Les thrombopathies congénitales sont des pathologies au phénotype hémorragique hétérogène dues à des anomalies fonctionnelles des plaquettes sanguines parfois associées à des anomalies morphologiques et/ou à une thrombopénie. Les outils diagnostic disponibles permettent de corréler une anomalie fonctionnelle plaquettaire à un déficit protéique voire à une mutation d’un gène spécifique. Cependant, en 2015, au moins 50% des thrombopathies restent non étiquetées. Dans ce contexte, le laboratoire d’hémostase de l’EFS Alsace et l’unité INSERM UMR S949 travaillent de concert à l’identification et à la caractérisation de ces pathologies. Ainsi, nous avons mis en évidence trois nouvelles mutations du récepteur P2Y12 dans trois familles différentes. La mutation p.His187Gln a été entièrement caractérisée sur des plaquettes fraiches du patient et dans un modèle cellulaire adapté. La reproduction des deux autres mutations (p.Tyr259Cys et p.Phe95Ser) est en cours. Nous avons par ailleurs identifié une famille dont trois membres sont atteints d’une maladie du pool vide non syndromique et nous suspectons grâce au séquençage d’exome effectué que le transporteur de nucléotides VNUT est impliqué dans la pathologie. / Inherited platelets disorders (IPD) are pathologies associated with heterogeneous bleedingphenotypes. They are due to functional platelets deficiency that may come with morphological abnormalities and/or thrombocytopenia. Available diagnosis tools are used to link a functional deficiency to a protein defect and in some instances to a specific genetic mutation. However, in 2015 at least 50% of IPD are still not identified. In this context, the haemostasis laboratory of the EFS Alsace together with INSERM UMR S949 team are working to diagnose and characterize these pathologies. Thus, we found three new P2Y12 receptor variants in three different families. The mutation p.His187Gln has been fully characterized on blood fresh platelets coming from one patient and in an appropriate cellular model. The reproduction of the two other mutations (p.Tyr259Cys and p.Phe95Ser) is currently ongoing. We also studied a family in which three members are carrying a delta storage pool disease. The full exome sequencing analysis leads us to suspect the involvement of the nucleotides transporter VNUT in this pathology.

Magister Scientiae - MSc / Traditionally in human genetic linkage analysis, extended families were only used in the analysis of dichotomous traits, such as Disease/No Disease. For quantitative traits, analyses initially focused on data from family trios (for example, mother, father, and child) or sib-pairs. Recently however, there have been two very important developments in genetics: It became clear that if the disease status of several generations of a family is known and their genetic information is obtained, researchers can pinpoint which pieces of genetic material are linked to the disease or trait. It also became evident that if a trait is quantitative (numerical), as blood pressure or viral loads are, rather than dichotomous, one has much more power for the same sample size. This led to the development of statistical mixed models which could incorporate all the features of the data, including the degree of relationship between each pair of family members. This is necessary because a parent-child pair definitely shares half their genetic material, whereas a pair of cousins share, on average, only an eighth. The statistical methods involved here have however been developed by geneticists, for their specific studies, so there does not seem to be a unified and general description of the theory underlying the methods. The aim of this dissertation is to explain in a unified and statistically comprehensive manner, the theory involved in the analysis of quantitative trait genetic data from extended families. The focus is on linkage analysis: what it is and what it aims to do. There is a step-by-step build up to it, starting with an introduction to genetic epidemiology. This includes an explanation of the relevant genetic terminology. There is also an application section where an appropriate human genetic family dataset is analysed, illustrating the methods explained in the theory sections. / South Africa

