A compreensão da fisiologia e dos mecanismos moleculares da sepse tem sido foco de muitos estudos. As infecções severas, como a sepse, são responsáveis por 10% do total de mortes registradas em Unidades de Tratamento Intensivo em todo o mundo. O desfecho da sepse ocorre devido a influência de fatores ambientais e genéticos, cuja expressão de variantes genéticas suportam ou não este desfecho. Muitos mecanismos estão envolvidos na sepse, incluindo a liberação de citocinas e a ativação de neutrófilos, de monócitos e de células endoteliais. Há associação entre superprodução de óxido nítrico, produção excessiva de radicais livres, depleção de antioxidantes, e déficit energético celular. Enzimas antioxidantes endógenas como a Superóxido Dismutase, a Glutationa Peroxidase e a Catalase protegem a célula do dano oxidativo. A enzima superóxido dismutase dependente de manganês é um potente antioxidante intracelular codificada por um gene (SOD2; 6q25-2) que tem sua expressão induzida por mediadores inflamatórios tais como interleucina 1, interleucina 4, interleucina 6, Fator de Necrose Tumoral – α, lipopolisacarídeos. O gene SOD2 apresenta um polimorfismo de mutação de base C47T no exon 2, o qual resulta na substituição do resíduo 16 (Ala16Val) pertencente ao peptídeo sinal da proteína. O objetivo deste trabalho foi estudar o efeito diferencial das variantes - 9Ala e -9Val da superóxido dismutase dependente de manganês sobre as células mononucleares de sangue periférico humano (in vitro) durante um processo infeccioso (induzido por lipopolisacarídeos), investigando sua implicação: (I) na produção de Espécies Reativas; (II) na atividade e imuno-conteúdo da Superóxido Dismutase dependente de Manganês; (III) na atividade e imuno- conteúdo da Catalase; (IV) na atividade e imunoconteúdo da Glutationa Peroxidase; (V) na produção de nitrotirosina; (VI) na produção de nitrito/nitrato; (VII) na liberação de Fator de Necrose Tumoral - α; (VIII) na produção de Carboximetil-lisina; (IX) dienos conjugados; (X) no imuno-conteúdo da Poli (ADP ribose) Polimerase; (XI) no imuno-conteúdo do Receptor de Produtos Avançados de Glicação; (XII) no imuno-conteúdo da Proteína de Choque Térmico; (XIII) no imuno-conteúdo do Fator Nuclear κB; (XIV) no dano ao DNA celular; (XV) na determinação das defesas antioxidantes totais não enzimáticas. Os resultados demonstraram que o polimorfismo Ala-9Val participa na regulação do ambiente redox celular, e que o alelo 47C permite que as células no estado basal (sem lipopolisacarídeos) respondam com mais eficiência ao estresse oxidativo celular. Este alelo apesar de produzir mais espécies reativas também aumenta o mecanismo de defesa antioxidante. Porém, quando em uma doença que produza estresse oxidativo, no caso a sepse, o alelo 47C torna o ambiente intracelular pró-oxidativo podendo agravar a condição celular. Em suma, os dados aqui apresentados sugerem que o polimorfismo Ala-9Val é um alvo promissor para novos estudos com o objetivo de usar marcadores genéticos para direcionar a terapia necessária para cada paciente. / The understanding of the physiology and of molecular mechanisms of sepsis has been focus of many studies. The severe infections, as the sepsis, are responsible for 10% of total of deaths registered in Intensive Care Units all over the world. The outcome of sepsis happens due to influence of environmental and genetic factors, whose the expression of genetic variants supports or not this outcome. Many mechanisms are involved in sepsis, including the cytokines liberation and the neutrophils activation, of monocytes and of endothelial cells. There is association among overproduction of nitric oxide, excessive production of free radicals, depletion of antioxidants, and cellular energy deficit. Endogenous antioxidant enzymes as Superoxide Dismutase, Glutathione Peroxidase and Catalase protect the cell of oxidative damage. The manganese superoxide dismutase enzyme it is a potent antioxidant intracellular codified by a gene (SOD2; 6q25-2) that has her expression induced by the inflammatory mediators such as interleukin 1, interleukin 4, interleukin 6, tumor necrosis factor – α, lipopolysaccharide. The SOD2 gene presents a single-nucleotide polymorphism C47T in the exon 2, which results in the substitution of the residue 16 (Ala16 Val) belonging to the signal peptide of the protein. The aim of this work was to study the differential effect of the variants -9Ala and -9Val of manganese superoxide dismutase on the Peripheral Blood Mononuclears Cells human (in vitro) during an infectious process (induced by lipopolysaccharide), investigating her implication: (I) in the production of Reactive Species; (II) in the activity and immunocontent of Manganese Superoxide Dismutase; (III) in the activity and immunocontent of Catalase; (IV) in the activity and immunocontent of Glutathione Peroxidase; (V) in the nitrotyrosine production; (VI) in the