Preliminary study of Porcine circovirus type 2 and Torque teno sus virus coinfection frequencies in Brazilian pig herds

Torque teno sus virus (TTSuV) is emergent in swine herds. Recent studies have shown an increased frequency of TTSuV2 in Porcine circovirus type 2 (PCV2)-associated diseases (PCVAD), which are endemic in many swine-producing countries, including Brazil. Coinfection with several other viral and bacterial agents results in an increased incidence of more severe PCVAD. Given the limited information on TTSuV and PCV2 coinfection, especially in Brazilian swine herds, this study made a preliminary estimation of the occurrence of coinfection in swine herds by testing samples from different categories. Between 2008 and 2009, 111 samples of feces and 23 serum samples from 5 swine herds were tested for PCV2 and TTSuVs and the results analyzed for associations between these agents. No significant differences in coinfection frequency were observed for PCV2 1 + TTSuV1 or for PCV2 1 TTSuV2 between nursery piglets (P = 0.730), growing pigs (P = 0.331), or sows (P = 0.472). However, a significant difference was observed for PCV2 1 TTSuV1 1 TTSuV2 between nursery piglets and growing pigs (P = 0.004; Fisher's exact test). Phylogenetic studies agreed with the grouping of TTSuV1 and TTSuV2 into 2 different clades, with no distinct pattern of clustering of these isolates with the animal categories.

Construction and validation of an instrument that breaks the silence: the impact of domestic and/or sexual violence on women's health, as shown during climacterium

Objective: The aim of this study was to construct and to validate a measure of the consequences of domestic violence on women's health during climacterium. Methods: A questionnaire was administered at the Outpatient Climacterium Clinic to 124 women aged 40 to 65 years who were the victims of domestic and/or sexual violence (experimental group). They were divided into three groups: (1) those who were victims of violence exclusively during childhood/adolescence, (2) those who were victims of violence exclusively during adulthood, and (3) those who were victims of violence throughout their lives. The instrument included 34 items evaluating the beginning, frequency, and type of violence; the search for health assistance and reporting of the violence; the violence and the number of comorbidities; and violence and the Kupperman Menopausal Index. We also included a control group composed of perimenopausal and postmenopausal women who did not experience any violence (n = 120). Results: The instrument presented a Cronbach alpha = 0.82, good reliability among the examiners (+0.80), and a good possibility of reproducibility. The mean age of menopause was 45.4 years, and the mean age in the control group was 48.1 years. Group 1 showed a mean of 5.1 comorbidities, Group 2 had 4.6, and Group 3 had 4.4. Sexual violence (43.5%) and other types of violence both presented average comorbidities (4.60) but represented a significant impairment in the victim's sexual life. There were significant associations in group 3 and a high Kupperman Menopausal Index score. In the experimental group, 80.6% did not seek health services for the violence they experienced. Conclusions: The questionnaire presented good internal consistency and a validated construction. It can be easily reproduced and is indicated to evaluate the consequences of domestic and/or sexual violence on women's health during climacterium.

Pre-and post-puberty physiological plasma oxytocin concentrations in male domestic cats (Felis silvestris catus)

The hormone oxytocin is released by the neuropituitary gland through stimulation of the neurons of the supraoptic and paraventricular nuclei of the hypothalamus. In order to determine the physiological concentrations of this hormone in domestic cats, blood samples were collected from 15 male animals (Felis silvestris catus) during the pre- and post-puberty periods (at four and eight months of age, respectively). Oxytocin determination was accomplished by radioimmunoassay. The average oxytocin concentrations measured in the pre- and post-puberty periods were 2.54±0.24 (μg/dL) and 2.53±0.28 (μg/dL), respectively, and there were no statistical differences between these measurements. Because there are few literature on the analysis of this hormone, especially in the case of male Felis silvestris catus, more studies on the influence of oxytocin on the physiology and reproduction of this species should be conducted under maintenance and situations of stress (such as transportation), and other routine events.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from São Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes, using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.

Molecular surveillance of the Newcastle disease virus in domestic and wild birds on the North Eastern Coast and Amazon biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

Isolation of Sporothrix schenckii from the claws of domestic cats (indoor and outdoor) and in captivity in São Paulo (Brazil)

