Plant viruses: the many aspects of fascinating nano-biotechnological tool

I capsidi virali sono strutture stabili e robuste composte da multiple copie di una o pi\uf9 tipi di subunit\ue0 proteiche organizzate con simmetrie ordinate (icosaedriche o filmentose). I capsidi virali possono essere prodotti in sistemi vegetali in consistenti quantit\ue0 sia tramite l\u2019infezione delle piante sia tramite l\u2019espressione delle subunit\ue0 capsidiche. Data la semplicit\ue0 e l\u2019elevata stabilit\ue0, le Chimeric Virus Particles (CVPs) e le empty Virus Like Particles (eVLPs) hanno attirato l\u2019attenzione della comunit\ue0 scientifica per lo sviluppo di reagenti che possono trovare impiego nell\u2019ambito delle nano-biotecnologie. In questo lavoro di tesi, le CVPs e le eVLPs sono state sfruttate per l\u2019espressione di peptidi funzionali, al fine di stabilizzarli e di produrli a basso costo. In particolare, le piattaforme di espressione virali scelte si basano sui seguenti virus vegetali: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) e il Turnip Mosaic Virus (TuMV). La prima applicazione descritta riguarda il trattamento di due malattie autoimmuni che hanno un forte impatto sociale: il Diabete Melito di tipo 1 (T1D) e l\u2019Artrite Reumatoide (AR). Attualmente, esistono trattamenti che sono solo in grado di arginare e gestire gli effetti di queste patologie senza per\uf2 bloccarne definitivamente l\u2019azione. In questo lavoro, virus vegetali che espongono peptidi associati al T1D e all\u2019AR sono stati utilizzati per lo sviluppo rispettivamente di un trattamento preventivo e terapeutico. Le particelle virali sono state geneticamente modificate in modo da esporre peptidi autoantigenici associati al T1D e all\u2019AR, espresse in pianta, purificate dal tessuto vegetale ed utilizzate per studi pre-clinici in animali modello. I risultati ottenuti mostrano che la struttura delle particelle virali \ue8 in grado di agire come adiuvante aumentando la capacit\ue0 immunomodulante dei peptidi autoantigenici selezionati ed esposti sui capsidi virali. La seconda parte di questo lavoro di tesi, si concentra sull\u2019utilizzo delle CVPs esprimenti peptidi associati alla Sindrome di Sj\uf6gren (SjS) e all\u2019AR per lo sviluppo di innovativi kit diagnostici. Queste due malattie autoimmuni sono attualmente difficili da diagnosticare principalmente per la presenza di un sottogruppo di pazienti che risulta essere negativo alla presenza dei pi\uf9 comuni marcatori sierologici utilizzati per la diagnosi. Questo progetto si \ue8 focalizzato sull\u2019espressione in pianta di CVPs di struttura filamentosa che, tramite l\u2019espressione di specifici peptidi, hanno permesso di migliorare notevolmente le performance diagnostiche di un test ELISA sviluppato con tali particelle in confrontato con lo stesso sviluppato utilizzo i peptidi sintetici. Inoltre, sono state espresse in pianta ulteriori CVPs che espongono due peptidi associati all\u2019AR. Tali CVPs saranno in futuro utilizzate per la messa a punto di un kit per la diagnosi dell\u2019AR. L\u2019ultima parte di questo lavoro, riguarda altre possibili applicazioni dei virus vegetali come strumenti nano-biotecnologici. Nello specifico, eVLPs sono state espresse in pianta al fine di esprimere un peptide antimicrobico (AMP) e un cell penetrating peptide (CPP) per ottenere rispettivamente un innovativo eco-pesticida e uno strumento biotecnologico per l\u2019internalizzazione di peptidi funzionali o proteine di interesse nelle cellule.The capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or few types of protein subunits arranged with either icosahedral or helical ordered symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunits. In view of their relative simplicity, stability and easy production, plant chimeric virus particles (CVPs) or empty virus-like particles (eVLPs) have attracted attention as potential reagents for applications in bionanotechnology. In this work CVPs and eVLPs have been exploited for the expression of functional peptides, in order to stabilize them and avoid peptide low intrinsic stability and susceptibility to degradation. In particular, the viral expression platforms chosen for the expression of target peptides are based on four plant viruses widely used as scaffold for peptide display: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) and Turnip Mosaic Virus (TuMV). The first application explored in this work regards the therapy of Type 1 diabetes (T1D) and Rheumatoid arthritis (RA), two autoimmune diseases that share a strong social impact. Currently, there are treatments able to manage and/or stem the effects of these disorders. In particular, plant viruses displaying peptides associated to T1D and RA have been used respectively for the development of a preventive and therapeutic drug. Virus particles displaying autoantigenic peptide specific for these diseases have been expressed and used for pre-clinical studies in T1D and RA animal models. The results observed suggest that the use of viral structure for peptide display works as an adjuvant by increasing peptide modulation capability. The second part of this work regards the use of plant viruses displaying peptide as reagents for the development of innovative kit for the Sj\uf6gren\u2019s Syndrome (SjS) and RA diagnosis. These two autoimmune diseases are difficult to be diagnosed and either for SjS of RA there are subgroups of patient seronegative to the main diagnostic serological markers. In this work, the use of filamentous particles for the display of specific SjS peptide allowed to increase the diagnostic performances of an ELISA kit in comparison to the use of the peptide alone. Moreover, autoantigenic peptides associated to RA were successfully expressed in plants on the surface of viral particles that will be exploited in the future for the development of a kit for seronegative RA diagnosis. A third part of this PhD thesis regards another possible application of plant viruses as tools for peptide display. In particular, viral particles have been used for the expression of a antimicrobial peptide (AMP) that could be exploited as eco-friendly pesticide and for \u201cnanoagriculture\u201d application. Finally, the possibility of developing a biotechnological tool for peptide internalisation into the cells has been exploited by fusing on the surface of an icosahedral plant virus a cell-penetrating peptide (CPP) derive from HIV. Regarding this third part, CVPs and eVLPs displaying the selected peptides have been successfully expressed in plants; however, several drawbacks have been encountered in the purification process

