Birth weight, current body mass index, and insulin sensitivity and secretion in young adults in two Latin American populations

Background and aims: Although studies have shown association of birth weight (BW) and adult body mass index (BMI) with insulin sensitivity in adults, there is limited evidence that BW is associated with insulin secretion. We assessed the associations between BW and current BMI with insulin sensitivity and secretion in young Latin American adults. Methods and results: Two birth cohorts, one from Ribeirao Preto, Brazil, based on 1984 participants aged 23-25 years, and another from Limache, Chile, based on 965 participants aged 22-28 years were studied. Weight and height at birth, and current fasting plasma glucose and insulin levels were measured. Insulin sensitivity (HOMA%S) and secretion (HOMA%beta) were estimated using the Homeostatic Model Assessment (HOMA2). Multiple linear regression analyses were carried out to test the associations between BW and adult BMI z-scores on log HOMA%S and log HOMA%beta. BW z-score was associated with HOMA%S in the two populations and HOMA%beta in Ribeirao Preto when adult BMI z-score was included in the model. BW z-score was associated with decreasing insulin secretion even without adjusting for adult BMI, but only in Ribeirao Preto. BMI z-score was associated with low HOMA%S and high HOMA%beta. No interactions between BW and BMI z-scores on insulin sensitivity were shown. Conclusions: This study supports the finding that BW may affect insulin sensitivity and secretion in young adults. The effect size of BW on insulin status is small in comparison to current BMI. (C) 2010 Elsevier B.V. All rights reserved.

Current perspectives in avian salmonellosis: Vaccines and immune mechanisms of protection

Salmonellosis is one of the most prevalent foodborne diseases worldwide. Food animals have been identified as reservoirs for nontyphoid Salmonella infections. in poultry, host-specific Salmonella infections cause fowl typhoid and pullorum diseases that produce economic losses in different parts of the world. Several measures have been used to prevent and control Salmonella infections in poultry, and vaccination is the most practical measure because it avoids contamination of poultry products and by-products and prevents disease in humans. Salmonella vaccines can decrease public health risk by reducing colonization and organ invasion, including invasion of reproductive tissues, and by diminishing fecal shedding and environmental contamination. We review available information on the host-specific and non-host-specific Salmonella serotypes found in poultry and the improved understanding of the pathogenesis of and immune responses to infection. We also include some approaches based on updated publications regarding killed and live attenuated vaccines and their immune mechanisms of protection.

Ageing exacerbates damage of systemic and salivary neutrophils from patients presenting Candida-related denture stomatitis

Abstract Background Ageing leads to a decline in the function of the immune system, increasing the body's susceptibility to infections through the impairment of T-cells, macrophages, neutrophils and dendritic cells Denture stomatitis is a primary oral disease affecting elderly denture wearers. The major etiologic factor involved in this pathology is the infection by Candida albicans, an opportunistic pathogen that causes local and disseminated diseases in immunosuppressed humans. Neutrophils play a critical role in the immune response against C. albicans and are continually present in the salivary fluid and in the blood. The aim of this study was to determine ageing-related changes in salivary and blood neutrophils and their potential implications in Candida-related denture stomatitis. Results Our results showed a lower number of neutrophils in the saliva from patients presenting Candida-related denture stomatitis in comparison to their matched controls. Furthermore, fewer neutrophils were isolated from the saliva of aged control individuals in comparison to matched younger subjects. CXCR1, CD62L and CD11b expression were significantly greater on systemic neutrophils from younger control individuals. Elderly individuals showed more apoptotic salivary neutrophils and lower GM-CSF levels than younger ones, regardless of the occurrence of Candida infection. On the other hand, CXCL-8 concentrations were higher in the saliva from elderly individuals. Besides, TNF-α was detected at elevated levels in the saliva from infected elderly subjects. Salivary neutrophils from elderly and young patients presented impaired phagocytic activity against C. albicans. However, just systemic neutrophils from elderly showed decreased phagocytosis when compared to the younger ones, regardless of the occurrence of infection. In addition, neutrophils from aged individuals and young patients presented low fungicidal activity. Conclusion The data suggests that the Candida related-denture stomatitis is associated to neutrophils function deficiency, and ageing drastically appears to alter important characteristics of such cells, facilitating the establishment of this infection.