Sporotrichosis is a subcutaneous mycosis and is also a zoonosis (sapro- and anthropozoonosis). The objective of the present study was to determine the occurrence of sporotrichosis in domestic cats and in wild or exotic felines in captivity through the isolation of Sporothrix spp. from claw impressions in a culture medium. The samples included 132 felines, of which 120 (91.0 %) were domestic cats, 11 (8.3 %) were wild felines, and one (0.7 %) was an exotic felid. Twenty-one (17.5 %) were outdoor cats. Of the total, 89 (67.4 %) had contact with other animals of the same species. It was possible to isolate Sporothrix schenckii from the claws of one (0.7 %) of the felids probed; this animal exhibited generalised sporotrichosis and had infected a female veterinarian. The potential pathogenic agents Microsporum canis and Malassezia pachydermatis were isolated in 12.1 and 5.3 % of the animals, respectively. The following anemophilous fungi, which were considered to be contaminants, were also isolated: Penicillium sp. (28 or 21.2 %), Aspergillus sp. (13 or 9.8 %), Rhodotorula sp. (5 or 3.8 %), Candida sp. (5 or 3.8 %), Trichoderma sp. (1 or 0.7 %), and Acremonium sp. (1 or 0.7 %). Due to the low magnitude of occurrence (0.7 %) of Sporothrix in feline claws, the potential of the cats evaluated in this study to be sources of infection in the city of São Paulo is considerably low.

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

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

A study on the fluid dynamics of domestic Gas burners

Domestic gas burners are investigated experimentally and numerically in order to further understand the fluid dynamics processes that drive the cooking appliance performances. In particular, a numerical simulation tool has been developed in order to predict the onset of two flame instabilities which may deteriorate the performances of the burner: the flame back and flame lift. The numerical model has been firstly validated by comparing the simulated flow field with a data set of experimental measurements. A prediction criterion for the flame back instability has been formulated based on isothermal simulations without involving the combustion modelization. This analysis has been verified by a Design Of Experiments investigation performed on different burner prototype geometries. On the contrary, the formulation of a prediction criterion regarding the flame lift instability has required the use of a combustion model in the numerical code. In this analysis, the structure and aerodynamics of the flame generated by a cooking appliance has thus been characterized by experimental and numerical investigations, in which, by varying the flow inlet conditions, the flame behaviour was studied from a stable reference case toward a complete blow-out.

Mechanosensitivity in the myenteric plexus of the guinea pig ileum

The enteric nervous system regulates autonomously from the central nervous system all the reflex pathways that control blood flow, motility, water and electrolyte transport and acid secretion. The ability of the gut to function in isolation is one of the most intriguing phenomenons in neurogastroenterology. This requires coding of sensory stimuli by cells in the gut wall. Enteric neurons are prominent candidates to relay mechanosensitivity. Surprisingly, the identity of mechanosensitive neurons in the enteric nervous system as well as the appropriate stimulus modality is unknown despite the evidence that enteric neurons respond to sustained distension. Objectives: The aim of our study was to record from mechanosensitive neurons using physiological stimulus modalities. Identification of sensory neurons is of central importance to understand sensory transmission under normal conditions and in gut diseases associated with sensorimotor dysfunctions, such as Irritable Bowel Syndrome. Only then it will be possible to identify novel targets that help to normalise sensory functions. Methods: We used guinea-pig ileum myenteric plexus preparations and recorded responses of all neurons in a given ganglion with a fast neuroimaging technique based on voltage sensitive dyes. To evoke a mechanical response we used two different kinds of stimuli: firstly we applied a local mechanical distortion of the ganglion surface with von Frey hair. Secondarily we mimic the ganglia deformation during physiological movements of myenteric ganglia in a freely contracting ileal preparation. We were able to reliably and reproducibly mimic this distortion by intraganglionic injections of small volumes of oxygenated and buffered Krebs solution using stimulus parameters that correspond to single contractions. We also performed in every ganglion tested, electrical stimulations to evoke fast excitatory postsynaptic potentials. Immunohistochemistry reactions were done with antibodies against Calbindin and NeuN, considered markers for sensory neurons. Results: Recordings were performed in 46 ganglia from 31 guinea pigs. In every ganglion tested we found from 1 to 21 (from 3% to 62%) responding cells with a median value of 7 (24% of the total number of neurons). The response consisted of an almost instantaneous spike discharge that showed adaptation. The median value of the action potential frequency in the responding neurons was 2.0 Hz, with a recording time of 1255 ms. The spike discharge lasted for 302 ± 231 ms and occurred only during the initial deformation phase. During sustained deformation no spike discharge was observed. The response was reproducible and was a direct activation of the enteric neurons since it remained after synaptic blockade with hexamethonium or ω-conotoxin and after long time perfusion with capsaicin. Muscle tone appears not to be required for activation of mechanosensory neurons. Mechanosensory neurons showed a response to mechanical stimulation related to the stimulus strength. All mechanosensory neurons received fast synaptic inputs. There was no correlation between mechanosensitivity and Calbindin-IR and NeuN-IR (44% of mechanosensitive neurones Calb-IR-/NeuN-IR-). Conclusions: We identified mechanosensitive neurons in the myenteric plexus of the guinea pig ileum which responded to brief deformation. These cells appear to be rapidly accommodating neurons which respond to dynamic change. All mechanosensitive neurons received fast synaptic input suggesting that their activity can be highly modulated by other neurons and hence there is a low stimulus fidelity which allows adjusting the gain in a sensory network. Mechanosensitivity appears to be a common feature of many enteric neurons belonging to different functional classes. This supports the existence of multifunctional enteric neurons which may fulfil sensory, integrative and motor functions.