nitrite/nitrate production; (VII) in the production of tumor necrosis factor - α; (VIII) in the production of carboxymethyl lysine; (IX) conjugated dienos; (X) in the immunocontent of the Poly (ADP-ribose) Polymerase; (XI) in the immunocontent of the Receptor for Advanced Glycation Endproducts; (XII) in the immunocontent of Heat Shock Protein; (XIII) in the immunocontent of the Nuclear Factor kappa B; (XIV) in the damage to cellular DNA; (XV) in the determination of the non-enzymatic antioxidant cellular defenses. The results demonstrated that the polymorphism Ala-9Val it participates in the regulation of the cellular redox environment, and that the 47C allele allows that the cells in the basal state (without lipopolysaccharide) they answer with more efficiency to the stress oxidative cellular. This allele in spite of producing more RS also increases the mechanism of antioxidant defense. However when in a disease that produces oxidative stress, in the case the sepsis, the 47C allele turns intracellular environmental pro-oxidative could worsen the cellular condition. In summary, the data presented here suggest that the polymorphism Ala- 9Val is a promising target for new studies with the goal of using genetic markers to guide therapy required for each patient.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Monoamine oxidase (MAO) is a target enzyme in the treatment of several pathologies, being that new molecules which inhibit of a selective, potent and reversible manner their isoforms and without adverse effects are searched. In this way, the first manuscript of this dissertation evaluated the in vitro inhibitory potential of the 4-organochalcogen-isoquinolines on cerebral MAO-A and B activities, elucidating their kinetics profile and the interaction compound x enzyme. The results demonstrated that all compounds were selective inhibitors of MAO-B, being compound 3-phenyl-4-(selenophenyl) isoquinoline the most potent. The kinetics profile revealed a mixed and reversible inhibition of enzyme, consistent to the results of molecular docking. It is known that both organic selenium compounds and isoquinolines are linked to pro-oxidants situations, thus, it was investigated the in vitro effect of 4-organoseleno-isoquinolines on cerebral activities of the enzymes δ- aminolevulinate dehydratase (δ-ALA-D) e Na+, K+-ATPase, which have easily oxidized cysteine residues. Data demonstrated that compounds substituted with chloro, fluoro and trifluoromethyl in the aromatic ring bonded to the selenium atom of compound 3-phenyl-4-(selenophenyl) isoquinoline inhibited both sulfhydryl enzymes, which was not observed in the compound substituted with methyl and in a nonsubstituted compound. Furthermore, since the inhibition of enzymes δ-ALA-D and Na+, K+-ATPase was restored by dithiothreitol it is possible to propose the oxidation of cysteine residues by compounds. The selective and reversible inhibition of MAO-B and the low toxicological potential demonstrated by compound 3-phenyl-4- (selenophenyl) isoquinoline become this compound a candidate for more studies, which aim this enzyme as a therapeutic target. / A monoamina oxidase (MAO) é uma enzima alvo no tratamento de diversas patologias, sendo que novas moléculas que a inibam de maneira seletiva, potente, reversível, e ausente de efeitos adversos suas isoformas são procuradas. Neste sentido, o primeiro manuscrito desta dissertação avaliou o potencial inibitório dos 4- organocalcogeno-isoquinolinas na atividade cerebral da MAO-A e B in vitro, elucidando seus perfis cinéticos e a interação composto e enzima. Os resultados demonstram que todos os compostos apresentam inibição seletiva da MAO-B, sendo o composto 3-fenil-4-(selenofenil) isoquinolina o mais potente. O perfil cinético revelou inibição do tipo mista e reversível da enzima, coerente aos resultados do docking molecular. Sabe-se que tanto compostos orgânicos de selênio quanto isoquinolinas relacionam-se a situações pró-oxidantes, deste modo, investigou-se o efeito in vitro dos 4-organoseleno-isoquinolinas na atividade cerebral das enzimas δ- aminolevulinato dehidratase (δ-ALA-D) e Na+, K+-ATPase, as quais possuem resíduos de cisteína facilmente oxidáveis. Os dados demonstram que os compostos substituídos com cloro, flúor e trifluormetil no anel aromático ligado ao átomo de Se do composto 3-fenil-4-(selenofenil) isoquinolina inibem ambas as enzimas sulfidrílicas, o que não foi observado com o composto substituído com metil e com o composto não substituído. Além disso, visto que a inibição das enzimas δ-ALA-D e Na+, K+-ATPase foi revertida por ditiotreitol é possível propor o envolvimento da oxidação dos resíduos de cisteína pelos compostos. Devido à inibição seletiva e reversível da MAO-B e ao baixo potencial toxicológico demonstrado, o composto 3- fenil-4-(selenofenil) isoquinolina torna-se um candidato a mais estudos que possuam esta enzima como alvo terapêutico.

