Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

The study of the maturation process that occurs to a protein is of pivotal importance for the understanding of its function. This is true also in the vaccine field but in this case is also important to evaluate if inappropriate protein conformation and maturation play roles in the impairment of the functional immunogenicity of protein vaccines. Mass spectrometry (MS) is the method of choice for the study of the maturation process since each modification that occurs during the maturation will lead to a change in the mass of the entire protein. Therefore the aim of my thesis is the development of mass spectrometry-based approaches to study the maturation of proteins and the application of these methods to proteic vaccine candidates. The thesis is divided in two main parts. In the first part, I focused my attention on the study of the maturation of different vaccine candidates using native mass spectrometry. The analyses in this case have been performed using recombinant proteins produced in E. coli. In the second part I applied different MS strategies for the identification of unknown PTMs on pathogenic bacteria surface proteins since modified surface proteins are now considered for vaccine candidate selection.

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

Nella presente tesi indaghiamo la potenzialità di LCM e Reverse Phase Protein microarray negli studi clinici. Si analizza la possibilità di creare una bio banca con line cellular primarie, al fine di conseguire drug test di sensibilità prima di decidere il trattamento da somministrare ai singoli pazienti. Sono stati ottenuti profili proteomici da biopsie pre e post terapia. I risultati dimostrano che questa piattaforma mostra il meccanismo di resistenza acquisito durante la terapia biologica. Questo ci ha portato ad analizzare una possibile stratificazione per pazienti con mCRC . I dati hanno rivelato distinti pathway di attivazione tra metastasi resecabile e non resecabili. I risultati mostrano inoltre due potenziali bersagli farmacologici. Ma la valutazione dell'intero tumore tramite singole biopsie sembra essere un problema a causa dell’eterogeneità intratumorale a livello genomico. Abbiamo indagato questo problema a livello dell'architettura del segnale in campioni di mCRC e ccRCC . I risultati indicano una somiglianza complessiva nei profili proteomici all'interno dello stesso tumore. Considerando che una singola biopsia è rappresentativa di un intera lesione , abbiamo studiato la possibilità di creare linee di cellule primarie, per valutare il profilo molecolare di ogni paziente. Fino ad oggi non c'era un protocollo per creare linee cellulari immortalizzate senza alcuna variazione genetica . abbiamo cosiderato, però, l'approccio innovativo delle CRCs. Ad oggi , non è ancora chiaro se tali cellule mimino il profilo dei tessuti oppure I passaggi in vitro modifichino i loro pathways . Sulla base di un modello di topo , i nostri dati mostrano un profilo di proteomica simile tra le linee di cellule e tessuti di topo LCM. In conclusione, i nostri dati dimostrano l'utilità della piattaforma LCM / RPPA nella sperimentazione clinica e la possibilità di creare una bio - banca di linee cellulari primarie, per migliorare la decisione del trattamento. / In the present thesis we investigate the potentiality of Laser Capture Microdissection (LCM) and Reverse Phase Protein microarray (RPPA) in clinical trials. Also we analyze the possibility to create a primary cell lines bio-bank in order to achieve sensitivity drug tests before to make a treatment decision. Proteomic profiles of pre and post therapy biopsies were obtained from the patients involved in a clinical trial. The results demonstrate that this platform shows the acquired resistance mechanism to the biological therapy. This had led us to discover a better stratification for patient with unresectable colorectal cancer liver metastasis. The RPPA data revealed distinct patterns activation between resectable and unresectable metastasis. Our results show two potential drug targets that could be combined for neo adjuvant treatment. But the evaluation of the whole tumor with single biopsies seems to be a problem due to the genomic intratumor heterogeneity. We investigate this problem in the functional signaling architecture of mCRC and ccRCC. The results indicated an overall similarity in the proteomic profiles within the same tumor. Based on the idea that a single biopsy is representative of a whole lesion, we evaluated the possibility to create primary cell lines, in order to evaluate the molecular profile for each patient for the best personalized therapy. To date there was not a protocol to create immortalized cell lines without any genetic change. We evaluate the innovative approach of the Conditionally Reprogrammed Cells. To date, it is still not clear if those cells mimic the tissue profile or not. Based on a CRC mouse model, our data show a similar proteomic profile between the cell lines and the mouse tissues. In conclusion, our data demonstrate the usefulness of LCM/RPPA platform in clinical trial and the possibility to create a primary cell lines bio-bank to improve treatment decision.

