Nas últimas décadas, diversos estudos têm demonstrado os efeitos nocivos dos ácidos graxos trans à saúde. Consequentemente, diversas agências reguladoras de saúde e sociedades responsáveis pela elaboração de diretrizes nutricionais recomendaram a redução do consumo desses ácidos graxos. Deste modo, a indústria de alimentos vem adequando seus produtos a fim de substituir os ácidos graxos trans por gorduras interesterificadas, porém seus efeitos sobre o desenvolvimento da aterosclerose não foram ainda totalmente elucidados. Portanto, o objetivo deste estudo foi avaliar o efeito de gorduras interesterificadas contendo principalmente ácido graxo palmítico ou esteárico sobre o desenvolvimento da aterosclerose. Desta forma, camundongos knockout para o receptor de LDL (LDLr-KO) recém-desmamados foram alimentados por 16 semanas com dietas hiperlipídicas (40% do valor calórico total sob forma de gordura) contendo principalmente ácidos graxos poli-insaturados (POLI), trans (TRANS), palmítico (PALM), palmítico interesterificado (PALM INTER), esteárico (ESTEAR) ou esteárico interesterificado (ESTEAR INTER) para determinação de concentrações plasmáticas de colesterol total e triglicérides; perfil de lipoproteínas; conteúdo de lípides (Oil Red O) e colágeno (Picrosirius Red) e infiltrado de macrófagos (imuno-histoquímica) na área de lesão aterosclerótica; expressão e conteúdo proteico de citocinas na aorta; dosagem das citocinas secretadas por macrófagos de peritônio estimulados ou não com lipopolissacarídeo (LPS); efluxo celular de colesterol mediado pela apo-AI e HDL2. Os resultados mostraram que os animais que consumiram a gordura interesterificada contendo ácido palmítico (PALM INTER) desenvolveram importante lesão aterosclerótica em comparação aos grupos PALM, ESTEAR, ESTEAR INTER e POLI, resultados confirmados pelo conteúdo de colágeno na lesão. Apesar do processo de interesterificação não ter alterado as concentrações plasmáticas de lípides, conforme verificado entre os grupos PALM vs PALM INTER e ESTEAR vs ESTEAR INTER, o acúmulo de colesterol na partícula de LDL foi similar entre os grupos PALM INTER e TRANS. Além desse efeito sobre o perfil de lipoproteínas, macrófagos do peritônio de camundongos que consumiram PALM INTER secretaram significativamente mais IL-1beta, IL-6 e MCP-1 em comparação aos demais grupos. Esse efeito pró-inflamatório foi confirmado na aorta, onde se observou maior expressão de TNF-alfa e IL-1beta para o grupo PALM INTER em comparação a PALM. Tal insulto inflamatório foi similar ao provocado por TRANS. Esses efeitos deletérios do PALM INTER podem ser parcialmente atribuídos ao acúmulo de colesterol nos macrófagos, promovido pelo prejuízo no efluxo de colesterol mediado pela apo-AI e HDL2, bem como aumento da expressão de receptores envolvidos na captação de LDL modificada (Olr-1) e diminuição daqueles envolvidos na remoção intracelular de colesterol (Abca1 e Nr1h3) na parede arterial. Como conclusão, as gorduras interesterificadas contendo ácido palmítico favorecem o acúmulo de colesterol nas partículas de LDL e em macrófagos, ativando o processo inflamatório, o que conjuntamente contribuiu para maior desenvolvimento de lesão aterosclerótica / In recent decades, several studies have shown harmful effects of trans fatty acids on human health. Consequently, simultaneous actions from Health Agencies and Societies responsible for the elaboration of Nutritional Guidelines recommended the reduction in the intake of trans fatty acids. Thus, the food industry is adapting their products to replace these fatty acids by substituting them for interesterified fats, however, the effects of the latter on atherosclerotic lesion development are unknown. Therefore, the aim of this study was to evaluate the effect of interesterified fats containing palmitic or stearic fatty acids on atherosclerosis development. For this purpose, weaning male LDL-c receptor knockout (LDLr-KO) mice were fed a high fat diet (40% of total daily energy intake from fat) enriched in polyunsaturated (PUFA), TRANS, palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER) fat for 16 weeks to determine several parameters: total cholesterol and triglycerides plasma concentrations; lipoprotein profile; lipid (Oil Red