Gastroesophageal reflux, dental erosion, and halitosis in epidemiological surveys: a systematic review

In published studies, it has been suggested that dental wear is associated with gastroesophageal reflux disease (GERD). This systematic review was carried out to evaluate the association of GERD, dental erosion, and halitosis and to compare the indices adopted in epidemiological surveys. The Medline database (until October, 2011) was searched systematically to identify studies evaluating the prevalence of oral alterations, such as dental erosion and halitosis, in patients with GERD symptoms. Two reviewers analyzed all reports and the selected studies were evaluated according to the quality of evidence, using the validated Newcastle–Ottawa Quality Assessment Scale. Full-text copies of a total of 32 publications were obtained in duplicate. Sixteen publications were identified among the citations in the Bibliographic lists of studies that fulfilled the exclusion/inclusion criteria and quality of evidence. The relationship between dental erosion and GERD patients was significant in only seven studies. According to three studies, halitosis could be one of several extraesophageal symptoms or manifestations in GERD patients. In one study, it was found that the mucosa of GERD patients was significantly more acidic in comparison with that of the control group. This systematic review showed that there is a relationship between GERD and oral diseases (dental erosion and halitosis). The epidemiological surveys used different indices to analyze GERD and dental erosion. Further research could investigate the best method for assessing the two diseases.

Cause-specific mortality and income inequality in São Paulo, Brazil

OBJECTIVE: To analyze cause-specific mortality rates according to the relative income hypothesis. METHODS: All 96 administrative areas of the city of São Paulo, southeastern Brazil, were divided into two groups based on the Gini coefficient of income inequality: high (>0.25) and low (<0.25). The propensity score matching method was applied to control for confounders associated with socioeconomic differences among areas. RESULTS: The difference between high and low income inequality areas was statistically significant for homicide (8.57 per 10,000; 95%CI: 2.60;14.53); ischemic heart disease (5.47 per 10,000 [95%CI 0.76;10.17]); HIV/AIDS (3.58 per 10,000 [95%CI 0.58;6.57]); and respiratory diseases (3.56 per 10,000 [95%CI 0.18;6.94]). The ten most common causes of death accounted for 72.30% of the mortality difference. Infant mortality also had significantly higher age-adjusted rates in high inequality areas (2.80 per 10,000 [95%CI 0.86;4.74]), as well as among males (27.37 per 10,000 [95%CI 6.19;48.55]) and females (15.07 per 10,000 [95%CI 3.65;26.48]). CONCLUSIONS: The study results support the relative income hypothesis. After propensity score matching cause-specific mortality rates was higher in more unequal areas. Studies on income inequality in smaller areas should take proper accounting of heterogeneity of social and demographic characteristics.

Mitochondrial translation in health and disease

Mitochondrial disorders have become the most common cause of inborn errors of metabolism. Impairments in mitochondrial protein synthesis are one of the causes of these diseases, which are clinically and genetically heterogeneous. The mitochondrial translation machinery decodes 13 polypeptides essential for the oxidative phosphorylation process. Mitochondria protein synthesis depends on the integrity of mitochondrial rRNAs and tRNAs genes, and at least one hundred of nuclear encoded products. Diseases caused by mutations in mitochondrial genes as well as in ribosomal proteins, translational factors, RNA modifying enzymes, and all other constituents of the translational machinery have been described in patients with combine respiratory chain deficiency, and are the object of this review.