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

No description available.

No description available.

<p>Photodynamic therapy (PDT) is a rapidly developing clinical treatment modality involving a light-activatable photosensitizer, tissue oxygen and light of an appropriate wavelength to generate cytotoxic reactive molecular species - primarily singlet oxygen (¹O₂). Singlet oxygen readily reacts with surrounding biomolecules leading to different biological effects and subsequent therapeutic outcomes. Over the last decades, many standard PDT treatments have been approved worldwide to treat different medical conditions ranging from a variety of cancer conditions to age-related macular degeneration (AMD). Meanwhile, many active clinical trials and pre-clinical studies are underway for other clinical indications. The therapeutic outcomes of PDT are difficult to predict reliably even with many years of research. The fundamental cause for this is the inherent complexity of PDT mechanisms. As PDT involves three main components, the outcomes of PDT are determined by the combination of all components. Each component varies temporally and spatially during PDT, and the variations are mutually dependent on each other. Moreover, components such as the photosensitizer can have great variations in their initial distribution among patients even before PDT treatment. Given this, no well accepted standard PDT dose metric method has been recognized in clinics, although different approaches including explicit, implicit and direct dosimetry have been studied. To tackle the inherently complicated PDT mechanism in order to provide insights into PDT and PDT dosimetry, a theoretical one-dimensional model for aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX)-PDT of human skin was developed and is presented in this thesis. The model incorporates major photophysical and photochemical reactions in PDT, and calculated temporal and spatial distributions of PDT components as well as the detectable emission signals including both sensitizer fluorescence and singlet oxygen luminescence (SOL) using typical clinical conditions. Since singlet oxygen is considered to cause PDT outcomes, the correlations of different PDT dose metrics to average reacted (¹O₂) "dose" and "dose" at different depths were examined and compared for a wide range of varied treatment conditions. The dose metrics included absolute fluorescence bleaching metric (AFBM), fractional fluorescence bleaching metric (FFBM) and cumulative singlet oxygen luminescence (CSOL), and the varied treatment conditions took into account different treatment irradiances and wavelengths, varied initial sensitizer concentration and distribution, and a wide range of optical properties of tissue. These investigations and comparisons provide information about the complicated dynamic process of PDT such as the induction of tissue hypoxia, photosensitizer photobleaching and possible PDT-induced vascular responses. It was also found that the CSOL is the most robust and could serve as a gold standard for the testing of other techniques. In addition to these theoretical studies, recent progress on the assessment of a novel, more efficient superconducting nanowire single photon detector (SNSPD) for singlet oxygen luminescence detection will be introduced and the current photomultiplier tubes (PMT) system will be briefly described as well. The author participated in the experimental assessments of the SNSPD and analyzed the results shown in this thesis.</p> / Doctor of Philosophy (PhD)

Die Tetrapyrrol-Biosynthese der Pflanzen ist ein hoch konservierter Prozess, indem sich die Häm- und Chlorophyllsynthese gemeinsame Syntheseschritte von der 5-Aminolävulinsäure (ALA)- bis hin zur Protoporphyrin IX (Proto)-Bildung teilen. Zur Hämsynthese sind in Arabidopsis thaliana zwei Isoformen der Ferrochelatase (FC) vorhanden, welche die Insertion von Eisenionen in Proto katalysieren. In dieser Arbeit wurden fc1 und fc2 Mutanten analysiert und für Komplementationsversuche mit nativen und modifizierten FC1/FC2-Sequenzen genutzt. Die in der fc1-2 Mutante gestörte Embryonalentwicklung infolge des FC1 Mangels konnte durch Expression eines pFC1::FC1 Genkonstruktes komplementiert werden. Die Expression von FC2 unter dem FC1 Promoter (pFC1::FC2) konnte die fc1-2 Mutante unter Standard-Wachstumsbedingungen vollständig komplementieren, jedoch nicht unter Salzstress. Zusätzlich zu den Komplementationsversuchen der fc1 Mutanten wurde auch eine fc2 Null-Mutante zur Expression der beiden genomischen FC Sequenzen