O) and collagen (Picrosirius Red) contents, as well as macrophage infiltration (immunohistochemistry) in the atherosclerotic lesion; cytokine transcription and protein content in the aorta; cytokines secreted from peritoneal macrophages stimulated or not with lipopolysaccharide (LPS); apo-AI and HDL2-mediated cellular cholesterol efflux. The results showed that mice fed PALM INTER presented greater atherosclerotic lesion as compared to PALM, STEAR, STEAR INTER and PUFA, which was confirmed by the collagen content in the aortic lesions. Although the interesterification process did not modify the plasma lipid concentration, as verified by the comparisons PALM vs PALM INTER and STEAR vs STEAR INTER, the cholesterol accumulation in LDL particle was similar in PALM INTER and TRANS. Peritoneal macrophages from PALM INTER-fed mice secreted significantly more IL-1beta, IL-6 and MCP-1 as compared to all other groups. This pro-inflammatory effect was confirmed in the aorta, in which PALM INTER presented higher TNF-alfa and IL-1beta expressions as compared to PALM. This inflammatory insult was similar to that elicited by TRANS. All these deleterious effects of PALM INTER can be partially attributed to macrophage cholesterol accumulation, due to impaired apo-AI and HDL2-mediated cholesterol efflux, as well as to higher expression of receptors involved in modified LDL uptake (Olr-1), together with a reduction in those involved in cholesterol removal (Abca1 e Nr1h3) in the arterial wall. In conclusion, interesterified fats containing palmitic acid promotes cholesterol accumulation in LDL particles and in macrophages, activating the inflammatory process, which contributed to atherosclerosis development

Introdução: as gorduras interesterificadas, ricas em ácidos graxos saturados, vêm sendo utilizadas pela indústria alimentícia em substituição aos ácidos graxos trans. Os principais ácidos graxos saturados empregados no processo de interesterificação são o palmítico e o esteárico, os quais podem, respectivamente, apresentar efeitos deletérios ou serem neutros, do ponto de vista cardiovascular e metabólico. A quantidade de ácido esteárico na dieta é menor em comparação ao palmítico, e não está elucidada a implicação de seu alto consumo. O propósito deste estudo foi avaliar o efeito das gorduras interesterificadas contendo ácido palmítico ou ácido esteárico sobre as principais vias de sinalização envolvidas no metabolismo do tecido hepático e adiposo. Métodos: camundongos machos LDLr-KO foram alimentados durante 16 semanas com dieta hiperlipídica (40% de energia em forma de gordura) contendo maior concentração de poli-insaturados (POLI), palmítico (PALM), palmítico interesterificado (PALM INTER), esteárico (ESTEAR) ou esteárico interesterificado (ESTEAR INTER). Determinaram-se a composição corporal, conteúdo de gordura no fígado, concentração plasmática de colesterol, triglicérides, glicose e insulina. A expressão de genes envolvidos no metabolismo lipídico e vias inflamatórias no tecido adiposo visceral e hepático foi determinada por RT-qPCR. Além disso, a síntese de proteínas e proteínas fosforiladas foi medida por Imunnoblotting. O infiltrado de células inflamatórias no tecido hepático e histologia do tecido adiposo, bem como o conteúdo de colágeno do fígado, foram determinados por coloração com eosina e hematoxilina e Sirius Red, respectivamente. Resultados: o processo de interesterificação não alterou parâmetros bioquímicos plasmáticos e as quantidades de CT e TG no tecido hepático. No entanto, ambas as gorduras interesterificadas induziram maior conteúdo de colágeno no fígado e fosforilação de JNK. Adicionalmente, o grupo ESTEAR INTER desenvolveu NASH, associada a maior infiltrado de neutrófilos. Por outro lado, PALM INTER induziu maior expansão do tecido adiposo, juntamente com hipertrofia de adipócitos, condições associadas à ativação da via de sinalização \"upstream\" do NFkB, observada para a fosforilação de IKK, o que contribuiu para maior teor de TNFalfa. Conclusão: as gorduras interesterificadas enriquecidas com ácido palmítico induziram fibrose hepática, bem como a expansão do tecido adiposo, com hipertrofia dos adipócitos; e as gorduras contendo ácido esteárico induziram NASH em camundongos LDLr-KO / Background: interesterified fats, rich in saturated fatty acids, have been used by the food industry as a substitute for trans fatty acids. The main saturated fatty acids used in the interesterification process are palmitic and stearic, which may, respectively, have deleterious effects or be cardiovascular and metabolic neutral. Objetive: to evaluate the effect of interesterified fats containing palmitic or stearic acids on signaling pathways involved in the hepatic and adipose tissue lipid metabolism. Methods: Weaning male LDLr-KO mice were fed a high-fat diet (40% energy as fat) containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER) fats for 16 weeks. Body composition, liver fat content, total plasma cholesterol, triglycerides, glucose and insulin concentrations were determined. Gene expression involved in lipid metabolism and inflammation process in liver and white adipose tissue was determined by quantitative RT-PCR. Furthermore, amount of proteins and activated proteins were measured by Western blot. Hepatic and adipose tissue inflammatory cells infiltration as well as liver collagen content were determined by eosin and hematoxylin and Sirius Red staining, respectively. Results: Interesterified process did not alter plasma biochemical parameters and amounts of TC and TG in hepatic tissue. However, both interesterified fats brought on a higher liver collagen content and JNK phosphorylation. Additionally, STEAR INTER group developed NASH associated with a higher neutrophil infiltration. On the other hand, PALM INTER induced a higher adipose tissue expansion together with the enlargement of adipocytes, conditions associated with the activation of upstream NFkB signaling pathway as observed for IKK phosphorylation, contributing to higher TNFalpha protein content. Conclusions: Interesterified fats containing stearic acid induced NASH and enriched with palmitic acid trigger on steatosis with fibrosis as well as hypertrophy and inflammatory signaling activation in the adipose tissue in LDLr-KO mice

A fertilização in vitro tem sido associada com um aumento na incidência das hipospádias, e alguns hormônios esteróides usados em seus protocolos têm sido implicados neste processo. Para testar essas hipóteses em um modelo animal, descrevemos neste trabalho as alterações morfológicas ocorridas no tubérculo genital de camundongos, expostos à progesterona durante a vida intrauterina. Foi administrado acetato de medroxiprogesterona por via subcutânea no período pré-natal em animais normais e animais desprovidos de receptores androgênicos (Tfm). A progesterona induziu a formação de hipospádia nos animais do sexo masculino, virilização nos do sexo feminino e não causou alterações nos animais Tfm / In vitro fertilization (IVF) has been associated with an increase incidence of hypospadias. IVF protocols require the maternal use of progesterone which may be a factor in causing hypospadias. To test these hypotheses in an animal model, we describe the effects of maternal progesterone exposure on genital development in mice. Medroxyprogesterone acetate (MPA) was administered by subcutaneous injection during the pre-natal period to wild type mice and animals knockout to androgen receptors (Tfm mice). Progesterone caused hypospadias in male mice fetuses, a virilizing effect in the female mice genitalia and didn't have any effect in Tfm animals

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Light and ultraestructural changes of central and peripheral nervous system lesions in mice KO for connexin-32 and submitted to the ethidium bromide gliotoxic demyelinating model are described. Their KO condition was tested with PCR and a negative connexin-32 labelling was performed by immunofluorescence. The experimental animals were C57BL/6 normal mice and C57BL/6 KO for connexin-32. For all groups the animals were maintained in cages of 5 individuals within a temperature controlled room and had ration and filtered water ad libitum. A single local injection of either 0,1% ethidium bromide in normal saline (5 μl in the brainstem and 1 μl in the sciatic nerve) or normal saline was performed as described for Wistar rats. The injected mice were observed daily until euthanasia was performed at 24, 48 hours and 3, 7, 15, 21 and 30 days after injection. The mice were perfused through the heart with either neutral 10% formalin or 2,5% glutaraldehyde. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopic methods were used to analyze the development of the lesions after differentiated processing. Hematoxylin- eosine, luxol fast blue and toluidine blue methods and immunolabelling with anti-GFAP, anti-CNPase, anti-S100 protein and anti-OSP, anti Cx32 and anti Cx43 antibodies were used. Within the CNS the lesions showed an acute degenerative phase with disappearance of glial cells, and myelin sheaths were withdrawn by a scant number of macrophages. In KO mice some granulocytes were detected within the lesions in tight contact with decaying myelin sheaths. Remyelination was carried out by oligodendrocytes since no Schwann cells were seen during the regenerating process of KO mice. Occasional remyelinating Scwann cells were seen in normal mice. For the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compaction and redundant loops (tomacula) were conspicuous. Mast cells degranulated or not were seen in all BE- induced lesions and after saline injection. It is concluded that the repair of the CNS demyelinated lesions differs from the observed in normal and immunosupressed rats because Schwann cells remyelination was absent; the absence of connexin-32 may have caused that absence. The regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other species. It is suggested that the absence of connexin-32 determined the different repair of the myelin sheaths within the CNS whereas for the PNS, the normal pattern of tissue response might be due to the early age of the injected mice. / São descritas as alterações de microscopia de luz e ultra-estruturais induzidas pelo brometo de etídio no sistema nervoso central e periférico de camundongos KO para conexina 32. O genótipo KO foi testado por PCR e confirmado por imunofluorescência negativa para conexina 32. Os animais dos experimentos foram camundongos C57BL-6 normais (controles) e KO para conexina 32. Todos os camundongos foram mantidos em gaiolas de 5 indivíduos em sala climatizada e receberam ração e água à vontade. Uma única injeção de BE 0,1% em salina 0,9% ou de salina 0,9% (5 μl na cisterna basal e 1μl no nervo ciático) foi realizada como descrita em ratos Wistar. Os camundongos eram observados diariamente até ser realizada a eutanásia às 24 e 48 h, 3, 7, 15, 21 e 30 dias após a injeção. Os camundongos foram perfundidos através do coração; um grupo com glutaraldeído 2,5% visando o processamento para microscopia eletrônica; um outro grupo com solução salina com EDTA e posterior fixação em metacarn para inclusão em parafina. As amostras incluídas em parafina foram analisadas através dos métodos de hematoxilina e eosina, luxol fast blue e azul de toluidina. Foram realizadas imunoistoquímica e imunofluorescência visando a marcação de GFAP, CNPase, OSP, S100, e Cx43 e Cx32, respectivamente. As lesões do SNC eram discretas e tiveram uma fase ativa com desaparecimento das células gliais; os debris celulares e de mielina foram retirados por um reduzido número de fagócitos. Nos camundongos KO foram vistos granulócitos em estreito contato com bainhas de mielina em degradação. A remielinização dos axônios desmielinizados foi realizada exclusivamente por oligodendrócitos nos camundongos KO; nos camundongos normais, ocasionais células de Schwann podiam ser encontradas remielinizando axônios do SNC. No nervo ciático, as células de Schwann intoxicadas rejeitaram seus internodos de mielina; após sete dias, finas bainhas reparadas eram encontradas, com compactação irregular da mielina e alças redundantes (tomacula). Mastócitos, desgranulados ou não, eram vistos nas lesões do BE e após a injeção de solução salina. Conclui-se que o reparo das lesões do SNC difere do observado em ratos normais e imunossuprimidos devido à ausência de remielinização por células de Schwann; a falta de expressão da Cx 32 e o tamanho reduzido das lesões podem ter contribuído para essa ausência. A regeneração das bainhas perdidas no SNP obedeceu ao padrão descrito para esse modelo em outras espécies. Sugere-se que a ausência da Cx 32 não afetou o reparo do SNP devido à idade precoce dos animais.