Inherited disorders of platelet function are a rare and heterogeneous group of diseases usually characterised by a mild to moderate bleeding tendency. Typical bleeding symptoms are easy bruising, epistaxis, menorrhagia as well as mucocutaneous and perioperative bleeding. The performance of platelet function diagnostics in children is hampered by age-dependent restriction of blood sample size, poor venous access, and the lack of reproducible test reference ranges for children of different age groups. Platelet function testing is limited to specialised centres, because platelet function test procedures are complicated and time-consuming, which most likely results in a relevant number of undiagnosed and incorrectly classified children with clinically relevant platelet function defects. Evaluation of bleeding history and bleeding symptoms is essential for a rational step-bystep approach to diagnosis. Platelet function diagnostics should be preceded by the exclusion of thrombocytopenia, von Willebrand disease, and secondary haemostasis defects. Light transmission aggregometry is still considered the standard for the assessment of platelet function. Every effort should be made to classify the specific platelet function defect in the patient, because this is essential for accurate treatment and counselling. / Angeborene Thrombozytenfunktionsstörungen stellen eine seltene und heterogene Gruppe von Erkrankungen dar, welche meist durch eine leichte bis mittelschwere Blutungsneigung auffallen. Typische Blutungssymptome sind Hämatomneigung, Epistaxis, Menorrhagien sowie Schleimhaut- und perioperative Blutungen. Die Durchführung der Thrombozytenfunktionsdiagnostik bei Kindern wird erschwert durch die altersabhängig begrenzte Blutprobenmenge, schwierige Venenverhältnisse und das Fehlen von Referenzbereichen für Kinder unterschiedlichen Alters. Aufgrund der meist komplizierten und zeitaufwendigen Tests ist die Thrombozytendiagnostik auf spezialisierte Zentren begrenzt. Mit hoher Wahrscheinlichkeit wird eine relevante Anzahl von Kindern mit nichtdiagnostizierten bzw. unkorrekt klassifizierten, klinisch relevanten Thrombozytopathien übersehen. Die Erhebung der Blutungsanamnese und die Bewertung der Blutungssymptome sind erforderlich für eine stufenweise erfolgreiche Gerinnungsdiagnostik. Vor Durchführung einer Thrombozytenfunktionsdiagnostik sollten das Vorliegen einer Thrombozytopenie, einer von-Willebrand-Erkrankung und sekundärer Gerinnungsstörungen ausgeschlossen werden. Die Lichttransmissionsaggregometrie gilt noch immer als Standardmethode für die Beurteilung der Thrombozytenfunktion. Nach Möglichkeit sollte stets versucht werden, den vorliegenden spezifischen Thrombozytenfunktionsdefekt zu klassifizieren, da dies für eine adäquate Behandlung und eine gezielte genetische Beratung notwendig ist. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Mitochondrial diseases are caused by gene mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) and they are among one of the most common forms of inherited disorders. It is estimated that 1 out of every 5000 individuals will develop a mitochondrial disease in their lifetime. Due to the crucial and widespread functionality of mitochondria in human cells, prolonged diseases of the mitochondria affect cells of the brain, heart, liver, muscles and kidneys and can lead to multi-organ failure in some patients. Inherited or acquired mitochondrial diseases can present at any stage of life, affecting both children and adults. Since its discovery, the mitochondrial genome has been analyzed and sequenced with increasing ease and this process has helped recognize various mitochondrial disorders as the root of genetic diseases. This paper will explore the unique properties of the mitochondrion and its genome, examine the relationship between mtDNA and some common myopathies such as Leigh syndrome (LS) or maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in order to explore commonalities and differences in their inheritance patterns and their effect on mitochondrial function. Although studies have shown that these conditions generally affect the process of oxidative phosphorylation in mitochondria, because of the wide variety of presentations of this disease, further research is needed to understand the different etiologies, as well as to explore novel therapies to treat them.

This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...