L’osservazione degli stili educativi padri-figli nella struttura penitenziaria

This contribution is a part of a wider research project carried out in Prison of Milan-Opera (Italy),whose aim was to orientate the interest on the role of father and on the most appropriate educationalstrategies to keep alive the role and the parental bond. To achieve this objective, a multidisciplinarymethodological approach was used: the risks correlated to the detention status withrespect to the emotional relationship of an imprisoned father (personal experiences, sense offrustration and/or guilt, feeling of failure/impotence, avoidance/denial of the problem, etc.);the ways in which the individual prisoner attempts to decline his or her parental role; the possiblepedagogical support that education professionals can offer

Methylation-dependent PAD2 upregulation in multiple sclerosis peripheral blood

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines - an event that may favour autoimmunity - while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling. Objectives: Our aim was to verify whether an altered epigenetic control of PAD2, as already shown in the MS brain, can be observed in peripheral blood mononuclear cells (PBMCs) of patients with MS since some of these cells also synthesize MBP. Methods: The expression of most suitable reference genes and of PAD2 and PAD4 was assessed by qPCR. Analysis of DNA methylation was performed by bisulfite method. Results: The comparison of PAD2 expression level in PBMCs from patients with MS vs. healthy donors showed that, as well as in the white matter of MS patients, the enzyme is significantly upregulated in affected subjects. Methylation pattern analysis of a CpG island located in the PAD2 promoter showed that over-expression is associated with promoter demethylation. Conclusion: Defective regulation of PAD2 in the periphery, without the immunological shelter of the blood-brain barrier, may contribute to the development of the autoimmune responses in MS

Poly(ADP-ribosyl)ation is involved in the epigenetic control of TET1 gene transcription

TET enzymes are the epigenetic factors involved in the formation of the Sixth DNA base 5-hydroxymethylcytosine, whose deregulation has been associated with tumorigenesis. In particular, TET1 acts as tumor suppressor preventing cell proliferation and tumor metastasis and it has frequently been found down-regulated in cancer. Thus, considering the importance of a tight control of TET1 expression, the epigenetic mechanisms involved in the transcriptional regulation of TET1 gene are here investigated. The involvement of poly(ADP-ribosyl)ation in the control of DNA and histone methylation on TET1 gene was examined. PARP activity is able to positively regulate TET1 expression maintaining a permissive chromatin state characterized by DNA hypomethylation of TET1 CpG island as well as high levels of H3K4 trimethylation. These epigenetic modifications were affected by PAR depletion causing TET1 downregulation and in turn reduced recruitment of TET1 protein on HOXA9 target gene. In conclusion, this work shows that PARP activity is a transcriptional regulator of TET1 gene through the control of epigenetic events and it suggests that deregulation of these mechanisms could account for TET1 repression in cancer

Validation of suitable internal control genes for expression studies in aging.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors

PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression

To overcome cancer cells resistance to pharmacological therapy, the development of new therapeutic approaches becomes urgent. For this purpose, the use of poly(ADP-ribose) polymerase (PARP) inhibitors in combination with other cytotoxic agents could represent an efficacious strategy. Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification that plays a well characterized role in the cellular decisions of life and death. Recent findings indicate that PARP-1 may control the expression of Snail, the master gene of epithelial-mesenchymal transition (EMT). Snail is highly represented in different resistant tumors, functioning as a factor regulating anti-apoptotic programmes. MDA-MB-231 is a Snail-expressing metastatic breast cancer cell line, which exhibits chemoresistance properties when treated with damaging agents. In this study, we show that the PARP inhibitor ABT-888 was capable to modulate the MDA-MB-231 cell response to doxorubicin, leading to an increase in the rate of apoptosis. Our further results indicate that PARP-1 controlled Snail expression at transcriptional level in cells exposed to doxorubicin. Given the increasing interest in the employment of PARP inhibitors as chemotherapeutic adjuvants, our in vitro results suggest that one of the mechanisms through which PARP inhibition can chemosensitize cancer cells in vivo, is targeting Snail expression thus promoting apoptosi

