Human papillomavirus (HPV) and associated diseases: between applied diagnostic and basic research

Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet. In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types. The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker. The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.

Effects of Global Warming on Berry Composition of cv. Sangiovese: Biochemical and Molecular Aspects and Agronomical Adaptation Approaches

Wine grape must deal with serious problems due to the unfavorable climatic conditions resulted from global warming. High temperatures result in oxidative damages to grape vines. The excessive elevated temperatures are critical for grapevine productivity and survival and contribute to degradation of grape and wine quality and yield. Elevated temperature can negatively affect anthocyanin accumulation in red grape. Particularly, cv. Sangiovese was identified to be very sensitive to such condition. The quantitative real-time PCR analysis showed that flavonoid biosynthetic genes were slightly repressed by high temperature. Also, the heat stress repressed the expression of the transcription factor “VvMYBA1” that activates the expression of UFGT. Moreover, high temperatures had repressing effects on the activity of the flavonoids biosynthetic enzymes “PAL” and “UFGT”.Anthocyanin accumulation in berry skin is due to the balance between its synthesis and oxidation. In grape cv. Sangiovese, the gene transcription and activity of peroxidases enzyme was elevated by heat stress as a defensive mechanism of ROS-scavenging. Among many isoforms of peroxidases genes, one gene (POD 1) was induced in Sangiovese under thermal stress condition. This gene was isolated and evaluated via the technique of genes transformation from grape to Petunia. Reduction in anthocyanins concentration and higher enzymatic activity of peroxidase was observed in POD 1 transformed Petunia after heat shock compared to untrasformed control. Moreover, in wine producing regions, it is inevitable for the grape growers to adopt some adaptive strategies to alleviate grape damages to abiotic stresses. Therefore, in this thesis, the technique of post veraison trimming was done to improve the coupling of phenolic and sugar ripening in Vitis vinifera L. cultivar Sangiovese. Trimming after veraison showed to be executable to slow down the rate of sugar accumulation in grape (to decrease the alcohol potential in wines) without evolution of the main berry flavonoids compounds.

Comparative DNA sequence and methylation analyses of orthologous genes in humans and non-human primates: Genetic and epigenetic footprints of evolution