Les dystrophies rétiniennes héréditaires (DRH) sont un ensemble de pathologies rétiniennes incurable provoquant la cécité. Les DRH sont caractérisées par le dysfonctionnement/dégénérescence des photorécepteurs et le remodelage de la structure de la rétine. Une des approches thérapeutiques envisagées pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Cependant, bien qu’efficace, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou quand le gène muté n’est pas connu. Afin de traiter tous les cas de DRH quelle que soit leur origine génétique et leur stade de progression, une approche de thérapie génique d’addition est envisagée : Le transfert d’optogène. Cela consiste à convertir les cellules encore présentes dans la rétine malgré la dégénérescence, en cellule photosensible suite à l’expression d’un optogène (protéine photosensible). Mon projet de thèse a consisté dans un premier temps à évaluer le transfert de gène avec un vecteur AAV2/2 dans les cellules ganglionnaires rétiniennes de chien et de primate non-humain. Cette première partie a permis d’initier un second projet qui a eu pour objectif d’évaluer l’efficacité du transfert d’optogène (Channelrhodopsin-2) pour la restauration de la fonction visuelle dans un modèle canin de dystrophie rétinienne (le chien Rpe65- /-). / Inherited retinal dystrophies (IRD), a group of incurable retinal pathologies, are associated with visual impairments due to a malfunction and/or degeneration of photoreceptors and/or retinal pigment epithelium (RPE). Significant progress in the field of gene therapy has allowed the development and the characterization of an innovative tool to treat IRD patients: recombinant adeno-associated viral vectors (AAV) that carry and deliver therapeutic nucleic acids. However, due to the heterogenic nature of IRD, gene supplementation will not allow to treat all forms of IRD because: (i) the numbers of mutated genes are unknown according to the state of art; (ii) the dominant forms of IRD in which mutations lead to negative effects are not eligible; (iii) the limit of AAV packaging excludes large-sized mutated genes and (iv) this approach is only applicable when photoreceptors are still alive. To treat all IRD patients, a novel therapeutic approach, independently of the mutated gene and the disease kinetic is suitable: the optogene transfer (light-sensitive protein) to restore photosensitivity in neurodegenerative retina by converting surviving retinal cells into photosensors. The primary goal of my research was to promote and characterize adeno-associated virus type 2-(AAV2) transduction in retinal ganglion cells of dog and non-human primate. A second aim was to investigate the feasibility of AAV2-mediated optogenes transfer in retinal ganglion cells as a therapeutic approach to restore visual function in RPE65 deficient dog, a canine model of IRDs.

The aim of this study was to investigate the basis to male mating decisions in two related species of bitterling: Rhodeus ocellatus and R. amarus. Bitterling have a resource-based mating system; females lay eggs in the gills of live freshwater mussels and males fertilize the eggs by releasing sperm into the inhalant syphon of the mussel. Male bitterling perform courtship behaviour and aggressively defend mussels in a territory from which they exclude other males. Using laboratory and field experiments it was shown that male aggressive behaviour is inherited through additive maternal genes. Male aggression is also influenced by the number of conspecific males encountered in competition for a mussel, and by the degree of clustering of mussels. Limited availability of mussels results in stronger selection on traits males use in mating context: hence they are more aggressive, larger and more colourful. The differences in mating behaviours in different environments may indicate a conflict between male dominance and female choice, but have not led to reproductive isolation. Resource availability during ontogenesis and male density during embryogenesis, however, do not exert an effect on male aggressive behaviour. Red carotenoid-based nuptial coloration functions as an inter- and intra-sexual signal and undergoes rapid variation in response to changes in mating context. Male bitterling do not modulate their courtship and aggressive behaviour in response to variation in female size, and their choice of mussel species is influenced by, and consistent with, female oviposition choice.