Estudo da estabilidade da cirurgia do tipo sagital da mandíbula com vista a correção de deformidades faciais

This research evaluated the surgical stabilily in patients with mandibular prognathism and retrognathism in which was used sagital split technic to correct those detormities. Twelve patients were selected from the clinic of only one experienced surgeon. Six patients presenter a Class III 6 a Class II molar relationship. A comparative cefalometric analysis using linear and angular measurements was performed of pre-surgery, imediate pós-surgery and 1 year follow-up. The following conclusions were obtained. 1 The Dal Pont sagital split technic modified by Epker to correct mandibular prognathisn and retroghnatism is a stable technic and must be indicated to correct those deformities. 2 Small relapses are easily corrected by the post-surgical orthodontic treatment. 3 A small over correction is advised in cases of large mandibular advancements or set bascks. 4 In those cases which a large amount of mandibular retrusion on advancement need to be performed, a combination of maxillary and mandibular surgery should be used. Rigid fixation technic is also indicated in those cases

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation

The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

Expressions- und Funktionsanalysen von Neuroglobin und Cytoglobin

Im Rahmen der vorliegenden Dissertation wurden Untersuchungen zur Expression und Funktion der respiratorischen Proteine Neuroglobin (Ngb) und Cytoglobin (Cygb) in Vertebraten durchgeführt. Beide Globine wurden erst kürzlich entdeckt, und ihre Funktionen konnten trotz vorliegender Daten zur Struktur und biochemischen Eigenschaften dieser Proteine bisher nicht eindeutig geklärt werden. Im ersten Abschnitt der vorliegenden Arbeit wurde die zelluläre und subzelluläre Lokalisation von Neuroglobin und Cytoglobin in murinen Gewebeschnitten untersucht. Die Expression von Ngb in neuronalen und endokrinen Geweben hängt offensichtlich mit den hohen metabolischen Aktivitäten dieser Organe zusammen. Insbesondere im Gehirn konnten regionale Unterschiede in der Ngb-Expression beobachtet werden. Dabei korrelierte eine besonders starke Neuroglobin-Expression mit Gehirnbereichen, die bekanntermaßen die höchsten Grundaktivitäten aufweisen. In Anbetracht dessen liegt die Funktion des Neuroglobins möglicherweise im basalen O2-Metabolismus dieser Gewebe, wobei Ngb als O2-Lieferant und kurzfristiger O2-Speicher den vergleichsweise hohen Sauerstoffbedarf vor Ort sicherstellen könnte. Weitere Funktionen in der Entgiftung von ROS bzw. RNS oder die kürzlich publizierte mögliche Rolle des Ngb bei der Verhinderung der Mitochondrien-vermittelten Apoptose durch eine Reduktion des freigesetzten Cytochrom c wären darüber hinaus denkbar. Die Cygb-Expression im Gehirn beschränkte sich auf relativ wenige Neurone in verschiedenen Gehirnbereichen und zeigte dort vorwiegend eine Co-Lokalisation mit der neuronalen NO-Synthase. Dieser Befund legt eine Funktion des Cytoglobins im NO-Metabolismus nahe. Quantitative RT-PCR-Experimente zur mRNA-Expression von Ngb und Cygb in alternden Säugern am Bsp. der Hamsterspezies Phodopus sungorus zeigten keine signifikanten Änderungen der mRNA-Mengen beider Globine in alten im Vergleich zu jungen Tieren. Dies widerspricht publizierten Daten, in denen bei der Maus anhand von Western Blot-Analysen eine Abnahme der Neuroglobin-Menge im Alter gezeigt wurde. Möglicherweise handelt es sich hierbei um speziesspezifische Differenzen. Die im Rahmen dieser Arbeit durchgeführte vergleichende Sequenzanalyse der humanen und murinen NGB/Ngb-Genregion liefert zum einen Hinweise auf die mögliche Regulation der Ngb-Expression und zum anderen eine wichtige Grundlage für die funktionellen Analysen dieses Gens. Es konnte ein minimaler Promotorbereich definiert werden, der zusammen mit einigen konservierten regulatorischen Elementen als Basis für experimentelle Untersuchungen der Promotoraktivität in Abhängigkeit von äußeren Einflüssen dienen wird. Bioinformatische Analysen führten zur Identifizierung des sog. „neuron restrictive silencer element“ (NRSE) im Ngb-Promotor, welches vermutlich für die vorwiegend neuronale Expression des Proteins verantwortlich ist. Die kontrovers diskutierte O2-abhängige Regulation der Ngb-Expression konnte hingegen anhand der durchgeführten komparativen Sequenzanalysen nicht bestätigt werden. Es wurden keine zwischen Mensch und Maus konservierten Bindestellen für den Transkriptionsfaktor HIF-1 identifiziert, der die Expression zahlreicher hypoxieregulierter Gene, z.B. Epo und VEGF, vermittelt. Zusammen mit den in vivo-Daten spricht dies eher gegen eine Regulation der Ngb-Expression bei verminderter Verfügbarkeit von Sauerstoff. Die Komplexität der Funktionen von Ngb und Cygb im O2-Stoffwechsel der Vertebraten macht den Einsatz muriner Modellsysteme unerlässlich, die eine sukzessive Aufklärung der Funktionen beider Proteine erlauben. Die vorliegende Arbeit liefert auch dazu einen wichtigen Beitrag. Die hergestellten „gene-targeting“-Vektorkonstrukte liefern in Verbindung mit den etablierten Nachweisverfahren zur Genotypisierung von embryonalen Stammzellen die Grundlage zur erfolgreichen Generierung von Ngb-knock out sowie Ngb- und Cygb-überexprimierenden transgenen Tieren. Diese werden für die endgültige Entschlüsselung funktionell relevanter Fragestellungen von enormer Bedeutung sein.