Welche genetische Unterschiede machen uns verschieden von unseren nächsten Verwandten, den Schimpansen, und andererseits so ähnlich zu den Schimpansen? Was wir untersuchen und auch verstehen wollen, ist die komplexe Beziehung zwischen den multiplen genetischen und epigenetischen Unterschieden, deren Interaktion mit diversen Umwelt- und Kulturfaktoren in den beobachteten phänotypischen Unterschieden resultieren. Um aufzuklären, ob chromosomale Rearrangements zur Divergenz zwischen Mensch und Schimpanse beigetragen haben und welche selektiven Kräfte ihre Evolution geprägt haben, habe ich die kodierenden Sequenzen von 2 Mb umfassenden, die perizentrischen Inversionsbruchpunkte flankierenden Regionen auf den Chromosomen 1, 4, 5, 9, 12, 17 und 18 untersucht. Als Kontrolle dienten dabei 4 Mb umfassende kollineare Regionen auf den rearrangierten Chromosomen, welche mindestens 10 Mb von den Bruchpunktregionen entfernt lagen. Dabei konnte ich in den Bruchpunkten flankierenden Regionen im Vergleich zu den Kontrollregionen keine höhere Proteinevolutionsrate feststellen. Meine Ergebnisse unterstützen nicht die chromosomale Speziationshypothese für Mensch und Schimpanse, da der Anteil der positiv selektierten Gene (5,1% in den Bruchpunkten flankierenden Regionen und 7% in den Kontrollregionen) in beiden Regionen ähnlich war. Durch den Vergleich der Anzahl der positiv und negativ selektierten Gene per Chromosom konnte ich feststellen, dass Chromosom 9 die meisten und Chromosom 5 die wenigsten positiv selektierten Gene in den Bruchpunkt flankierenden Regionen und Kontrollregionen enthalten. Die Anzahl der negativ selektierten Gene (68) war dabei viel höher als die Anzahl der positiv selektierten Gene (17). Eine bioinformatische Analyse von publizierten Microarray-Expressionsdaten (Affymetrix Chip U95 und U133v2) ergab 31 Gene, die zwischen Mensch und Schimpanse differentiell exprimiert sind. Durch Untersuchung des dN/dS-Verhältnisses dieser 31 Gene konnte ich 7 Gene als negativ selektiert und nur 1 Gen als positiv selektiert identifizieren. Dieser Befund steht im Einklang mit dem Konzept, dass Genexpressionslevel unter stabilisierender Selektion evolvieren. Die meisten positiv selektierten Gene spielen überdies eine Rolle bei der Fortpflanzung. Viele dieser Speziesunterschiede resultieren eher aus Änderungen in der Genregulation als aus strukturellen Änderungen der Genprodukte. Man nimmt an, dass die meisten Unterschiede in der Genregulation sich auf transkriptioneller Ebene manifestieren. Im Rahmen dieser Arbeit wurden die Unterschiede in der DNA-Methylierung zwischen Mensch und Schimpanse untersucht. Dazu wurden die Methylierungsmuster der Promotor-CpG-Inseln von 12 Genen im Cortex von Menschen und Schimpansen mittels klassischer Bisulfit-Sequenzierung und Bisulfit-Pyrosequenzierung analysiert. Die Kandidatengene wurden wegen ihrer differentiellen Expressionsmuster zwischen Mensch und Schimpanse sowie wegen Ihrer Assoziation mit menschlichen Krankheiten oder dem genomischen Imprinting ausgewählt. Mit Ausnahme einiger individueller Positionen zeigte die Mehrzahl der analysierten Gene keine hohe intra- oder interspezifische Variation der DNA-Methylierung zwischen den beiden Spezies. Nur bei einem Gen, CCRK, waren deutliche intraspezifische und interspezifische Unterschiede im Grad der DNA-Methylierung festzustellen. Die differentiell methylierten CpG-Positionen lagen innerhalb eines repetitiven Alu-Sg1-Elements. Die Untersuchung des CCRK-Gens liefert eine umfassende Analyse der intra- und interspezifischen Variabilität der DNA-Methylierung einer Alu-Insertion in eine regulatorische Region. Die beobachteten Speziesunterschiede deuten darauf hin, dass die Methylierungsmuster des CCRK-Gens wahrscheinlich in Adaption an spezifische Anforderungen zur Feinabstimmung der CCRK-Regulation unter positiver Selektion evolvieren. Der Promotor des CCRK-Gens ist anfällig für epigenetische Modifikationen durch DNA-Methylierung, welche zu komplexen Transkriptionsmustern führen können. Durch ihre genomische Mobilität, ihren hohen CpG-Anteil und ihren Einfluss auf die Genexpression sind Alu-Insertionen exzellente Kandidaten für die Förderung von Veränderungen während der Entwicklungsregulation von Primatengenen. Der Vergleich der intra- und interspezifischen Methylierung von spezifischen Alu-Insertionen in anderen Genen und Geweben stellt eine erfolgversprechende Strategie dar.

Nanoparticles for efficient uptake by human dendritic cells and T lymphocytes to be used in adoptive immunotherapy

Dendritic cells (DCs) are the most potent cell type for capture, processing, and presentation of antigens. They are able to activate naïve T cells as well as to initiate memory T-cell immune responses. T lymphocytes are key elements in eliciting cellular immunity against bacteria and viruses as well as in the generation of anti-tumor and anti-leukemia immune responses. Because of their central position in the immunological network, specific manipulations of these cell types provide promising possibilities for novel immunotherapies. Nanoparticles (NP) that have just recently been investigated for use as carriers of drugs or imaging agents, are well suited for therapeutic applications in vitro and also in vivo since they can be addressed to cells with a high target specificity upon surface functionalization. As a first prerequisite, an efficient in vitro labeling of cells with NP has to be established. In this work we developed protocols allowing an effective loading of human monocyte-derived DCs and primary antigen-specific T cells with newly designed NP without affecting biological cell functions. Polystyrene NP that have been synthesized by the miniemulsion technique contained perylenmonoimide (PMI) as a fluorochrome, allowing the rapid determination of intracellular uptake by flow cytometry. To confirm intracellular localization, NP-loaded cells were analyzed by confocal laser scanning microscopy (cLSM) and transmission electron microscopy (TEM). Functional analyses of NP-loaded cells were performed by IFN-γ ELISPOT, 51Chromium-release, and 3H-thymidine proliferation assays. In the first part of this study, we observed strong labeling of DCs with amino-functionalized NP. Even after 8 days 95% of DCs had retained nanoparticles with a median fluorescence intensity of 67% compared to day 1. NP loading did not influence expression of cell surface molecules that are specific for mature DCs (mDCs) nor did it influence the immunostimulatory capacity of mDCs. This procedure did also not impair the capability of DCs for uptake, processing and presentation of viral antigens that has not been shown before for NP in DCs. In the second part of this work, the protocol was adapted to the very different conditions with T lymphocytes. We used leukemia-, tumor-, and allo-human leukocyte antigen (HLA) reactive CD8+ or CD4+ T cells as model systems. Our data showed that amino-functionalized NP were taken up very efficiently also by T lymphocytes, which usually had a lower capacity for NP incorporation compared to other cell types. In contrast to DCs, T cells released 70-90% of incorporated NP during the first 24 h, which points to the need to escape from intracellular uptake pathways before export to the outside can occur. Preliminary data with biodegradable nanocapsules (NC) revealed that encapsulated cargo molecules could, in principle, escape from the endolysosomal compartment after loading into T lymphocytes. T cell function was not influenced by NP load at low to intermediate concentrations of 25 to 150 μg/mL. Overall, our data suggest that NP and NC are promising tools for the delivery of drugs, antigens, and other molecules into DCs and T lymphocytes.