Dietary intake of environmentally persistent plaguicides in the european population

[EN] Global pollution has become one of the most important problems of the modern societies and pesticides play a major role among the chemical contaminants that are released to the environment every year. Organochlorine compounds have been widely used in the past all over the world as pesticides and some of them are still used for the control of vectors of infectious diseases. Due to their high lipophilicity, stability and resistance to degradation, most of them have been banned or strictly regulated but their levels remain high in the environment as persistent organic pollutants (POPs). Their presence in the environment leads to their introduction into the food chain, especially affecting food of animal origin with higher fat content. Due to their liposolubility, these substances tend to be bioaccumulated into the fat tissues of living beings along their entire lives, and to be biomagnificated across trophic levels in the food chain. Thus, the main human source of these pesticides comes from the daily intake of contaminated food from environmental sources. It has been established the role of many of the POPs as endocrine disruptors and even as carcinogenic agents, being thus considered as important risk factors for health. Moreover, chronic exposure to pesticides, even at very low levels, has been related with cancer, fertility problems, immunosuppression and other diseases. For this reason, it is important to know the level of POPs contamination and to identify the main sources contributing to it. Fish, meat and dairy products are the food groups with the highest levels of pollution. However, the residue levels vary by geographic area, and are influenced by several factors as food security policies, local pesticide use patterns, environmental practices, or the time when the study is conducted. Food habits, conditioned by different social and economic factors, as well as several biological factors (i.e. gender, sex and age), determine the levels of POPs exposure in the population. The purpose of this chapter is to give an overview of the dietary intake of environmentally persistent pesticides by the European population, and the potential adverse consequences of this exposure on human health.

The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Characterization of primary biogenic aerosol particles by DNA analysis: Diversity of airborne Ascomycota and Basidiomycota

Primary biogenic aerosol (PBA) particles account for large proportions of air particulate matter, and they can influence the hydrological cycle and climate as nuclei for water droplets and ice crystals in clouds, fog, and precipitation. Moreover, they can cause or enhance human, animal, and plant diseases. The actual abundance and properties of PBA particles and components in the atmosphere are, however, still poorly understood and quantified. rnIn this study, the identity, diversity, and frequency of occurrence of PBA particles were investigated by DNA analysis. Methods for the extraction, amplification, and analysis of DNA from aerosol filter samples were developed and optimized for different types of organisms, including fungi, bacteria, and plants. The investigations were focused on fungal DNA, and over 2500 sequences were obtained from air samples collected at different locations and climatic zones around the world (tropical, mid-latitude, sub-polar; continental, marine). rnNearly all fungal DNA sequences could be attributed to the phyla of Ascomycota and Basidiomycota. With regard to species richness, the ratio of Basidiomycota to Ascomycota was much higher in continental air samples (~60:40) than in marine air samples (~30:70). Pronounced differences in the relative abundance and seasonal cycles of various groups of fungi were detected in coarse and fine particulate matter from continental air, with more plant pathogens in the coarse and more human pathogens and allergens in the respirable fine particle fraction (<3 µm). The results of this study provide new information and insights into the sources of PBA particles and the interactions of the biosphere with the atmosphere, climate, and public health. rn

Bioinformatische Identifizierung und Charakterisierung von Zyklin-abhängigen Kinasen und Zyklinen des Parasiten Eimeria tenella und deren Nutzung im rationalen Wirkstoffdesign