herangezogen, um die spezifischen Funktionen der FC2-Varianten zu untersuchen. Während die pFC1FC2 (fc2/fc2) Pflanzen unter Dauerlicht eine vollständige Komplementation zeigten, konnte unter Kurztagbedingungen nur eine partielle Komplementation beobachtet werden. Versuche geben erste wichtige Hinweise, dass auch FC2 an der Regulation der ALA-Synthese infolge ihrer Interaktion mit PORB beteiligt ist. Dies deutet darauf hin, dass der Häm- und der Chlorophyllzweig eine gemeinsame Regulation der ALA-Synthese teilen, um das Gleichgewicht der TBS zu wahren. Neben der Funktion der FC2 in der Regulation der TBS konnte die vorliegende Arbeit ebenfalls die Rolle der FC2 in der Assemblierung der PSII-LHCII Superkomplexe offenlegen. Basierend auf den Ergebnissen, dieser Studie können Modelle für die funktionale Verteilung der beiden FC-Isoformen in unterschiedlichen Geweben und Entwicklungsstadien, sowie die Funktionen in verschiedenen biologischen Prozessen postuliert werden. / In plants, heme and chlorophyll synthesis share the common synthetic steps from 5- aminolevulinic acid (ALA) formation to Protoporphyrin IX (Proto) production in the conserved Tetrapyrrole biosynthesis (TBS) pathway. Arabidopsis thaliana utilizes two ferrochelatses (FC) to catalyse the insertion of ferrous iron into Proto to yield heme. In this study, the fc1 and fc2 defective mutants have been re-analysed and used for complementation tests with expression of a native or modified FC1/FC2 sequence. The pFC1FC1 (fc1/fc1) complementation plants confirmed that the defective embryo maturation in homozygous fc1-2 seeds is attributed to a lack of FC1. Expression of FC2 under the FC1 promoter contributed to a full complementation of fc1-2 under standard growth conditions, but not under salt stress. A fc2 null mutant has been used to express the two FC genomic sequences to substantiate the specific functions of FC2. Expression of FC2 under its own promoter was able to rescue fc2-2 mutants under both SD and CL conditions. However, pFC2FC1 (fc2/fc2) plants showed a partial complementation under SD condition. Via multiple interaction assays and mutant analyses, this thesis uncovered a mechanism of FC2 action on ALA synthesis regulation via interaction of FC2 and PORB. The results indicate that both branches of heme and chlorophyll synthesis share a common regulation to balance the TBS pathway. Apart from a role of FC2 involved in the regulation of TBS pathway, the presented study also revealed FC2 function in the assembly of the PSII-LHCII supercomplexes. Based on all the results obtained in this study, the functional distribution models of the two FC in different tissues and development stages, as well as diverse biological processes, have been proposed. In addition, to which extent that FC1/FC2 could compensate the function of the other isoform has been discussed.

Fluorescence in blue light (FLU) ist ein negativer Feedbackregulator der Chlorophyllbiosynthese, welcher an der Dunkelrepression der 5-Aminolävulinsäure (ALA)-Synthese beteiligt ist. FLU ist Teil eines Komplexes, der die Enzyme umfasst, welche an der Katalyse der finalen Schritte der Chlorophyllbiosynthese beteiligt sind. Drei funktionelle Domänen wurden für das Arabidopsis FLU Protein postuliert: eine Tetratricopeptid-Wiederholungsdomäne (TPR) befindet sich am C-Terminus; eine Transmembrandomäne (TM) ist am N-Terminus lokalisiert; eine Coiled-coil-Domäne (linker) liegt dazwischen. Die TPR-Domäne von FLU Domäne interagiert mit dem C-terminalen Ende der Glutamyl-tRNA Reduktase (GluTR), dem geschwindigkeitsbestimmenden Enzym der ALA-Synthese. Diese Arbeit zur Erweiterung des Wissen über die Funktion von FLU im Licht sowie über die Rolle der funktionellen Domänen von FLU bei der Inaktivierung der ALA-Synthese bei. / Fluorescence in blue light (FLU), a negative feedback regulator of chlorophyll biosynthesis, is involved in dark repression of 5-aminolevulinic acid (ALA) synthesis. FLU is part of a complex comprising the enzymes catalyzing the final steps of chlorophyll synthesis. Three functional domains were proposed in the Arabidopsis FLU protein: a tetratricopeptide repeat (TPR) domain is at the C-terminus; a transmembrane domain (TM) is at the N-terminus; a coiled-coil domain (linker) is in between. The TPR(FLU) domain interacts with the C-terminal end of glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of ALA synthesis. This thesis contributes to the extended knowledge about the function of FLU in light as well as the role of the structural domains of FLU in the inactivation of ALA synthesis.

