cannot recover the neural loss, which instead continues to advance. In the present thesis both motor and cognitive aspects of Parkinson’s disease and basal ganglia circuitry were investigated, at first focusing on Parkinson’s disease sensory and balance issues by means of a new instrumented method based on inertial sensor to provide further information about postural control and postural strategies used to attain balance, then applying this newly developed approach to assess balance control in mild and severe patients, both ON and OFF levodopa replacement. Given the inability of levodopa to recover balance issues and the new physiological findings than underline the importance in Parkinson’s disease of non-dopaminergic neurotransmitters, it was therefore developed an original computational model focusing on acetylcholine, the most promising neurotransmitter according to physiology, and its role in synaptic plasticity. The rationale of this thesis is that a multidisciplinary approach could gain insight into Parkinson’s disease features still unresolved.

Effect of host genotype and prion strain on the phenotypic heterogeneity of Creutzfeldt-Jakob disease: the peripheral nervous system involvement and the spectrum of the genetic form

The first study was designed to assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). To this aim, we reviewed medical records of 117 sCJDVV2, 65 sCJDMV2K, and 121 sCJDMM(V)1 subjects for symptoms/signs and neurophysiological data. We looked for the presence of PrPSc in postmortem PNS samples from 14 subjects by western blotting and real-time quaking-induced conversion (RT-QuIC) assay. Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms/signs suggestive of PNS involvement and neuropathy was documented in half of the VV2-MV2K patients tested. RT-QuIC was positive in all PNS samples, whereas western blotting detected PrPSc in the sciatic nerve in only one VV2 and one MV2K. These results support the conclusion that peripheral neuropathy, likely related to PrPSc deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2, the two variants linked to the V2 strain. The second study aimed to characterize the genetic/molecular determinants of phenotypic variability in genetic CJD (gCJD). To this purpose, we compared 157 cases of gCJD to 300 of sCJD. We analyzed: demographic aspects, neurological symptoms/signs, histopathologic features and biochemical characteristics of PrPSc. The results strongly indicated that the clinicopathological phenotypes of gCJD largely overlap with those of sCJD and that the genotype at codon 129 in cis with the mutation (i.e. haplotype) contributes more than the latter to the disease phenotype. Some mutations, however, cause phenotypic variations including haplotype-specific patterns of PrPSc deposition such as the “dense” synaptic pattern (E200K-129M), the intraneuronal dots (E200K-129V), and the linear stripes perpendicular to the surface in the molecular layer of cerebellum (OPRIs-129M). Overall, these results suggest that in gCJD PRNP mutations do not cause the emergence of novel prion strains, but rather confer increased susceptibility to the disease in conjunction with “minor” clinicopathological variations.

Malattie immunomediate del cane e del gatto: aspetti diagnostici e terapeutici

La presente tesi di dottorato affronta alcune delle più comuni malattie immunomediate del cane e del gatto. Il manoscritto è incentrato sugli aspetti diagnostici e terapeutici in corso di: anemia emolitica immunomediata (Immune-mediated hemolytic anemia, IMHA), trombocitopenia immunomediata (Immune-mediated thrombocytopenia, ITP) e poliartrite immunomediata (Immune-mediated polyarthritis, IMP). Il capitolo 1 costituisce un’introduzione all’argomento delle malattie immunologiche; vengono sottolineati alcuni aspetti patogenetici delle singole malattie immunomediate e riassunte le difficoltà diagnostiche e terapeutiche. Il capitolo 2 riporta uno studio riguardante una popolazione di gatti con diagnosi, o sospetto diagnostico, di IMHA che evidenziava una discrepanza tra i test diagnostici per il virus della leucemia felina (Feline Leukemia Virus, FeLV). La positività FeLV al test point of care, non confermata dalla PCR del DNA provirale, lascia spazio a diverse interpretazioni. Il capitolo 3 mostra i dati relativi al confronto tra tre diversi protocolli immunosoppressivi (glucocorticoidi, glucocorticoidi+ciclosporina, glucocorticoidi+micofenolato mofetile) in una popolazione di cani con IMHA non associativa. Il confronto verteva, principalmente, sulla risposta ematologica dei pazienti, che non si è dimostrata differente tra i tre gruppi terapeutici. Il capitolo 4 riporta una revisione della letteratura riguardante l’ITP del cane e del gatto. Si tratta di una malattia eterogenea in cui le manifestazioni cliniche appaiono variabili: alcuni pazienti sono asintomatici, altri presentano dei sanguinamenti spontanei. La mancanza di criteri diagnostici standardizzati, porta il clinico a considerare l’ITP una diagnosi “ad esclusione”. Le strategie terapeutiche non si basano purtroppo su linee guida condivise, pertanto il target della terapia rimane, ad oggi, sconosciuto. Nel capitolo 5 viene posta l’attenzione su alcuni interrogativi diagnostici e terapeutici che riguardano l’IMP del cane e del gatto. La sintomatologia clinica, caratterizzata da zoppia, febbre e riluttanza al movimento, talvolta può essere subdola. Anche in questa malattia, non vi sono evidenze scientifiche circa il regime immunosoppressivo più corretto ed indicato.

Bright-field multiplex immunohistochemistry for tumor. Microenvironment evaluation in mucosal melanoma of the head and neck region: technical aspects, results, and correlation with clinicopathologic features and molecular landscape

Mucosal melanoma of the head and neck region (MM-H&N) is a rare disease, characterized by a poor prognosis and limited therapeutic strategies, especially regarding targeted therapy (lower rate of targetable mutations compared to cutaneous melanoma) and immunotherapy (lack of diagnostic tools able to predict the response). Meanwhile, bright-field multiplex immunohistochemistry (BF-mIHC) is emerging as a promising tool for characterizing tumor microenvironment (TME) and predicting response to immunotherapy in several tumors, including melanoma. This PhD project aims to develop a BF-mIHC protocol to evaluate the TME in MM-H&N, analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular), and find new biomarkers useful for prognostic-therapeutic stratification of these patients. Specific aims are: (I) describe the clinicopathological features of MM-H&N; (II) analyze the molecular status of MM-H&N and correlate it with the clinicopathological features; (III) analyze the molecular status of multiple specimens from the same patient to verify whether molecular heterogeneity of MM-H&N could affect the results with relevant prognostic-therapeutic implications; (IV) develop a BF-mIHC protocol to study TME in MM-H&N; (V) analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular) to test whether BF-mIHC could be a promising tool for prognostic-therapeutic characterization of these patients.