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required

DNA hydroxymethylation levels are altered in blood cells from Down syndrome persons enrolled in the MARK-AGE project

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the ageing process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work we investigated the levels of 5-hydroxymethylcytosine (5hmC) and of the TET dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in ageing. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease of 5hmC, TET1 and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS

Factors associated with variation in intracranial pressure in a model of intra-abdominal hypertension with acute lung injury

OBJETIVO: Avaliar o efeito de alterações hemodinâmicas, respiratórias e metabólicas sobre a pressão intracraniana em um modelo de lesão pulmonar aguda e síndrome compartimental abdominal. MÉTODOS: Oito porcos Agroceres foram submetidos, após a instrumentação, a cinco cenários clínicos: 1) estado basal com baixa pressão intra-abdominal e pulmão sadio; 2) pneumoperitôneo, com pressão intra-abdominal de 20 mm Hg; 3) lesão pulmonar aguda induzida por lavagem pulmonar e desativação de surfactante; 4) pneumoperitôneo com pressão intra-abdominal de 20 mm Hg na vigência de lesão pulmonar aguda e com PEEP baixo; e 5) PEEP ajustado a 27 cm H2O na vigência de pneumoperitôneo e lesão pulmonar aguda. Variáveis respiratórias e hemodinâmicas foram coletadas. Análise multivariada foi realizada buscando as variáveis associadas com elevação da pressão intracraniana nos cinco cenários estudados. RESULTADOS: Após a análise multivariada, nas situações não associadas com lesão pulmonar aguda apenas a pressão de platô das vias aéreas se correlacionou positivamente com a pressão intracraniana. Nos modelos associados com lesão pulmonar aguda, a pressão de platô de vias aéreas, a pressão arterial de CO2, o CO2 no final da expiração e a pressão venosa central se correlacionaram positivamente com incrementos da pressão intracraniana. CONCLUSÃO: Em um modelo de disfunção orgânica múltipla com situações clínicas associadas com aumento da pressão torácica e abdominal, o incremento da pressão intracraniana desencadeado pela elevação da pressão abdominal parece ser decorrente da piora da complacência do sistema respiratório e da redução do gradiente para drenagem venosa cerebral ocasionado pela elevação da pressão venosa central.OBJECTIVE: To evaluate the effects of hemodynamic, respiratory and metabolic changes on intracranial pressure in a model of acute lung injury and abdominal compartment syndrome. METHODS: Eight Agroceres pigs were submitted to five different clinical scenarios after instrumentation: 1) a baseline condition with low intra-abdominal pressure and healthy lungs; 2) pneumoperitoneum with 20 mmHg intra-abdominal pressure; 3) acute lung injury induced by pulmonary lavage with surfactant deactivation; 4) pneumoperitoneum with 20 mmHg intra-abdominal pressure with lung pulmonary injury and low positive end-expiratory pressure; and 5) 27 cmH2O positive end-expiratory pressure with pneumoperitoneum and acute lung injury. Respiratory and hemodynamic variables were collected. A multivariate analysis was conducted to search for variables associated with increased intracranial pressure in the five scenarios. RESULTS: Only plateau airway pressure showed a positive correlation with intracranial pressure in the multivariate analysis. In the models with acute lung injury, plateau airway pressure, CO2 arterial pressure, end tidal CO2 and central venous pressure were positively correlated with increased intracranial pressure. CONCLUSION: In a model of multiple organ dysfunction with associated clinical conditions causing increased intra-thoracic and abdominal pressure, increased intracranial pressure triggered by elevated intra-abdominal pressure is apparently caused by worsened respiratory system compliance and a reduced brain venous drainage gradient due to increased central venous pressure

A computational procedure to identify significant overlap of differentially expressed and genomic imbalanced regions in cancer datasets†

The integration of high-throughput genomic data represents an opportunity for deciphering the interplay between structural and functional organization of genomes and for discovering novel biomarkers. However, the development of integrative approaches to complement gene expression (GE) data with other types of gene information, such as copy number (CN) and chromosomal localization, still represents a computational challenge in the genomic arena. This work presents a computational procedure that directly integrates CN and GE profiles at genome-wide level. When applied to DNA/RNA paired data, this approach leads to the identification of Significant Overlaps of Differentially Expressed and Genomic Imbalanced Regions (SODEGIR). This goal is accomplished in three steps. The first step extends to CN a method for detecting regional imbalances in GE. The second part provides the integration of CN and GE data and identifies chromosomal regions with concordantly altered genomic and transcriptional status in a tumor sample. The last step elevates the single-sample analysis to an entire dataset of tumor specimens. When applied to study chromosomal aberrations in a collection of astrocytoma and renal carcinoma samples, the procedure proved to be effective in identifying discrete chromosomal regions of coordinated CN alterations and changes in transcriptional levels