Introduction: Stenotic nares are one of the most important characteristics of brachycephalic obstructive airway syndrome (BOAS), which was first described in 1949. However, although they have been mentioned in most texts about brachycephalic malformations, few studies have attempted to characterize the nasal entrance of dogs morphologically or functionally, and none of them have described it objectively with landmarks. Recently, as the popularity of brachycephalic dogs has exploded worldwide, innumumberable dogs are suffering from BOAS due to structural malformations. In the point of view of animal welfare, stenotic nares should be objectively evaluated to provide evidence of torture breeding. Objectives: The aim of this study was to objectively measure and thus characterize the nasal entrance stenosis of brachycephalic dogs in comparison to non-brachycephalic dogs. To this end, the following objectives were defined: 1. Our aim is to use three-dimensional (3D) soft-tissue morphometry to objectively measure the externally visible part of the nasal entrance of healthy and brachycephalic dogs. 2. Our aim is to define specific soft-tissue landmarks and parameters to objectively characterize the nasal entrance of dogs. 3. Compare 3D high-resolution scanning with computed tomography (CT) using the newly defined parameters. 4. Reexamine the nasal entrance with 3D scan six months after surgical correction of nasal entrance stenosis (Ala-vestibuloplasty). Animals, Material and Methods: Forty-five brachycephalic and 45 non-brachycephalic dogs were included in this study between 2018 and 2020. All the animals were referred to the Ear, Nose, and Throat Unit of the Small Animal Department at the University of Leipzig, either for surgical correction of BOAS or for endoscopic examination. Animals were excluded if their nasal entrance was potentially affected due to disease or previous surgery. All dogs were scanned with a 3D scanner under general anesthesia using a standardized anesthetic protocol before endoscopic intervention. The 3D scans and CT images were later imported into two advanced software (Amira, Thermo Fisher and Facial Analysis Tool) for objective measurement of the nasal entrance. Intra-observer error and ala-vestibuloplasty (AVP) efficacy of the CT images and 3D scans were tested and compared. All the data were normalized using Shapiro–Wilk normality test. All statistical analyses were later performed using Pearson or Spearman test for correlation, paired t-test, or t-test. Paired t-test was used to test whether the mean difference between pairs of measurements was zero. If it was not the same group of animals, t-test was used instead. Results: We defined new specific soft-tissue landmarks to calculate the nasal opening area (NOA) and stenotic angle (SA) to objectively describe the canine nasal entrance. The NOA and SA were significantly smaller in brachycephalic dogs than in non-brachycephalic dogs, and the NOA was significantly correlated with body weight, whereas SA was not. After nasal entrance correction via AVP, the nasal entrances were 295%left and 233%right larger than before surgery in brachycephalic dogs. Intra-observer reliability was tested with excellent significance through NOA measurements. Comparison between the 3D scanner and CT was measured with the NOA, and it did not show a significant difference between the methods. Conclusion: Obtaining the NOA and SA using 3D surface scans seems to be a reliable and reproducible tool for precise objective evaluation of the visible canine nasal entrance. With the measurement of derived NOA and SA, all the brachycephalic dogs in the study were found to have stenotic nares, and all they