Clostridium difficile is an obligate anaerobic, Gram-positive, endospore-forming bacterium. Although an opportunistic pathogen, it is one of the important causes of healthcare-associated infections. While toxins TcdA and TcdB are the main virulence factors of C. difficile, the factors or processes involved in gut colonization during infection remain unclear. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Little is known about biofilm formation by anaerobic gut species. Biofilm formation by C. difficile could play a role in virulence and persistence of C. difficile, as seen for other intestinal pathogens. We demonstrate that C. difficile clinical strains, 630, and the strain isolated in the outbreak, R20291, form structured biofilms in vitro. Biofilm matrix is made of proteins, DNA and polysaccharide. Strain R20291 accumulates substantially more biofilm. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella and a putative quorum sensing regulator, LuxS, Spo0A, are required for maximal biofilm formation by C. difficile. Moreover we demonstrate that bacteria in C. difficile biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI, and that inhibitory and sub-inhibitory concentrations of the same antibiotic induce biofilm formation. Surprisingly, clinical C. difficile strains from the same out-break, but from different origin, show differences in biofilm formation. Genome sequence analysis of these strains showed presence of a single nucleoide polymorphism (SNP) in the anti-σ factor RsbW, which regulates the stress-induced alternative sigma factor B (σB). We further demonstrate that RsbW, a negative regulator of alternative sigma factor B, has a role in biofilm formation and sporulation of C. difficile. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

This study deals with the discovery and characterization of EXN6 and EXN11 as novel tumor-associated proteins. EXN6 is mainly present in breast and ovary cancers (40 and 35%) while EXN11 is mainly detected in primary and metastatic colon cancer (40%). A characterization of the two proteins confirmed that they could be novel targets for cancer therapy.

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Esta tesis doctoral investiga la noción de necesidad física. Concretamente, estudia si es posible explicar las regularidades no accidentales sin la asunción habitual de un conjunto preexistente de leyes que gobiernan la Naturaleza. Al dispensar de dicha asunción se pone del lado de las llamadas teorías deflacionistas sobre leyes de la Naturaleza, como la humeana o la antirealista. El propósito principal de la tesis es el de complementar dichas teorías con una explicación —ausente a día de hoy— sobre la apariencia de necesidad física. Para proveer dicha explicación, recurro a campos a los que no se había recurrido en la literatura sobre metafísica de leyes: a la teoría de sistemas complejos y a los fundamentos de mecánica estadística. Concretamente, la explicación propuesta está inspirada por la forma en que la teoría de sistemas complejos da cuenta de la emergencia de patrones y por las explicaciones probabilísticas de la 2a ley de la termodinámica. Más en detalle, esta tesis estudia cómo ciertas restricciones —que no hagan referencia directa a cómo debe ser la dinámica del sistema— puedan ser suficientes para obtener a largo plazo, y con alta probabilidad, regularidades estables. Espero mostrar cómo ciertas teorías metafísicas sobre leyes pueden beneficiarse de los resultados obtenidos en estos otros campos. Según esta propuesta, la razón por la cual ciertas regularidades son no accidentales no es debido a una necesidad física subyacente. Es sólo debido a su inmensa estabilidad, fruto de un proceso de convergencia a regularidades estables. Así pues, el objetivo viene a ser el de conseguir explicar la estabilidad de patrones temporales sin presuponer un conjunto de leyes preexistente. Si esta propuesta es correcta no haría falta postular una (misteriosa) categoría intermedia entre la necesidad lógica y la contingencia pura. Del mismo modo, no haría falta postular un (misterioso) conjunto de leyes preexistentes. La parte I de la tesis motiva la parte II argumentando por qué las nociones de necesidad física y de leyes gobernantes son efectivamente misteriosas, y que una explicación debería ser bienvenida (capítulo 1), y estudiando la plausibilidad de un nivel fundamental sin leyes (capítulos 2 y 3). La parte II desarrolla la explicación de la formación de comportamiento estable más simple desde un nivel subyacente más complejo (en última instancia, caótico o aleatorio). / This doctoral dissertation investigates the notion of physical necessity. Specifically, it studies whether it is possible to account for non-accidental regularities without the standard assumption of a pre-existent set of governing laws. Thus, it takes side with the so called deflationist accounts of laws of nature, like the humean or the antirealist. The specific aim is to complement such accounts by providing a missing explanation of the appearance of physical necessity. In order to provide an explanation, I recur to fields that have not been appealed to so far in discussions about the metaphysics of laws. Namely, I recur to complex systems’ theory, and to the foundations of statistical mechanics. The explanation proposed is inspired by how complex systems’ theory has elucidated the way patterns emerge, and by the probabilistic explanations of the 2nd law of thermodynamics. More specifically, this thesis studies how some constraints that make no direct reference to the dynamics can be a sufficient condition for obtaining in the long run, with high probability, stable regular behavior. I hope to show how certain metaphysical accounts of laws might benefit from the insights achieved in these other fields. According to the proposal studied in this thesis, some regularities are not accidental not in virtue of an underlying physical necessity. The non-accidental character of certain regular behavior is only due to its overwhelming stability. Thus, from this point of view the goal becomes to explain the stability of temporal patterns without assuming a set of pre-existent guiding laws. It is argued that the stability can be the result of a process of convergence to simpler and stable regularities from a more complex lower level. According to this project, if successful, there would be no need to postulate a (mysterious) intermediate category between logical necessity and pure contingency. Similarly, there would be no need to postulate a (mysterious) set of pre-existent governing laws. Part I of the thesis motivates part II, mostly by arguing why further explanation of the notions of physical necessity and governing laws should be welcomed (chapter 1), and by studying the plausibility of a lawless fundamental level (chapters 2 and 3). Given so, part II develops the explanation of formation of simpler and stable behavior from a more complex underlying level.