Parasiten der Apicomplexa umfassen sowohl humanpathogene, als auch tierpathogene Protozoen. Beispiele für wichtige Vertreter human- und tierpathogener Parasiten sind Plasmodium falciparum und Eimeria tenella. E. tenella verursacht die Kokzidiose des Hühnchens, eine Darmerkrankung die weltweit für Verluste in einer geschätzten Höhe von bis zu 3 Milliarden US$ verantwortlich zeichnet. Eine prophylaktische Vakzinierung gegen diese Krankheit ist ökonomisch meist ineffizient, und eine Behandlung mit Kokzidiostatika wird durch häufige Resistenzbildung gegen bekannte Wirkstoffe erschwert. Diese Situation erfordert die Entwicklung neuer kostengünstiger Alternativen. Geeignete Zielproteine für die Entwicklung neuartiger Arzneistoffe zur Behandlung der Kokzidiose sind die Zyklin-abhängigen Kinasen (CDKs), zu denen auch die CDK-related Kinase 2 (EtCRK2) aus E. tenella gehört. Diese Proteine sind maßgeblich an der Regulation des Zellzyklus beteiligt. Durch chemische Validierung mit dem CDK Inhibitor Flavopiridol konnte nachgewiesen werden, dass ein Funktionsverlust von CDKs in E. tenella die Vermehrung des Parasiten in Zellkultur inhibiert. E. tenella CDKs sind daher als Zielproteine für die Entwicklung einer Chemotherapie der Kokzidiose geeignet. Mittels bioinformatischer Tiefenanalysen sollten CDK Proteine im Parasiten E. tenella identifiziert werden. Das Genom von E. tenella liegt in Rohfassung vor [ftp://ftp.sanger.ac.uk]. Jedoch waren zum Zeitpunkt dieser Arbeiten viele Sequenzen des Genoms noch nicht annotiert. Homologe CDK Proteine von E. tenella konnten durch den Vergleich von Sequenzinformationen mit anderen Organismen der Apicomplexa identifiziert und analysiert werden. Durch diese Analysen konnten neben der bereits bekannten EtCRK2, drei weitere, bislang nicht annotierte CDKs in E. tenella identifiziert werden (EtCRK1, EtCRK3 sowie EtMRK). Darüber hinaus wurde eine Analyse der entsprechenden Zykline – der Aktivatoren der CDKs – bezüglich Funktion und Struktur, sowie eine Datenbanksuche nach bisher nicht beschriebenen Zyklinen in E. tenella durchgeführt. Diese Suchen ergaben vier neue potentielle Zykline für E. tenella, wovon EtCYC3a als Aktivator der EtCRK2 von María L. Suárez Fernández (Intervet Innovation GmbH, Schwabenheim) bestätigt werden konnte. Sequenzvergleiche lassen vermuten, dass auch EtCYC1 und EtCYC3b in der Lage sind, EtCRK2 zu aktivieren. Außerdem ist anzunehmen, dass EtCYC4 als Aktivator der EtCRK1 fungiert. Ein weiterer Schwerpunkt der vorliegenden Arbeit war die Suche und Optimierung nach neuen Inhibitoren von CDKs aus E. tenella. In vorangegangenen Arbeiten konnten bereits Inhibitoren der EtCRK2 gefunden werden [BEYER, 2007]. Mittels Substruktur- und Ähnlichkeitssuchen konnten im Rahmen dieser Arbeit weitere Inhibitoren der EtCRK2 identifiziert werden. Vier dieser Strukturklassen erfüllen die Kriterien einer Leitstruktur. Eine dieser Leitstrukturen gehört zur Strukturklasse der Benzimidazol-Carbonitrile und ist bislang nicht als Inhibitor anderer Kinasen beschrieben. Diese neu identifizierte Leitstruktur konnte in silico weiter optimiert werden. Im Rahmen dieser Arbeit wurden Bindungsenergien von Vertretern dieser Strukturklasse berechnet, um einen wahrscheinlichen Bindemodus vorherzusagen. Für die weiterführende in silico Optimierung wurde eine virtuelle kombinatorische Substanzbibliothek dieser Klasse erstellt. Die Auswahl geeigneter Verbindungen für eine chemische Synthese erfolgte durch molekulares Docking unter Nutzung von Homologiemodellen der EtCRK2. Darüber hinaus wurde ein in silico Screening nach potentiellen Inhibitoren der PfMRK und EtMRK durchgeführt. Dabei konnten weitere interessante virtuelle Hit-Strukturen aus einer Substanzdatenbank kommerziell erhältlicher Verbindungen gefunden werden. Durch dieses virtuelle Screening konnten jeweils sieben Verbindungen als virtuelle Hits der PfMRK sowie der EtMRK identifiziert werden. Die Häufung von Strukturklassen mit bekannter CDK Aktivität deutet darauf hin, dass während des virtuellen Screenings eine Anreicherung von CDK Inhibitoren stattgefunden hat. Diese Ergebnisse lassen auf eine Weiterentwicklung neuer Wirkstoffe gegen Kokzidiose und Malaria hoffen.