Detection and localization of GLUTs in spermatozoa from different domestic species

Sperm cells need hexoses as a substrate for their function, for both the maintenance of membrane homeostasis and the movement of the tail. These cells have a peculiar metabolism that has not yet been fully understood, but it is clear that they obtain energy from hexoses through glycolisis and/or oxidative phosphorylation. Spermatozoa are in contact with different external environments, beginning from the testicular and epididymal fluid, passing to the seminal plasma and finally to the female genital tract fluids; in addition, with the spread of reproductive biotechnologies, sperm cells are diluted and stored in various media, containing different energetic substrates. To utilize these energetic sources, sperm cells, as other eukaryotic cells, have a well-constructed protein system, that is mainly represented by the GLUT family proteins. These transporters have a membrane-spanning α-helix structure and work as an enzymatic pump that permit a fast gradient dependent passage of sugar molecules through the lipidic bilayer of sperm membrane. Many GLUTs have been studied in man, bull and rat spermatozoa; the presence of some GLUTs has been also demonstrated in boar and dog spermatozoa. The aims of the present study were - to determine the presence of GLUTs 1, 2, 3, 4 and 5 in boar, horse, dog and donkey spermatozoa and to describe their localization; - to study eventual changes in GLUTs location after capacitation and acrosome reaction in boar, stallion and dog spermatozoa; - to determine possible changes in GLUTs localization after capacitation induced by insulin and IGF stimulation in boar spermatozoa; - to evaluate changes in GLUTs localization after flow-cytometric sex sorting in boar sperm cells. GLUTs 1, 2, 3 and 5 presence and localization have been demonstrated in boar, stallion, dog and donkey spermatozoa by western blotting and immunofluorescence analysis; a relocation in GLUTs after capacitation has been observed only in dog sperm cells, while no changes have been observed in the other species examined. As for boar, the stimulation of the capacitation with insulin and IGF didn’t cause any change in GLUTs localization, as well as for the flow cytometric sorting procedure. In conclusion, this study confirms the presence of GLUTs 1, 2 ,3 and 5 in boar, dog, stallion and donkey spermatozoa, while GLUT 4 seems to be absent, as a confirmation of other studies. Only in dog sperm cells capacitating conditions induce a change in GLUTs distribution, even if the physiological role of these changes should be deepened.

The EU and Dispute Settlement within the WTO: a strategy for the protection of the Union’s autonomy in the domestic regulation of goods?

Oggetto della ricerca è l’accertamento dell’esistenza, nonché la definizione, della strategia dall’UE in materia di controversie commerciali aventi ad oggetto l’interpretazione e l’applicazione di norme facenti capo agli Accordi OMC in materia di misure sanitarie e di barriere tecniche al commercio. Nella prima parte della tesi, si ricostruiscono gli obbiettivi perseguiti dall’UE in materia di controversie SPS e TBT. In questo contesto, un’importanza di primo piano è attribuita alla difesa dell’autonomia regolamentare dell’Unione. Ad essa si riconduce la prassi UE finalizzata a prevenire il sorgere di controversie sul piano bilaterale attraverso la conclusione di accordi di mutuo riconoscimento, la cui portata ella sottolinea essere tuttavia limitata. L’analisi di cinque controversie sorte in ambito OMC di cui l’Unione è o è stata parte convenuta e che si fondano su presunte o accertate violazioni delle norme facenti capo ai due accordi menzionati consente di classificare gli argomenti giuridici avanzati dall’Unione nel contesto di tali controversie. Nella seconda parte della ricerca, la candidata identifica i mezzi a servizio della strategia UE, in primo luogo, attraverso l’analisi del quadro giuridico relativo alla partecipazione dell’Unione e degli Stati Membri al sistema OMC di risoluzione delle controversie; in secondo luogo, attraverso lo studio, da un lato, dello status delle norme OMC nell’ordinamento UE e, dall’altro, degli effetti delle pronunce dell’Organo di Risoluzione delle Controversie e della questione della responsabilità dell’Unione per violazione del diritto OMC. Sulla base del lavoro di ricerca svolto, si conclude che una strategia dell’UE esiste nella misura in cui l’Unione persegue l’obbiettivo di preservare la propria autonomia regolamentare attraverso, anche se non esclusivamente, gli strumenti afferenti all’ordine giuridico interno analizzati nella seconda parte. La candidata conclude altresì che la riforma del diritto delle relazioni esterne operata dal Trattato di Lisbona può indurre un cambiamento di tale strategia.