required AVP to relieve breathing difficulty. The brachycephalic nasal entrances were increased significantly after AVP, the modified nare correction surgery. We consider the results of our study strong evidence that the breeding of brachycephalic dogs is torture breeding. The restriction of nasal breathing due to the extreme reduction of the nasal entrance to a fraction of the size of that of non-brachycephalic dogs is obvious evidence of this.:1 INTRODUCTION - 1 - 1.1 General Introduction - 1 - 1.2 Objectives - 2 - 2 OVERVIEW OF THE LITERATURE - 3 - 2.1 Brachycephalic Obstructive Airway Syndrome (BOAS) - 3 - 2.1.1 General Introduction of BOAS - 3 - 2.1.2 How to Define Brachycephalic Dogs - 3 - 2.1.3 Commonly Affected Breeds - 4 - 2.2 The Canine Nasal Entrance - 4 - 2.2.1 Anatomy of the Canine Nasal Entrance and Stenosis - 4 - 2.2.2 Pathophysiology of Stenotic Nares in Brachycephalic Dogs - 5 - 2.3 Diagnosis of Stenotic Nares - 9 - 2.3.1 Visual Assessment - 9 - 2.3.2 Nostril Ratio - 11 - 2.3.3 Air Volume of the Nasal Entrance - 11 - 2.4 Development of Morphometry in Brachycephalic Dogs - 11 - 2.5 3D Scanners for 3D Model Reconstruction - 12 - 2.6 Landmarks - 14 - 2.6.1 Introduction of Landmarks - 14 - 2.6.2 Development of Landmarks in Human Medicine - 15 - 2.6.3 Development of Landmarks in Veterinary Medicine - 16 - 2.6.4 Cephalometric Analysis Software - 17 - 3 ANIMALS AND METHODS - 19 - 3.1 Animals - 19 - 3.2 Methods - 19 - 3.2.1 3D Scanning Process and Set-ups of Canine Nasal Entrance - 19 - 3.2.2 Cephalomorphometric Software - 20 - 3.2.3 Cephalometric Landmarks - 20 - 3.2.4 Advanced Morphometric Parameters for Nares - 22 - 3.2.5 Efficacy of Ala-Vestibuloplasty (AVP) - 24 - 3.2.6 Comparison between CT and the 3D Scanning Tool - 24 - 3.2.7 Intra-observer Reliability - 25 - 3.2.8 Statistics - 25 - 4 RESULTS - 26 - 4.1 Brachycephalic Dogs and Non-brachycephalic Dogs - 26 - 4.2 The Nasal Opening Area (NOA) - 28 - 4.3 Efficacy of Ala-Vestibulopasty - 30 - 4.4 Stenotic Angle (SA) - 31 - 4.5 Comparison between CT and the 3D Scanning Tool - 32 - 4.6 Intra-observer Reliability - 34 - 5 DISCUSSION - 36 - 5.1 The Nasal Entrance - 36 - 5.2 Method: the 3D Scanner - 37 - 5.3 Method: Comparison between CT and the 3D Scanner - 38 - 5.4 Method: Landmarks and Reproducibility, Intra-observer Reliability - 39 - 5.5 Nasal Opening Area (NOA) and Efficacy of Ala-Vestibuloplasty (AVP) - 42 - 5.6 Stenotic Angle (SA) - 44 - 5.7 Animal Welfare - 45 - 6 SUMMARY - 47 - 7 ZUSAMMENFASSUNG - 49 - 8 REFERENCES - 51 - / Einleitung: Die Naseneingangstenose ist eine der wichtigsten Merkmale des brachyzephalen Syndroms (BOAS), das erstmals 1949 beschrieben wurde. Obwohl diese komplexe Stenose in den meisten Texten über brachyzephale Fehlbildungen erwähnt wird, haben nur wenige Studien versucht, den Naseneingang von Hunden morphologisch oder funktionell zu charakterisieren. Eine objektive Beschreibung mit Landmarken ist bisher nicht bekannt. In den letzten Jahren ist die Popularität brachyzephaler Hunde weltweit explodiert. Unzählige Hunde leiden aufgrund struktureller Fehlbildungen an BOAS. Unter dem Gesichtspunkt des Tierschutzes sollte die einzige, mit bloßem Auge sichtbare Stenose der oberen Atemwege brachyzephaler Hunde, die Stenose des Naseneingangs objektiv bewertet werden können, um Hinweise auf eine Qualzucht zu belegen. Zielsetzung: Ziel dieser Studie war es, den Naseneingang von brachyzephalen Hunden im Vergleich zu nicht-brachyzephalen Hunden objektiv zu messen und damit die Stenose des Naseneingangs zu charakterisieren und eine chirurgische Therapie, die Ala-Vestibuloplastie (AVP), zu evaluieren. Zu diesem Zweck wurden die folgenden Ziele definiert: 1. Anwendung der dreidimensionalen (3D) Weichteilmorphometrie zur objektiven Messung des äußerlich sichtbaren Teils des Naseneingangs von gesunden und brachyzephalen Hunden. 