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In one research, cases of congenital lymphedema were observed affecting 17 Red Angus calves and their crosses from two farms in the central region of the state of Rio Grande do Sul, Brazil. Affected calves presented variable degrees of subcutaneous edema at birth, involving mainly the hind limbs, but also other body regions. The injection of methylene blue dye in a 5% solution into the interdigital space of the hind limbs of three affected calves and one normal control indicated an interruption of the lymph flow in the affected calves; this suggests a failure of the distal peripheral lymphatics to connect with the central trunks. At necropsy of three affected calves, subcutaneous edema was variably observed in the hind limbs, prepuce and ventral abdomen. Edema of body cavities was not observed. In two cases there was hipoplasia of the popliteal lymph nodes associated to dilatation of lymphatic vessels. DNA tests demonstrated that all affected calves were born from cows sired by the same bull suggesting an inherited basis for the disease. Furthermore unrelated cows of different breeds sired by Bull 1 delivered affected calves which suggest that condition is an autosomic dominant trait. Considering the large numbers of cows sired by Bull 1 relative few calves were affected; this could be explained by a low penetrance of the trait. In another research, cases of congenital defects (CDs) in cattle diagnosed at the Laboratory of Veterinary Pathology of the Federal University of Santa Maria from 1964 to 2010 were reviewed. During the studied period, tissues collected from the necropsy of 7,132 cattle were examined and 31 calves (0.4%) with CDs were found. These CDs were classified into 34 different types and ascribed to the body system primarily affected. The results of this survey indicate that the majority o CDs in cattle in the central Rio Grande do Sul are sporadic; nevertheless their continued study is important for determining the etiology and control. / Em uma pesquisa, casos de linfedema congênito foram observados acometendo 17 bezerros cruza Red Angus na região Central do Rio Grande do Sul. Os bezerros afetados apresentavam graus variáveis de edema subcutâneo ao nascimento, envolvendo principalmente os membros pélvicos, mas também outras regiões do corpo. A injeção de solução a 5% de azul de metileno no espaço interdigital dos membros pélvicos de três bezerros afetados e um controle, indicaram uma interrupção no fluxo da linfa, sugerindo uma falha dos linfáticos periféricos distais em conectar os troncos centrais. Na necropsia de três bezerros afetados, edema subcutâneo foi observado de modo variável nos membros pélvicos, prepúcio e abdômen ventral. Não foi observado edema nas cavidades orgânicas. Em dois casos, observou-se hipoplasia dos linfonodos poplíteos associadas à dilatação dos vasos linfáticos. Testes de DNA demonstraram que todos os bezerros afetados nasceram de vacas inseminadas pelo mesmo touro (Touro 1), sugerindo uma base genética para a doença. Além disso, vacas não relacionadas e de outras raças cobertas pelo Touro 1 pariram bezerros afetados indicando uma condição autossômica dominante. Considerando o grande número de vacas cobertas pelo Touro 1, relativamente poucos bezerros foram afetados; isso pode ser explicado por uma condição hereditária de baixa penetrância. Em outra pesquisa, foram revisados casos de defeitos congênitos (DCs) diagnosticados em bovinos no Laboratório de Patologia da Universidade Federal de Santa Maria entre 1964-2010. Durante o período estudado, foram examinados materiais provenientes da necropsia de 7.132 bovinos e foram encontrados 31 bezerros (0,4%) com DCs, os quais foram classificados em 34 tipos e alocados nos sistemas orgânicos primariamente afetados. Os resultados indicam que a maioria dos DCs em bovinos na Região Central do Rio Grande do Sul é esporádica. No entanto, seu estudo continuado é importante para o estabelecimento de sua etiologia e controle.