Identifizierung, Expression und Charakterisierung des Nanos-verwandten Proteins aus dem marinen Schwamm Suberites domuncula

Im Rahmen dieser Arbeit konnte in dem marinen Schwamm Suberites domuncula ein Gen identifiziert werden, dessen C-terminale konservierte Domäne eine hohe Sequenzähnlichkeit zu den Zinkfingerdomänen der Nanos-Proteine aufweist. Weiter konnte ein N-terminales Sequenzmotiv identifiziert werden, das eine hohe Sequenzidentität zu den konservierten NIM Motiven (CNOT1-interagierendes-Motiv) von Nanos zeigt. Nach der Klonierung der cDNA erfolgte die Expression des als Sd_nrp bezeichneten Proteins in E. coli Bakterien, für dessen 231 Aminosäuren umfassende Polypeptidkette eine theoretische Molekülmasse von 25.8 kDa berechnet wurde. Anschließend gelang ein Nachweis des Proteins mithilfe eines polyklonalen, gegen Sd_nrp gerichteten Antikörpers in drei Gewebetypen, dem Pinacoderm, den Primmorphen (3D-Zellaggregate) und den Gemmulae (Dauerstadien der Schwämme). Dabei konnte die höchste Expression von Sd_nrp in den als totipotent geltenden Stammzellen der Schwämme, den Archaeocyten innerhalb der Gemmulae beobachtet werden. Die Identifizierung der Zellstrukturen, erfolgte dabei aufgrund morphologischer Vergleiche. Speziell die Merkmale der Zellen in den Gemmulae, der große Nukleus, die amöboide Form sowie die granulären Reservesubstanzen, entsprechen den typischen morphologischen Eigenschaften der Archaeocyten, und bestätigen die Interpretation der Ergebnisse. Weiter konnte mit Hilfe des Anti-Sd_nrp Antikörpers das native Protein in Proteinextrakten aus Gewebe adulter Tiere nachgewiesen werden. Die vergleichende Sequenzanalyse von Sd_nrp mit dem Nanos-verwandten Protein der Schwammspezies Ephydatia fluviatilis und die phylogenetische Stammbaum-Analyse mit Nanos-Homologen unterschiedlicher Invertebraten und Vertebraten lässt die Schlussfolgerung zu, dass es sich bei dem hier identifizierten Protein Sd_nrp um ein Nanos-verwandtes Protein handelt. Darüber hinaus konnte anhand eines Homologiemodells bestätigt werden, dass es sich bei der konservierten C-terminalen Domäne des Proteins Sd_nrp um die für Nanos-Proteine spezifische Zinkfingerstruktur mit dem konservierten Sequenzmotiv CCHC handelt. Dieses Ergebnis konnte auch bei einem Vergleich der Zinkfingerdomäne von Sd_nrp mit den Zinkfingerdomänen der Nanos-Homologen unterschiedlicher Invertebraten- und Vertebratenspezies bestätigt werden.

Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.

An autochthonous case of cutaneous bovine leishmaniasis in Switzerland

The present case report describes a novel etiological agent of cutaneous leishmaniasis that appears for the first time in a cow. A similar agent had recently been described as causing autochthonous infections in horses of Germany and Switzerland. The infection in the cow was initially diagnosed upon clinical and immunohistological findings. Subsequent comparative sequence analysis of diagnostic PCR products from the internal transcribed spacer 1 (ITS1) of ssrRNA classified the respective isolate as neither Old World nor New World Leishmania species, but yielded complete identity of the analysed sequence with the above mentioned horse cases and 98% identity to Leishmania sp. siamensis, an organism recently identified in a visceral leishmaniasis patient from Thailand. The potential transmitting vectors for all these cases have not yet been identified. Future investigations will have to elucidate the veterinary-epidemiological relevance of this etiological agent, as well as biological parameters such as transmission mode and geographical origin and distribution.

Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromosome 18

PURPOSE: To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep. METHODS: Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia-anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites. RESULTS: The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations. CONCLUSIONS: This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

Occurrence of Leishmania sp. in cutaneous lesions of horses in Central Europe

The present report describes a novel etiological agent of cutaneous leishmaniasis in horses that, at least for some cases, sporadically appeared as autochthonous infections in geographically distant regions of Germany and Switzerland. The infection was initially diagnosed upon clinical and immunohistological findings. Subsequent comparative sequence analysis of diagnostic PCR products from the internal transcribed spacer 1 (ITS1) of ssrRNA classified the respective isolates as neither Old World nor New World Leishmania species. However, four isolates subjected to molecular analyses all exhibited a close phylogenetic relationship to Leishmania sp. siamensis, an organism recently identified in a visceral leishmaniasis patient from Thailand. Future investigations will demonstrate if this form of leishmaniasis represents an emerging, and perhaps zoonotic, disease of European, or even global, importance.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

Monitoring the temporal development of natural hazard risks as a basis indicator for climate change adaptation.

The potential effects of climatic changes on natural risks are widely discussed. But the formulation of strategies for adapting risk management practice to climate changes requires knowledge of the related risks for people and economic values. The main goals of this work were (1) the development of a method for analysing and comparing risks induced by different natural hazard types, (2) highlighting the most relevant natural hazard processes and related damages, (3) the development of an information system for the monitoring of the temporal development of natural hazard risk and (4) the visualisation of the resulting information for the wider public. A comparative exposure analysis provides the basis for pointing out the hot spots of natural hazard risks in the province of Carinthia, Austria. An analysis of flood risks in all municipalities provides the basis for setting the priorities in the planning of flood protection measures. The methods form the basis for a monitoring system that periodically observes the temporal development of natural hazard risks. This makes it possible firstly to identify situations in which natural hazard risks are rising and secondly to differentiate between the most relevant factors responsible for the increasing risks. The factors that most influence the natural risks could be made evident.