Le récepteur opioïde mu est responsable des propriétés analgésiques et addictives puissantes de la morphine et de l’héroïne, mais son mode d’action à l’échelle des circuits neuronaux est mal connu et a été peu étudié par des approches génétiques. Le récepteur mu est largement exprimé dans le système nerveux, essentiellement dans des neurones GABAergiques. Le premier objectif de mon projet a été d’inactiver le gène codant pour le récepteur mu dans les neurones GABAergiques du cerveau antérieur et d’en étudier les conséquences comportementales. Notre étude montre que ces récepteurs ne sont pas impliqués dans l’analgésie et la dépendance physique à la morphine, mais qu’ils sont essentiels à l’effet hyperlocomoteur de l’héroïne. De plus, nos résultats indiquent que ces récepteurs limitent la motivation à consommer de l’héroïne et du chocolat, révélant un rôle entièrement nouveau pour cette population particulière de récepteurs (Manuscrit 1 : Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Aussi, cette population de récepteurs mu n’est pas responsable du syndrome autistique décrit chez les souris knockout totales (Manuscrit 2 : Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Enfin, nous avons développé un nouveau modèle transgénique visant l’inactivation génétique du récepteur mu dans les neurones glutamatergiques, mais qui n’a pas abouti à un knockout conditionnel détectable. Nous avons aussi initié la création d’une lignée transgénique Cre pour l’inactivation de gènes d’intérêt dans l’amygdale étendue, qui permettra notamment d’étudier le rôle du récepteur mu dans ce microcircuit. / Mu opioid receptors mediate the strong analgesic and addictive properties of morphine and heroin;however mu receptor function at circuit levels is not well understood and has been poorly studied by genetic approaches. These receptors are widely expressed throughout the nervous system, essentially in GABAergic neurons. The first aim of my project was to genetically inactivate the mu receptor gene in GABAergic forebrain neurons and study the behavioral consequences. Our study shows that these mu receptors are not implicated in morphine-induced analgesia and physical dependence, but are essential for locomotor effects of heroin. Moreover, our data show that these receptors inhibit motivation to consume heroin and chocolate, revealing an entirely new role for this particular population of mu receptors (Manuscript 1: Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Also, mu receptors expressed in forebrain GABAergic neurons are not responsible for the autistic syndrome described in total mu receptor knockout mice (Manuscript 2: Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Finally, we developed a new transgenic model targeting the mu receptor gene in glutamatergic neurons, but receptor deletion was not detectable in conditional mice. We also initiated the creation of a transgenic Cre driver line to knockout genes of interest in the extended amygdala, and this tool will enable us to study mu receptor function within this microcircuit.

Les facteurs spectroscopiques nous renseignent sur l'occupation des couches nucléaires et peuvent être extraits par des réactions directes comme le transfert à basse énergie et le knockout aux énergies intermédiaires. L’étude récente de noyaux radioactifs de la couche sd ayant une large différence d'énergie de séparation, DeltaS=Sp-Sn~20 MeV, montre que les sections efficaces d’arrachage d’un nucléon très lié sont considérablement réduites par rapport aux prédictions théoriques. Cette tendance n’est pas observée pour des noyaux moins exotiques, jusqu’à DeltaS~12 MeV, par réaction de transfert (d,p).Pour comprendre l'origine de cette réduction, nous avons réalisé deux expériences complémentaires sur un noyau présentant une large différence d’énergie de séparation l’14O : (i) le knockout d'un nucléon au NSCL, 14O (53 MeV/n) et 16C (75 MeV/n), sur une cible de 9Be; (ii) le transfert d'un nucléon avec le faisceau d’14O à 18 MeV/n de SPIRAL, 14O(d,t)13O et 14O(d,3He)13N, étudié avec le dispositif MUST2. L'analyse des données présentée en détails dans ce manuscrit conduit à des facteurs spectroscopiques incompatibles entre ces deux expériences lorsqu'un neutron fortement lié est enlevé ou transféré de l'14O. Dans le cas du knockout, la section efficace mesurée est fortement réduite par rapport aux prédictions basées sur un modèle eikonal et le modèle en couches. La distribution en moment parallèle de l’13O mesurée après l’arrachage d’un neutron présente par ailleurs une forme qui n’est pas reproduite par le modèle eikonal. Dans le cas du transfert, une telle réduction n'est pas observée et le rapport entre expérience et théorie est compatible avec ce qui est obtenu pour les noyaux stables. Ces résultats suscitent de nouveaux développements théoriques pour la modélisation du mécanisme de réaction dans ces cas extrêmes où le nucléon enlevé est fortement lié. / The distribution of spectroscopic strength in nuclei can be extracted from direct-reaction cross section measurements, as one-nucleon knockout at intermediate energy or transfer at low energy. The study of deeply-bound nucleon removal from several sd-shell nuclei having a large difference of proton-neutron separation energies, DeltaS=Sp-Sn~20 MeV, exhibits experimental cross sections about four times smaller than theoretical predictions from state-of-the-art calculations. This trend is not observed from (d,p) transfer reactions with nuclei having smaller separation energy asymmetry, DeltaS~12 MeV.To investigate the origin of this reduction, we have performed two complementary experiments for the 14O case having a large energy asymmetry: (i) one-nucleon knockout from 14O (53 MeV/n) and 16C (75 MeV/n) on a 9Be target at the NSCL; (ii) one-nucleon transfer reaction using SPIRAL beam, 14O(d,t)13O and 14O(d,3He)13N at 18 MeV/n, and the MUST2 array. The analysis of the data presented in this document leads to a discrepancy between the spectroscopic factors extracted from these two experiment when a deeply-bound neutron is removed from 14O. In the neutron knockout from 14O, the cross section is strongly reduced compared to predictions based on an eikonal model and shell model spectroscopic factors. Moreover, several deviations from the eikonal prediction are observed on the shape of the parallel momentum distribution of the ejectile 13O. For the transfer 14O(d,t)13O, such a reduction is not observed and results are in agreement with stable nuclei values. These results call for new theoretical developments concerning the description of the reaction mechanism when a deeply-bound nucleon is removed from a nucleus.

No description available.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age).

1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...

Tau ist ein Mikrotubuli-assoziiertes Protein, das die Polymerisation von Tubulin fördert und die Mikrotubuli stabilisiert. Folglich wird angenommen, dass Tau essentiell für die neuronale Morphogenese ist, vor allem für die Axonenausbildung und -aufrechterhaltung. Mittels tangentieller Nissl-gefärbter Schnitte von Mäusegehirnen konnte in der vorliegenden Arbeit gezeigt werden, dass Tau-knockout Mäuse die regelhafte thalamokortikale Barthaar-projektion („Barrel“ Konfiguration) entwickeln. Der Einfluss von Tau auf die Entstehung von Dendriten wurde anhand von Golgi-gefärbten Präparaten untersucht. Die Sholl-Analyse der gefärbten CA1-Pyramidenzellen zeigte, dass die Komplexität apikaler Dendriten durch das Fehlen von Tau reduziert wurde, während die Basaldendriten unbeeinflusst blieben. Das Tau-Protein scheint demzufolge unwesentlich für die Entstehung von axonalen Verbindungen im embryonalen Gehirn zu sein, ist aber beteiligt an der Steuerung des