Le déficit héréditaire en Carnitine Palmitoyl Transférase 2 (CPT2), l’un des déficits de l’oxydation mitochondriale des acides gras (OAG) les plus fréquents, est caractérisé dans sa forme adulte par une myopathie métabolique avec des épisodes récurrents de douleurs musculaires, de myoglobinurie et de rhabdomyolyse, habituellement déclenchés par un exercice prolongé ou un jeûne. A l’heure actuelle, il n’y a pas de traitement pharmacologique efficace pour la correction de ce déficit à l’exception de prise en charge nutritionnelle. Mon travail de thèse a porté sur l’étude du potentiel thérapeutique du composé GSK773 un activateur direct de l’AMP-activated Protein Kinase (AMPK), un senseur énergétique de la cellule, dans des myotubes de quatre patients déficitaires en CPT2. En effet, l'AMPK est considérée comme une cible thérapeutique potentielle dans de nombreux troubles métaboliques ou neurodégénératifs courants associés aux dysfonctionnements mitochondriaux. Nous montrons que le composé GSK773 est capable de stimuler les capacités résiduelles de l’OAG et de corriger le flux d’OAG dans des myotubes déficitaires en CPT2 (n=4) après un traitement par 30µM pendant 48h. L’étude par western-blot et par immunofluorescence montre que le GSK773 augmente la quantité de protéine mutante CPT2. L'analyse des intermédiaires C16-acylcarnitines montre que les myotubes déficients en CPT2 présentent, comme prévu, une accumulation de C16-acylcarnitines significativement diminuée après le traitement par le GSK773. De manière intéressante, l'IF et l’xCELLigence, une nouvelle technique basée sur la mesure de l’impédance électrique en temps réel, montrent un processus de différenciation altéré dans les myotubes de patients déficitaires en CPT2 par rapport aux cellules témoins, qui est corrigé par le GSK773. Nous avons également montré que le GSK773 induit une conversion des fibres musculaires vers les fibres de type I lentes/oxydatives, mais aussi une amélioration générale de la qualité du réseau mitochondrial accompagnée d’une biogenèse mitochondriale et une augmentation du niveau de ROS suggérant que le GSK773 agirait sur la plasticité musculaire. D’un point de vue mécanistique, nous avons montré que les effets du GSK773 passent par l’AMPK, PGC-1α, p38 MAPK et les ROS. Ainsi, ces résultats suggèrent que le GSK773 améliore les paramètres métaboliques et structuraux dans les myotubes déficients en CPT2 et que l'AMPK pourrait représenter une cible thérapeutique hautement pertinente pour la correction pharmacologique du déficit en CPT2. / Carnitine Palmitoyl Transferase 2 (CPT2) deficiency is among the most common inherited defects of mitochondrial fatty acid oxidation (FAO). A frequent phenotype is an early adult-onset myopathy characterized by recurrent episodes of muscle pain, myoglobinuria and rhabdomyolysis usually triggered by prolonged exercise or fasting. To date, there is no treatment of this disorder other than dietary management. AMPK is considered as a potential therapeutic target in many common metabolic or neurodegenerative disorders associated to mitochondrial dysfunctions. Thus, we tested the therapeutic potential of the direct AMPK activator GSK773 in myotubes from four CPT2-deficient patients. We show that GSK773 is able to stimulate residual FAO capacities in a dose- and time-dependent manner. Correction of CPT2 defect is achieved after treatment with GSK773 at 30µM for 48h. Western-blots analysis and Immunostaining shows that GSK773 increases the amount of CPT2 mutant protein. Analysis of acylcarnitine intermediates in the culture media shows that CPT2-deficient myotubes exhibit, as expected, an accumulation of C16-acylcarnitines that is significantly decreased after GSK773 treatment. Surprisingly, immunofluorescence and xCELLigence (a real-time monitoring of cell culture technic) show an impaired differentiation process in CPT2-deficient myotubes that is corrected by GSK773. We also show that GSK773 induces a shift in myosin-heavy-chain isoforms toward slow oxidative fiber types, improves the quality of mitochondrial network with an induction of mitochondrial biogenesis and increases ROS levels, suggesting that GSK773 might induce muscle plasticity. Finally, from a mechanistic point of view, siRNAs experiments showed that the effects triggered by GSK773 implicate AMPK, PGC-1α, ROS and p38 MAPK. Altogether these results suggest that GSK773 improves metabolic and structural parameters in CPT2-deficient myotubes and that AMPK might represent a highly relevant therapeutic target for pharmacological correction of inborn CPT2 deficiency.