Study of molecular mechanisms in cardio- and neuroprotection and possibility of modulation by nutraceutical phytocomponents

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules. This adaptation occurs in a biphasic pattern: an early phase which develops after 1-2 h, and a late phase that develops after 12-24 h. While it is widely accepted that reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. Methylglyoxal, a metabolic compound formed mainly from the glycolytic intermediate glyceraldehyde-3-phosphate., is a precursor of advanced glycation end product (AGEs) .It is more reactive than glucose and shows a stronger ability to cross-link with protein amino groups to form AGEs. Methylglyoxal induced cytotoxicity may be at least partially responsible for cardiovascular and Alzheimer diseases. Methylglyoxal omeostasis is controlled by the glyoxalase system that consists of two enzyme, glyoxalase 1 (GLO1) and glyoxalase 2. In a recent study it was demonstrated that the transcriptional levels of GLO1 are controlled by NF-E2-related factor 2 (Nrf2). The isothiocyanate sulforaphane, derived from the hydrolysis of glucoraphanin abundantly present in broccoli, represents one of the most potent inducers of phase II enzymes through the Keap1–Nrf2 pathway. The aim of this thesis was evaluated molecular mechanisms in cardio- and neuroprotection and the possibility of modulation by nutraceutical phytocomponents This thesis show to one side that the protection induced by H2O2 is mediated by detoxifying and antioxidant phase II enzymes induction, regulated, not only by transcriptional factor Nrf2, but also by Nrf1; on the other side our data represent an innovative result because for the first time it was demonstrated the possibility of inducing GLO1 by SF supplementation.

Detection and molecular characterization of viruses infecting Actinidia spp.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

New analytical LC-mass spectrometry methodologies for the quali-quantitative determination of natural substances and drugs in complex matrices

This thesis reports an integrated analytical and physicochemical approach for the study of natural substances and new drugs based on mass spectrometry techniques combined with liquid chromatography. In particular, Chapter 1 concerns the study of Berberine a natural substance with pharmacological activity for the treatment of hepatobiliary and intestinal diseases. The first part focused on the relationships between physicochemical properties, pharmacokinetics and metabolism of Berberine and its metabolites. For this purpose a sensitive HPLC-ES-MS/MS method have been developed, validated and used to determine these compounds during their physicochemical properties studies and plasma levels of berberine and its metabolites including berberrubine(M1), demethylenberberine(M3), and jatrorrhizine(M4) in humans. Data show that M1, could have an efficient intestinal absorption by passive diffusion due to a keto-enol tautomerism confirmed by NMR studies and its higher plasma concentration. In the second part of Chapter 1, a comparison between M1 and BBR in vivo biodistribution in rat has been studied. In Chapter 2 a new HPLC-ES-MS/MS method for the simultaneous determination and quantification of glucosinolates, as glucoraphanin, glucoerucin and sinigrin, and isothiocyanates, as sulforaphane and erucin, has developed and validated. This method has been used for the analysis of functional foods enriched with vegetable extracts. Chapter 3 focused on a physicochemical study of the interaction between the bile acid sequestrants used in the treatment of hypercholesterolemia including colesevelam and cholestyramine with obeticolic acid (OCA), potent agonist of nuclear receptor farnesoid X (FXR). In particular, a new experimental model for the determination of equilibrium binding isotherm was developed. Chapter 4 focused on methodological aspects of new hard ionization coupled with liquid chromatography (Direct-EI-UHPLC-MS) not yet commercially available and potentially useful for qualitative analysis and for “transparent” molecules to soft ionization techniques. This method was applied to the analysis of several steroid derivatives.