2. Definition spezifischer Weichteil-Landmarken und Parameter zur objektiven Charakterisierung des Naseneingangs von Hunden. 3. Methoden-Vergleich zwischen den hochauflösenden 3D-Scans und der Computertomographie (CT) unter Verwendung der neu definierten Parameter. 4. Kontrolluntersuchung mit 3D-Scan sechs Monate nach der chirurgischen Korrektur der Naseneingangsstenose (Ala-Vestibuloplastik). Tiere und Methoden: 45 brachyzephale und 45 nicht-brachyzephale Hunde wurden zwischen 2018 und 2020 in diese Studie aufgenommen und wurden an die Hals-Nasen-Ohren-Abteilung der Kleintierklinik der Universität Leipzig überwiesen, zur endoskopischen Untersuchung der oberen Atemwege und gegebenenfalls zur chirurgischen Korrektur der BOAS-assozierten Stenosen. Ausgeschlossen wurden Tiere, deren Naseneingang aufgrund von Krankheiten oder einer früheren Operation verändert war. Alle Hunde wurden nach einem standardisierten Anästhesieprotokoll anästhesiert, ein Computertomogramm des Kopfes erstellt und vor dem endoskopischen Eingriff wurde der Kopf mit einem 3D-Scanner gescannt. Die 3D-Scans und CT-Bilder wurden in zwei Softwareprogramme (Amira, Thermo Fisher und Facial Analysis Tool) zur objektiven Messung des Naseneingangs importiert. Zur genauen Charakterisierung wurden neue Landmarken am Naseneingang definiert und zur Berechnung von zwei neuen, abgeleiteten Parametern, der Nasenöffnungsfläche (NOA) und dem stenotischen Winkel (SA) verwendet. Der Intraobserver-Fehler und die Wirksamkeit der Ala-Vestibuloplastie wurden anhand der CT-Bilder und 3D-Scans mit demselben Verfahren geprüft und verglichen. Alle statistischen Analysen wurden später mit dem Pearson-Test oder dem Spearman-Test für die Korrelation, dem gepaarten t-Test oder dem t-Test durchgeführt. Der gepaarte t-Test wurde verwendet, um zu prüfen, ob der Mittelwertunterschied zwischen Paaren von Messungen gleich Null war. Handelte es sich nicht um dieselbe Gruppe von Tieren, wurde stattdessen der t-Test verwendet. Ergebnisse: Mit den neu definierten spezifischen Weichteil-Landmarken und den abgeleiteten Parametern NOA und SA konnte der Naseneingang aller brachyzephalen und nicht-brachyzephalen Hunde objektiv beschrieben werden. NOA und SA waren bei brachyzephalen Hunden signifikant kleiner als bei nicht-brachyzephalen Hunden. NOA korrelierte in beiden Gruppen signifikant mit dem Körpergewicht. Dagegen zeigte SA keine Korrelation mit dem Körpergewicht. Nach der chirurgischen AVP waren die Naseneingänge bei allen brachyzephalen Hunden größer als vor der Operation, mit einer Zunahme der NOA links um 295 % und rechts um 233 %. Die Intraobserver-Zuverlässigkeit wurde durch NOA-Messungen getestet und war hoch signifikant. Zum Methoden-Vergleich zwischen 3D-Scanner und CT wurde die Nasenöffnungsfläche jeweils am gemessen und zeigte keinen signifikanten Unterschied zwischen den Methoden. Schlussfolgerungen: Morphometrische Messungen mit 3D-Oberflächenscans scheinen ein zuverlässiges und reproduzierbares Instrument zur präzisen, objektiven Bewertung des Naseneingangs des Hundes zu sein. Bei der Messung der abgeleiteten Flächen (NOA) und Winkel (SA) wurde bei allen brachyzephalen Hunden dieser Studie eine Naseneingangsstenose festgestellt. Nach der chirurgischen Korrektur der brachyzephalen Naseneingangsstenose, der AVP, waren die Nasenöffnungen deutlich vergrößert. Die Einschränkung der Nasenatmung durch die extreme Verkleinerung des Naseneingangs auf einen Bruchteil der Größe von nicht-brachyzephalen Hunden, betrachten wir als eindeutigen Beweis dafür, dass