R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features.

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.

Role of SP-A gene polymorphism in lung transplantation

Lung transplantation is a widely accepted therapeutic option for end stage lung disease. Clinical outcome is yet challenged by primary graft failure responsible for the majority of the early mortality, by chronic allograft dysfunction and chronic rejection accounting for more than 30% of deaths after the third postoperative year. Pulmonary surfactant proteins (SP) A, B, C and D are one of the first host defense mechanisms the lung can mount. SP-A in particular, produced by the type II pneumocytes, is active in the innate and adaptive immune system being an opsonin, but also regulating the macrophage and lymphocyte response. The main hypothesis for this project is that pulmonary surfactant protein A polymorphism may determine the early and long term lung allograft survival. Of note SP-A biologic activity seems to be genetically determined and SP-A polymorphisms have been associated to various lung disease. The two SP-A genes SP-A1 and SP-A2 have several polymorphisms within the coding region, SP-A1 (6A, 6A2-20), and SP-A2(1A, 1A0-13). The SP-A gene expression is regulated by cAMP, TTF-1 and glucocorticoids. In vitro studies have indicated that SP-A1 and SP-A2 gene variants may have a variable response to glucocorticoids. We proposed to determine if SP-A gene polymorphism predicts primary graft dysfunction and/or chronic lung allograft dysfunction and if SP-A may serve as a biomarker of lung allograft dysfunction. We also proposed to study the interaction between immunosuppressive drugs and SP-A expression and determine whether this is dependent on SP-A polymorphisms. This study will generate novel information improving our understanding of lung allograft dysfunction. It is conceivable that the information will stimulate the interest for a multi centre study to investigate if SP-A polymorphism may be integrated in the donor lung selection criteria and/or to implement post transplant tailored immunosuppression.

Isolamento, variabilità ed espressione del gene PIMT in Girasole

Il vigore è un aspetto rilevante della qualità delle sementi, strettamente connesso al loro status fisiologico, al genotipo e alle condizioni di stoccaggio. Stress ossidativi e danni alle macromolecole sono alla base del deterioramento del seme, che è equipaggiato con sistemi protettivi e di riparazione. Uno di questi coinvolge l’L-isoaspartil metiltransferasi (PIMT) che ripara i misfolding proteici catalizzando la riconversione in aspartato dell’isoaspartile anomalo accumulato. Scopo di questo studio era valutare il possibile ruolo del meccanismo di riparazione di PIMT nel vigore del seme in girasole. Per questo il relativo gene è stato isolato e caratterizzato, la variabilità allelica determinata su un campione di linee inbred e l’espressione genica misurata in risposta all’invecchiamento accelerato (aging) e al priming. La sequenza codificante ottenuta è costituita da 4 esoni e contiene i 5 domini caratteristici delle metiltransferasi. Il gene mostra elevata similarità con gli ortologhi vegetali e scarsa diversità nucleotidica nei genotipi coltivati rappresentativi della variabilità della specie. Nella sequenza aminoacidica, comunque, sono state rinvenute tre sostituzioni che potrebbero influenzare la funzionalità enzimatica. Dal punto di vista fisiologico i genotipi considerati hanno esibito notevole variabilità di risposte ai trattamenti, sia in termini di vigore che di espressione genica. Aging e priming hanno prodotto generalmente gli effetti attesi, rispettivamente negativi e positivi, sulla germinabilità e sulla sua velocità. In generale l’espressione di PIMT è risultata massima nel seme secco, come riportato altrove, e ridotta dall’aging. Anche il priming ha diminuito l’espressione rispetto al seme quiescente, mentre il suo effetto dopo l’aging è risultato genotipo-dipendente. Tuttavia, nelle condizioni descritte, non si sono evidenziate correlazioni significative tra vigore ed espressione di PIMT, tali da suggerire un chiaro ruolo di questo meccanismo nella qualità fisiologica del seme.

Staphylococcus aureus bones and joints infections: in vivo studies and host immune response

Abstract The aim of this work was the development of a murine model of septic arthrosynovitis and osteomyelitis caused by Staphylococcus aureus, which could mimic the natural disease occurring in humans and which could be suitable for testing preventive and therapeutic interventions. This model could be particularly useful since S. aureus-mediated joints and bones infections are relevant in humans, both in terms of frequency and severity. Our attention focused in tracking bacterial infiltration in joints and bones over time using different microbiological and hystopathological tools, which allowed us to have a complete overview of the situation and to evaluate the immunological actions undertaken by the host to contain or eradicate the bacterial infection. Antibodies and cytokines profiles, as well as recruitment of host immune cells at joints of immunized and infected mice were therefore monitored for a time period that allowed us to study both the acute and the chronic phases of the disease in situ. Finally the Novartis vaccine formulation proposed against S. aureus infections was tested for its capacity to protect immunized mice from joints infections, and the preventive immunization was compared to a standard antibiotic prophylaxis. The availability of powerful tools to study specific bacterial-mediated diseases is nowadays an important requirement for the scientific community to shed light on the complex interactions between host and pathogens and to test treatments for preventing or contrasting infections. We believe that our work significantly contributes to the overall knowledge in the field of S. aureus-dependent pathologies, opening the possibility for further investigations in several fields of study.