Einfluss des Treg-Aktivierungsmarkers GARP auf die Differenzierung und Funktion von T-Zellen

CD4+CD25+FoxP3+ regulatorische T-Zellen (Treg) spielen eine essentielle Rolle bei der Unterdrückung von schädlichen Immunreaktionen. Da aktivierte CD4+ T-Helferzellen auch CD25 und FoxP3 exprimieren, können diese nicht als spezifische Marker zur Identifikation von Treg verwendet werden. Die Analyse der Membranproteinexpression beider Populationen führte zur Identifikation von GARP (glycoprotein A repetitions predominant) als spezifischer Marker auf aktivierten Treg. GARP bindet LAP und TGF-beta, welches für die Unterdrückung von entzündlichen T-Zellantworten von Bedeutung ist. Um die Funktion von GARP unabhängig von Treg zu untersuchen, wurde ein lösliches GARP Protein (sGARP) synthetisiert und sein Effekt auf die Aktivierung und Differenzierung von humanen T-Zellen untersucht. Die Ergebnisse zeigen, dass sGARP die Proliferation von naiven CD4+ T-Zellen supprimiert und zu einer Phosphorylierung von SMAD2/3 sowie zu der Induktion von FoxP3 führt. Zusätzlich inhibiert sGARP die Produktion von Effektorzytokinen wie IL-2 und IFN-gamma. Die Stimulation von naiven CD4+ T-Zellen mit sGARP induziert die Differenzierung zu Treg, welche in Kokultur die Aktivierung von T-Effektorzellen supprimieren. Die Wirkung war vergleichbar in naiven CD4+ und ruhenden CD4+CD45RA+ T-Zellen, konnte aber in differenzierten CD4+CD45RO+ T-Zellen nicht nachgewiesen werden. Die Induktion von FoxP3 und die Phosphorylierung von SMAD2/3 konnte durch eine Blockade des TGF-beta-Signalweges inhibiert werden. Dies lässt vermuten, dass die Funktion von sGARP zumindest teilweise von TGF-beta abhängig ist. Zusätzlich zu seiner passiven Rolle als TGF-beta-Transporter, induzierte sGARP die TGF-beta-Produktion in naiven T-Zellen und trägt so zum Mechanismus der infektiösen Toleranz bei. Des Weiteren fördert die Stimulation von sGARP in Anwesenheit von IL-6 und IL-23 die Differenzierung zu Th17 Zellen. rnNeben dem Einfluss von sGARP auf die Differenzierung von CD4+ T-Zellen, supprimiert sGARP die Proliferation und Granzyme B-Expression in CD8+ T-Zellen. rnFür die Analyse der immunmodulatorischen Funktion von sGARP in vivo wurde ein Modell einer xenogenen GvHD (graft-versus-host disease) verwendet. Der Transfer von humanen PBMC in neugeborene, immundefiziente Rag2-/-gamma-chain-/--Mäuse führt zu einer letalen GvHD, welche durch die Applikation von humanen Treg dosisabhängig unterdrückt werden kann. In diesem Modell konnte die repetitive Gabe von sGARP, ohne zusätzliche Zugabe von Treg, ebenfalls die GvHD unterdrücken. Dies lässt auf einen synergistischen Effekt von sGARP und Treg bei der Suppression inflammatorischer T-Zellantworten schließen. rnZusammengefasst lassen die Ergebnisse auf eine entscheidende Rolle von GARP in der Modulation der peripheren Toleranz folgern und zeigen sGARP als potentes Biological für die Behandlung von unerwünschten inflammatorischen Immunantworten.