die Zucht von brachyzephalen Hunden eine Qualzucht ist.:1 INTRODUCTION - 1 - 1.1 General Introduction - 1 - 1.2 Objectives - 2 - 2 OVERVIEW OF THE LITERATURE - 3 - 2.1 Brachycephalic Obstructive Airway Syndrome (BOAS) - 3 - 2.1.1 General Introduction of BOAS - 3 - 2.1.2 How to Define Brachycephalic Dogs - 3 - 2.1.3 Commonly Affected Breeds - 4 - 2.2 The Canine Nasal Entrance - 4 - 2.2.1 Anatomy of the Canine Nasal Entrance and Stenosis - 4 - 2.2.2 Pathophysiology of Stenotic Nares in Brachycephalic Dogs - 5 - 2.3 Diagnosis of Stenotic Nares - 9 - 2.3.1 Visual Assessment - 9 - 2.3.2 Nostril Ratio - 11 - 2.3.3 Air Volume of the Nasal Entrance - 11 - 2.4 Development of Morphometry in Brachycephalic Dogs - 11 - 2.5 3D Scanners for 3D Model Reconstruction - 12 - 2.6 Landmarks - 14 - 2.6.1 Introduction of Landmarks - 14 - 2.6.2 Development of Landmarks in Human Medicine - 15 - 2.6.3 Development of Landmarks in Veterinary Medicine - 16 - 2.6.4 Cephalometric Analysis Software - 17 - 3 ANIMALS AND METHODS - 19 - 3.1 Animals - 19 - 3.2 Methods - 19 - 3.2.1 3D Scanning Process and Set-ups of Canine Nasal Entrance - 19 - 3.2.2 Cephalomorphometric Software - 20 - 3.2.3 Cephalometric Landmarks - 20 - 3.2.4 Advanced Morphometric Parameters for Nares - 22 - 3.2.5 Efficacy of Ala-Vestibuloplasty (AVP) - 24 - 3.2.6 Comparison between CT and the 3D Scanning Tool - 24 - 3.2.7 Intra-observer Reliability - 25 - 3.2.8 Statistics - 25 - 4 RESULTS - 26 - 4.1 Brachycephalic Dogs and Non-brachycephalic Dogs - 26 - 4.2 The Nasal Opening Area (NOA) - 28 - 4.3 Efficacy of Ala-Vestibulopasty - 30 - 4.4 Stenotic Angle (SA) - 31 - 4.5 Comparison between CT and the 3D Scanning Tool - 32 - 4.6 Intra-observer Reliability - 34 - 5 DISCUSSION - 36 - 5.1 The Nasal Entrance - 36 - 5.2 Method: the 3D Scanner - 37 - 5.3 Method: Comparison between CT and the 3D Scanner - 38 - 5.4 Method: Landmarks and Reproducibility, Intra-observer Reliability - 39 - 5.5 Nasal Opening Area (NOA) and Efficacy of Ala-Vestibuloplasty (AVP) - 42 - 5.6 Stenotic Angle (SA) - 44 - 5.7 Animal Welfare - 45 - 6 SUMMARY - 47 - 7 ZUSAMMENFASSUNG - 49 - 8 REFERENCES - 51 -

Pre-treatment with polyunsaturated fatty acids or bioactive lipid mediators has been shown to reduce neuronal injury in rodent models of focal ischaemia, but the molecular mechanisms underlying this neuroprotection are unclear. In this study, we aimed to investigate whether systemic administration of alpha linolenic acid (ALA) leads to changes in the profile of mouse brain phospholipid and bioactive lipid mediators in both mouse brain and plasma within the previously determined neuroprotection time window. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) allowed us to detect and identify 47 phospholipids in mouse cerebral cortex, including several phospholipid species not previously reported in brain lipidomic studies. These included a phosphatidylethanolamine species with m/z 720 that has been associated with retinal stem cells. No widespread changes in cerebral cortex phospholipid composition were observed following intravenous ALA. Several significant changes in lipid mediators (P<0.05 with two-way ANOVA and post hoc Dunnett's t test) were detected in ALA-treated animals compared to untreated and vehicle-injected animals. Many of the affected lipid mediators are ligands for prostanoid receptors which have been demonstrated to play a role in the development of brain injury following cerebral ischaemia, implying that changes in bioactive lipid mediators or modulation of prostanoid receptors may occur following ALA pre-treatment in mice. This study illustrates the potential of advanced lipidomic analysis as a novel tool for neurochemists.