Do domestic interests matter? – Der Einfluss nationaler Interessengruppen auf die Position von Staaten zur Umweltpolitik auf EU-Ebene

Wie kann man Ergebnisse internationaler Umweltpolitik erklären? Wie hilfreich ist dabei die liberale Theorie der Internationalen Beziehungen (IB) von Andrew Moravcsik? Die vorliegende Arbeit versucht diese Fragen anhand eines Fallbeispiels internationaler Umweltpolitik – der Position der Bundesrepublik Deutschland bezüglich der einzelnen Streitfragen der EU-Verordnung 443/2009 über den CO2-Ausstoß von Automobilen – zu beantworten. Es wird eine theoriegeleitete Außenpolitikanalyse durchgeführt, deren Hauptaugenmerk auf der spezifischen nationalen Präferenzbildung in einem bestimmten Fall liegt. Hier weist Moravcsiks Theorie eine „Lücke“ auf. Wessen Interessen sich aus welchen Gründen in einer bestimmten Situation wie durchsetzen und damit Politik beeinflussen bleibt unklar. Deshalb erweitert die Arbeit Moravcsiks liberale Theorie der IB mithilfe von Annahmen und Erkenntnissen aus der Verbändeforschung nach innen. Auf diese Weise werden die situationsspezifischen Interessen und die situationsspezifische Durchsetzungsfähigkeit der betroffenen Akteure – nationale Interessengruppen – erhoben und untersucht, inwiefern man mit ihrer Hilfe die deutsche Position zur EU-Verordnung 443/2009 erklären kann. Empirisch erweist sich dabei, dass die Position der BRD zu acht von neun Streitfragen der EU-Verordnung 443/2009 den Interessen einer Koalition aus Industriegewerkschaft (IG) Metall und Verband der Automobilindustrie (VDA) entsprach, weil diese im vorliegenden Fall mit Abstand die größte Durchsetzungsfähigkeit aufwiesen. Lediglich bezüglich einer Streitfrage wich die Position der BRD von den Interessen von IG Metall und VDA ab. Damit lässt sich festhalten: Die Position der BRD zur EU-Verordnung 443/2009 kann weitgehend mithilfe der nach innen erweiterten liberalen Theorie nach Andrew Moravcsik erklärt werden. Trotz möglicher Schwierigkeiten bei der Übertragung erscheint daher eine Anwendung des nach innen erweiterten Liberalismus auf weitere erklärungsbedürftige Phänomene der internationalen Umweltpolitik und damit eine Überprüfung der Theorie insgesamt eine interessante und sinnvolle Aufgabe zu sein.

Genomic and proteomic approaches in pig meat quality research field

Pig meat and carcass quality is a complex concept determined by environmental and genetic factors concurring to the phenotypic variation in qualitative characteristics of meat (fat content, tenderness, juiciness, flavor,etc). This thesis shows the results of different investigations to study and to analyze pig meat and carcass quality focusing mainly on genomic; moreover proteomic approach has been also used. The aim was to analyze data from association studies between genes considered as candidate and meat and carcass quality in different pig breeds. The approach was used to detect new SNP in genes functionally associated to the studied traits and to confirm as candidate other genes already known. Five polymorphisms (one new SNP in Calponin 1 gene and four additional polymorphism already known in other genes) were considered on chromosome 2 (SSC2). Calponin 1 (CNN1) was associated to the studied traits and furthermore the results reported confirmed the data already known for Lactate dehydrogenase A (LDHA), Low density lipoprotein receptor (LDLR), Myogenic differentiation 1 (MYOD1) e Ubiquitin-like 5 (UBL5), in Italian Large White pigs. Using an in silico search it was possible to detect on SSC2 a new SNP of Deoxyhypusine synthase (DHPS) gene partially overlapping with WD repeat domain 83 (WDR83) gene and significant for the meat pH variation in Italian Large White (ILW) pigs. Perilipin 1 (PLIN1) mapping on chromosome 7 and Perilipin 2 (PLIN2) mapping on chromosome 1 were studied and the results obtained in Duroc breed have shown significant associations with carcass traits. Moreover a study of protein composition of porcine LD muscle, indicated an effect of temperature treatment of carcass, on proteins of the sarcoplasmic fraction and in particular on PGM1 phosphorylation. Future studies on pig meat quality should be based on the integration of different experimental approaches (genomics, proteomics, transcriptomics, etc).

Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential

Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims: (I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group; (II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs. It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.