Classical and quantum aspects of black hole dynamics

Il contenuto fisico della Relatività Generale è espresso dal Principio di Equivalenza, che sancisce l'equivalenza di geometria e gravitazione. La teoria predice l'esistenza dei buchi neri, i più semplici oggetti macroscopici esistenti in natura: essi sono infatti descritti da pochi parametri, le cui variazioni obbediscono a leggi analoghe a quelle della termodinamica. La termodinamica dei buchi neri è posta su basi solide dalla meccanica quantistica, mediante il fenomeno noto come radiazione di Hawking. Questi risultati gettano una luce su una possibile teoria quantistica della gravitazione, ma ad oggi una simile teoria è ancora lontana. In questa tesi ci proponiamo di studiare i buchi neri nei loro aspetti sia classici che quantistici. I primi due capitoli sono dedicati all'esposizione dei principali risultati raggiunti in ambito teorico: in particolare ci soffermeremo sui singularity theorems, le leggi della meccanica dei buchi neri e la radiazione di Hawking. Il terzo capitolo, che estende la discussione sulle singolarità, espone la teoria dei buchi neri non singolari, pensati come un modello effettivo di rimozione delle singolarità. Infine il quarto capitolo esplora le ulteriori conseguenze della meccanica quantistica sulla dinamica dei buchi neri, mediante l'uso della nozione di entropia di entanglement.

Procedural and dosimetric aspects in peripheral angiography using CO2 as contrast medium

Lo scopo di questa tesi è lo studio degli aspetti procedurali e dosimetrici in angiografie periferiche che utilizzano la CO2 come mezzo di contrasto. La tecnica angiografica consiste nell’imaging radiologico di vasi sanguigni tramite l’iniezione di un mezzo di contrasto, e il suo uso è in costante incremento a causa dell’aumento di pazienti con malattie vascolari. I mezzi di contrasto iodati sono i più comunemente utilizzati e permettono di ottenere immagini di ottima qualità, ma presentano il limite di una elevata nefrotossicità. La CO2 è considerata un’interessante alternativa al mezzo iodato, per la sua acclarata biocompatibilità, soprattutto per pazienti con elevati fattori di rischio (diabete e/o insufficienza renale). Il suo utilizzo presenta comunque alcuni aspetti problematici, dovuti allo stato gassoso e al basso contrasto intrinseco rispetto alla soluzione iodata. Per quest’ultimo motivo si ritiene generalmente che l’utilizzo della CO2 comporti un aumento di dose rispetto ai mezzi di contrasto tradizionali. Il nostro studio, effettuato su diversi apparati radiologici, ha dimostrato che i parametri di emissione radiologica sono gli stessi per i protocolli di angiografia tradizionale, con iodio, e quelli che utilizzano CO2. Questa evidenza suggerisce che i protocolli CO2 operino solo sul trattamento delle immagini ottenute e non sulla modalità di acquisizione, e dal punto di vista dosimetrico l’angiografia con CO2 è riconducibile all’angiografia tradizionale. L’unico fattore che potrebbe portare a un effettivo incremento di dose al paziente è un allungamento dei tempi di scopia e di procedura, che andrebbe verificato con una campagna di misure in ambito clinico. Sulla base della stessa evidenza, si ritiene che la visualizzazione della CO2 possa essere ulteriormente migliorata attraverso l’ottimizzazione dei parametri di emissione radiologica (kVp, frame rate e durata degli impulsi) attualmente predisposti per l’uso di mezzi di contrasto iodati.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Gender, age and ethnic aspects of analgesia in acute abdominal pain: is analgesia even across the groups?