Cancer and aging: a multidisciplinary medicinal chemistry approach on relevant biological targets such as proteasome, sirtuins and interleukin 6

It is well known that ageing and cancer have common origins due to internal and environmental stress and share some common hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury. Moreover, ageing is involved in a number of events responsible for carcinogenesis and cancer development at the molecular, cellular, and tissue levels. Ageing could represent a “blockbuster” market because the target patient group includes potentially every person; at the same time, oncology has become the largest therapeutic area in the pharmaceutical industry in terms of the number of projects, clinical trials and research and development (R&D) spending, but cancer remains one of the leading causes of mortality worldwide. The overall aim of the work presented in this thesis was the rational design of new compounds able to modulate activity of relevant targets involved in cancer and aging-related pathologies, namely proteasome and immunoproteasome, sirtuins and interleukin 6. These three targets play different roles in human cells, but the modulation of its activity using small molecules could have beneficial effects on one or more aging-related diseases and cancer. We identified new moderately active and selective non-peptidic compounds able to inhibit the activity of both standard and immunoproteasome, as well as novel and selective scaffolds that would bind and inhibit SIRT6 selectively and can be used to sensitize tumor cells to commonly used anticancer agents such gemcitabine and olaparib. Moreover, our virtual screening approach led us also to the discovery of new putative modulators of SIRT3 with interesting in-vitro and cellular activity. Although the selectivity and potency of the identified chemical scaffolds are susceptible to be further improved, these compounds can be considered as highly promising leads for the development of future therapeutics.

Molecular simulations and modeling of pharmaceutically relevant RNA-processing enzymes

The two-metal-ion architecture is a structural feature found in a variety of RNA processing metalloenzymes or ribozymes (RNA-based enzymes), which control the biogenesis and the metabolism of vital RNAs, including non-coding RNAs (ncRNAs). Notably, such ncRNAs are emerging as key players for the regulation of cellular homeostasis, and their altered expression has been often linked to the development of severe human pathologies, from cancer to mental disorders. Accordingly, understanding the biological processing of ncRNAs is foundational for the development of novel therapeutic strategies and tools. Here, we use state-of the-art molecular simulations, complemented with X-ray crystallography and biochemical experiments, to characterize the RNA processing cycle as catalyzed by two two-metal-ion enzymes: the group II intron ribozymes and the RNase H1. We show that multiple and diverse cations are strategically recruited at and timely released from the enzymes’ active site during catalysis. Such a controlled cations’ trafficking leads to the recursive formation and disruption of an extended two-metal ion architecture that is functional for RNA-hydrolysis – from substrate recruitment to product release. Importantly, we found that these cations’ binding sites are conserved among other RNA-processing machineries, including the human spliceosome and CRISPR-Cas systems, suggesting that an evolutionarily-converged catalytic strategy is adopted by these enzymes to process RNA molecules. Thus, our findings corroborate and sensibly extend the current knowledge of two-metal-ion enzymes, and support the design of novel drugs targeting RNA-processing metalloenzymes or ribozymes as well as the rational engineering of novel programmable gene-therapy tools.

Gene expression and genome-wide association studies analysis to select heavy pigs for protected designation of origin cured meats production

The high quality of protected designation of origin (PDO) dry-cured pork products depends largely on the chemical and physical parameters of the fresh meat and their variation during the production process of the final product. The discovery of the mechanisms that regulate the variability of these parameters was aided by the reference genome of swine adjuvant to genetic analysis methods. This thesis can contribute to the discovery of genetic mechanisms that regulate the variability of some quality parameters of fresh meat for PDO dry-cured pork production. The first study is of gene expression and showed that between low and high glycolytic potential (GP) samples of Semimembranosus muscle of Italian Large White (ILW) pigs in early postmortem, the differentially expressed genes were all but one over expressed in low GP. These were involved in ATP biosynthesis processes, calcium homeostasis, and lipid metabolism including the potential master regulator gene Peroxisome Proliferator-Activated Receptor Alpha (PPARA). The second is a study in commercial hybrid pigs to evaluate correlations between carcass and fresh ham traits, including carcass and fresh ham lean meat percentages, the former, a potential predictor of the latter. In addition, a genome-wide association study allowed the identification of chromosome-wide associations with phenotypic traits for 19 SNPs, and genome-wide associations for 14 SNPs for ferrochelatase activity. The latter could be a determinant for color variation in nitrite-free dry-cured ham. The third study showed gene expression differences in the Longissimus thoracis muscle of ILW pigs by feeding diets with extruded linseed (source of polyunsaturated fatty acids) and vitamin E and selenium (diet three) or natural (diet four) antioxidants. The diet three promoted a more rapid and massive immune system response possibly determined by improvement in muscle tissue function, while the diet four promoted oxidative stability and increased the anti-inflammatory potential of muscle tissue.