dendritischen Verzweigungsmusters. Ferner wurde beobachtet, dass sowohl die adulte Neurogenese, als auch die Mikrotubuli-Stabilität in den Apikal- und Basaldendriten und die Synapsen von dem Fehlen des Tau-Proteins unbeeinflusst blieben. In primären Zellkulturen aus dem Kleinhirn von Tau-knockout und Tau-wildtyp Mäusen konnten zwischen den zwei Genotypen keine signifikanten Unterschiede in der Länge oder im Verzweigungsmuster der Dendriten und der Axone von Körnerzellen beobachtet werden. Die Untersuchung der Effekte einzelner Tau-Isoformen auf die Morphologie von N2A-Zellen zeigte, dass es Unterschiede sowohl zwischen Tau-defizienten und Tau-positiven Zellen, als auch zwischen Zellen mit den verschiedenen Tau-Isoformen gibt. Das Tau-Protein übt demnach in vivo einen wichtigen Einfluss auf die Morphologie der Nervenzellen und besonders der Dendriten aus, welcher in vitro weiter analysiert wurde.:Abkürzungsverzeichnis 1 Einleitung 1 1.1 Das Mikrotubuli-assoziierte Protein Tau 1 1.2 Bedeutung des Tau-Proteins beim Menschen: Tauopathien 3 1.3 Bedeutung des Tau-Proteins beim Menschen: Mikrodeletion des MAPT-Lokus 4 1.4 Ergebnisse aus bisherigen Studien mit Tau-knockout Tieren 6 1.5 Aufgabenstellung 7 2 Material und Methoden 9 2.1 Material 9 2.1.1 Versuchstiere 9 2.1.2 Chemikalien 9 2.1.3 Häufig verwendete Lösungen 10 2.1.4 Geräte 10 2.2 Histologie 11 2.2.1 Fixierung 11 2.2.2 Golgi-Einzelschnittimprägnierung 11 2.2.3 Gefrierschnitte 11 2.2.4 Nissl-Färbung 12 2.2.5 Immunhistochemische Markierungen 13 2.3 Morphometrie 15 2.3.1 Sholl-Analyse 15 2.3.2 Volumenbestimmung 16 2.3.3 Zellzahl (Neurogenese) 17 2.3.4 Synapsenzahl 17 2.4 Proteinbiochemie 19 2.4.1 Proben 19 2.4.2 SDS-Polyacrylamid-Gelelektrophorese 19 2.4.3 Western Blot 20 2.4.4 Immundetektion am Western Blot 21 2.5 Transfektion von Nervenzellen in primärer Zellkultur 25 2.5.1 Primäre Zellkultur 25 2.5.2 Transfektion von primären Zellkulturen 25 2.5.3 Morphometrische Analyse von Körnerzellen des Kleinhirns 26 2.6 Klonierung von Tau-Protein-Isoformen 27 2.6.1 Klonierungsstrategie zur Herstellung eines pIRES-DsRed-Tau Vektors 27 2.6.2 Agarose-Gelelektrophorese 30 2.6.3 Gelextraktion der verschiedenen Tau-Isoform-Sequenzen 30 2.6.4 Herstellung chemisch kompetenter E.coli Zellen 31 2.6.5 Chemische Transformation kompetenter E. coli Zellen 32 2.6.6 Animpfen 32 2.6.7 Plasmid-DNA Purifikation aus 15ml Medium („Miniprep”) 32 2.6.8 Schneiden mit Restriktionsendonukleasen 33 2.6.9 Plasmidpräparation aus 100 ml Medium („Midiprep“) 33 2.6.10 Transfektion von N2A-Zellen mit pIRESRed-Tau 34 2.6.11 Rekonstruktion der transfizierten N2A-Zellen 35 2.7 Statistische Auswertung 36 3 Ergebnisse 37 3.1 Thalamokorticale Projektionen 37 3.2 Komplexität der Dendriten von CA1-Pyramidenzellen 37 3.3 Adulte Neurogenese im Hippocampus 41 3.4 Volumen des Hippocampus 43 3.5 Glia 45 3.6 Synapsen 46 3.7 Stabilität der Mikrotubuli 48 3.8 Mikrotubuli-assoziierte Proteine 50 3.9 Entwicklung in vitro 52 3.9.1 Primärkulturen 52 3.9.2 N2A-Zellkultur 54 4 Diskussion 58 4.1 Diskussion der Methoden 58 4.1.1 Herstellung von Tau-knockout Mäusen 58 4.1.2 Golgi-Einzelschnittimprägnierung und die dreidimensionale Zellrekonstruktion 59 4.1.3 Neurogenese 60 4.1.4 Volumenbestimmung von Hippocampus und Gyrus dentatus 61 4.1.5 Synaptische Marker 61 4.1.6 Western Blot 62 4.1.7 Transfektion von primären Zellkulturen 62 4.1.8 Transfektion von N2A-Zellen mit humanen Tau-Isoformen 63 4.2 Vergleich mit bekannten Daten aus der Forschungsliteratur 64 4.2.1 Axonogenese 64 4.2.2 Dendritogenese 64 4.2.3 Mikrotubuli-assoziierte Proteine und Mikrotubuli-Stabilität 67 4.2.4 Neurogenese 67 4.2.5 Synaptogenese 68 4.2.6 Rolle der einzelnen Isoformen 68 4.3 Bedeutung für die Medizin 71 4.4 Fazit 72 5 Zusammenfassung 73 6 Literaturverzeichnis 76 Posterpräsentationen 88 Danksagung 89 Erklärung über die eigenständige Abfassung der Arbeit 90 Lebenslauf 91