Numerous studies have shown differences in pain perception between men and women, which may affect pain management strategies.

The plasmin-antiplasmin system: structural and functional aspects

The plasmin-antiplasmin system plays a key role in blood coagulation and fibrinolysis. Plasmin and (2)-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into soluble fragments. However, besides plasmin(ogen) and (2)-antiplasmin the system contains a series of specific activators and inhibitors. The main physiological activators of plasminogen are tissue-type plasminogen activator, which is mainly involved in the dissolution of the fibrin polymers by plasmin, and urokinase-type plasminogen activator, which is primarily responsible for the generation of plasmin activity in the intercellular space. Both activators are multidomain serine proteases. Besides the main physiological inhibitor (2)-antiplasmin, the plasmin-antiplasmin system is also regulated by the general protease inhibitor (2)-macroglobulin, a member of the protease inhibitor I39 family. The activity of the plasminogen activators is primarily regulated by the plasminogen activator inhibitors 1 and 2, members of the serine protease inhibitor superfamily.

Cultural and migration aspects in functional abdominal pain

Compared to Europe's mean immigrant contingent of 7.3 to 8.6 % Switzerland holds the highest contingent of foreign population with 23.5 %. Therefore it is of utmost importance that physicians have a knowledge of the specific characteristics of immigrant patients. The influence of personality factors (experience, behavior) is not independent from the influence of culturally-related environmental factors (regional differences in diet, pollutants, meanings, etc.). In addition, different cultural groups rate their quality of life differently. Psychological reasons for recurrent abdominal pain are stress (life events), effects of self-medication (laxatives, cocaine) and sexual abuse but also rare infectious diseases are more common among immigrants (e.g. tuberculosis, histoplasmosis, etc.). Migration-specific characteristics are mainly to find in the semiotics of the symptoms: not every abdominal pain is real pain in the abdomen. Finally, it is crucial to make the distinction between organic, functional and psychological-related pain. This can, however, usually only be accomplished in the context of the entire situation of a patient and, depending on the situation, with the support of a colleague from the appropriate cultural group or an experienced interpreter. In this review we limit ourselves to the presentation of the working population of the migrants, because these represent the largest group of all migrants. The specific situation of asylum seekers will also be refrained to where appropriate.

Dental erosion--an overview with emphasis on chemical and histopathological aspects

The quality of dental care and modern achievements in dental science depend strongly on understanding the properties of teeth and the basic principles and mechanisms involved in their interaction with surrounding media. Erosion is a disorder to which such properties as structural features of tooth, physiological properties of saliva, and extrinsic and intrinsic acidic sources and habits contribute, and all must be carefully considered. The degree of saturation in the surrounding solution, which is determined by pH and calcium and phosphate concentrations, is the driving force for dissolution of dental hard tissue. In relation to caries, with the calcium and phosphate concentrations in plaque fluid, the 'critical pH' below which enamel dissolves is about 5.5. For erosion, the critical pH is lower in products (e.g. yoghurt) containing more calcium and phosphate than plaque fluid and higher when the concentrations are lower. Dental erosion starts by initial softening of the enamel surface followed by loss of volume with a softened layer persisting at the surface of the remaining tissue. Dentine erosion is not clearly understood, so further in vivo studies, including histopathological aspects, are needed. Clinical reports show that exposure to acids combined with an insufficient salivary flow rate results in enhanced dissolution. The effects of these and other interactions result in a permanent ion/substance exchange and reorganisation within the tooth material or at its interface, thus altering its strength and structure. The rate and severity of erosion are determined by the susceptibility of the dental tissues towards dissolution. Because enamel contains less soluble mineral than dentine, it tends to erode more slowly. The chemical mechanisms of erosion are also summarised in this review. Special attention is given to the microscopic and macroscopic histopathology of erosion.

Soft-Tissue Simulation for Cranio-Maxillofacial Surgery: Clinical Needs and Technical Aspects

Computerized soft-tissue simulation can provide unprecedented means for predicting facial outlook pre-operatively. Surgeons can virtually perform several surgical plans to have the best surgical results for their patients while considering corresponding soft-tissue outcome. It could be used as an interactive communication tool with their patients as well. There has been comprehensive amount of works for simulating soft-tissue for cranio-maxillofacial surgery. Although some of them have been realized as commercial products, none of them has been fully integrated into clinical practice due to the lack of accuracy and excessive amount of processing time. In this chapter, state-of-the-art and general workflow in facial soft-tissue simulation will be presented, along with an example of patient-specific facial soft-tissue simulation method.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.