In dieser Studie wurden erstmals die Auswirkungen von Zucht auf Kurzköpfigkeit auf ein breites Spektrum von Lebensbereichen brachyzephaler Hunde strukturiert untersucht. Hierzu wurde ein gegliederter Fragebogen für Tierbesitzer entwickelt. Erstmalig werden Validität und Reliabilität eines solchen Instruments zur Untersuchung von Symptomen einbezogen. Das Ziel der Arbeit war (1) die Konstruktion und Validierung eines internetbasierten Fragebogens für Tierbesitzer, der alle wichtigen Bereiche berücksichtigt, in denen brachyzephale Hunde Probleme zeigen; sowie die Bestimmung der Reliabilität des Fragebogens, (2) die Durchführung der Besitzerbefragung mit dem validierten Fragebogen und (3) die Analyse der Ergebnisse der Besitzerbefragung und Bestimmung von Häufigkeit und Schwere der Symptome bei den betroffenen Hunden. An der Befragung nahmen 100 Tierbesitzer teil, deren Hunde zur chirurgischen Behandlung des Brachyzephalen Syndroms an die Klinik für Kleintiere der Universität Leipzig überwiesen worden waren (61 Besitzer von Möpsen, 39 Besitzer von Französischen Bulldoggen). Die wichtigsten Ergebnisse dieser Studie sind: 100 % der Hunde haben bei Belastung laute Atemgeräusche, zwei Drittel sogar in Ruhe. Fast die Hälfte der Hunde hat in Ruhe Anzeichen für angestrengte Atmung. 41 % haben mindestens einmal wöchentlich Erstickungsanfälle und über ein Drittel ist schon mindestens einmal in ihrem Leben aufgrund von Atemnot umgefallen. Betroffene Tiere leiden an hochgradiger Belastungs- und Hitzeintoleranz. Die Belastbarkeit bei warmen Umgebungstemperaturen nimmt ab und die nach Belastung zur Erholung erforderliche Zeit steigt an. 88 % der Hunde sind nur eingeschränkt belastbar, 87 % beim Spielen; im Sommer können über ein Drittel der Hunde maximal 10 Minuten am Stück spazieren gehen, im Winter können über zwei Drittel der Hunde mindesten 30 Minuten spazieren gehen; bei 94 % der Hunde verschlimmern sich die Atembeschwerden bei warmen Temperaturen und zwar ab einer mittleren Temperatur von 19°C. Da diese Ergebnisse die hohe Temperaturabhängigkeit der Belastbarkeit zeigen, sollten für die Zuchtzulassung vorgeschriebene Belastungstests zukünftig bei eindeutig definierten Umgebungstemperaturen durchgeführt werden. Die Befragung der Tierbesitzer identifizierte ausgeprägte Atemnot während des Schla-fes als eine häufige schwerwiegende Beeinträchtigung. Ein Teil dieser Hunde entwickelt spezielle Strategien, um eine Verschlimmerung der obstruktiven Atemprobleme im Schlaf zu vermeiden. 31 % aller Hunde können nur schlafen, wenn sie ihr Kinn erhöht ablegen können; 24 % aller Hunde versuchen zeitweise im Sitzen zu schlafen und ver-meiden das Hinlegen, 6 % können nur mit geöffnetem Maul schlafen, da sie durch die pathologisch verkürzte Nase beim Schlafen keine Luft bekommen. Französische Bulldoggen haben signifikant häufiger Probleme beim Fressen als Möpse. 46 % aller Hunde zeigen Probleme im Zusammenhang mit der Futteraufnahme (Französische Bulldoggen zu 59 %, Möpse zu 38 %). Verschlucken und Erbrechen bzw. Regurgitieren sind sehr häufig genannte Probleme. Rückwärtsniesen (reverse sneezing) scheint ein weiteres durch Brachyzephalie ausge-löstes Problem zu sein. 73% der brachyzephalen Hunde zeigen Rückwärtsniesen, 25% davon täglich. Bisher ist vermehrtes Rückwärtsniesen als Symptom des Brachyzepha-len Syndroms in der Literatur nicht beschrieben. Alle brachyzephalen Hunde dieser Studie litten an tierschutzrelevanten Einschränkungen elementarer Grundbedürfnisse. Die teilweise hochgradigen Störungen in den Be-reichen Atmung, Bewegung, soziale Interaktion, Futteraufnahme, Schlafen und Thermo-regulation reduzieren die Lebensqualität erheblich. Alle Tiere hatten in mindestens einem der untersuchten Bereiche deutliche Einschränkungen. Diese Untersuchung wirft die Frage auf, ob die Zucht brachyzephaler Hunde mit den Ansprüchen eines modernen Tierschutzes überhaupt vereinbar ist.