Delivery of macromolecules into mammalian cells in vitro and in vivo has enabled new areas of research and offers the potential for powerful new treatment options. Recent research has generated many delivery platforms but these solutions remain limited by scope, potency and safety. We have reported a superpositively charged green fluorescent protein (+36 GFP) with the ability to deliver nucleic acids into a variety of mammalian cell lines in vitro and to potently deliver protein in vitro and in vivo without toxicity. These results have directed us to identify a subset of naturally occurring human proteins with similar but previously unknown cell-penetrating and protein delivery properties. Preliminary efforts have been made towards establishing the therapeutic potential for supercharged proteins replacement of the cytosolic enzyme, argininosuccinate synthase. Preliminary efforts have also been made towards enhancing endosomal escape with \(His_{39} GFP\).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20661号 / 医博第4271号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 柳田 素子, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O balanço redox em mitocôndrias de mamíferos é realizado pelo transportador de aspartato-glutamato (AGC), o qual é o principal mecanismo para o movimento de equivalentes redutores na forma de NADH. A citrulinemia do tipo II (CTLN2) é uma doença autossômica recessiva de início tardio, causada por mutações no gene SLC25A13 que codifica a citrina. A citrina é uma isoforma do transportador AGC e catalisa o transporte de glutamato citosólico através da troca com o aspartato mitocondrial, o qual será utilizado no ciclo da ureia. A CTLN2 promove uma deficiência no ciclo da ureia e consequente hiperamonemia. A deficiência da citrina promove um aumento da razão NADH/NAD+ citosólica. O aumento dessa razão inibe a glicólise e a gliconeogênese. O desenvolvimento de modelo in vitro da CTLN2 é importante para estudos do mecanismo da doença e de novas terapias. A expressão heteróloga de proteínas entre diferentes reinos tem sido utilizado como uma forma de corrigir algumas doenças mitocondriais. Estudos bioquímicos e moleculares em nosso laboratório demonstraram a presença de um transportador mitocondrial de nicotinamida adenina dinucleotídeo (Ndt1) em Aspergillus fumigatus. Ndt1 realiza o transporte de NAD+ citosólico para a matriz mitocondrial, sendo dessa forma uma proteína importante para manter o balanço redox em A. fumigatus. Assim, o objetivo deste trabalho foi obter uma linhagem de células de mamífero HEK293 com knockdown para o gene SLC25A13, ou seja, um modelo in vitro de CTLN2 e a expressão heteróloga da proteína Ndt1 como uma forma de recuperação do metabolismo. As células com knockdown para o gene SLC25A13 apresentaram um aumento da razão NADH/NAD+ citosólico, redução da glicólise, redução da concentração da ureia e aumento da concentração de amônia. A expressão de Ndt1 foi capaz de reduzir a razão NADH/NAD+ citosólico e recuperou a atividade glicolítica. Entretanto, a expressão de Ndt1 não foi capaz de aumentar a concentração de ureia e reduzir a concentração de amônia causadas pela CTLN2. Dessa forma, nossos resultados sugerem que a expressão da proteína Ndt1 em células de mamíferos recupera o metabolismo mitocondrial e atividade glicolítica das células com CTLN2, mas não melhora o ciclo da ureia e o aumento da concentração de amônia. / Redox balance in mammalian mitochondria is performed by the aspartate-glutamate carrier (AGC), which is the main mechanism for the movement of reducing equivalents in the form of NADH.Type II citrullinemia (CTLN2) is an adult-onset autosomal recessive disease caused by mutations in SLC25A13 gene, and that coding citrin. Citrin is an isoform of AGC and catalyzes the transport of cytosolic glutamate through exchange with mitochondrial aspartate. It will be used in the urea cycle. CTLN2 causes urea cycle deficiency and hyperammonemia. Citrin deficiency cause an increase in the cytosolic NADH/NAD+ ratio. The increase in this ratio inhibits glycolysis and gluconeogenesis. The development an in vitro CTLN2 model is important for new studies about the disease mechanism and new therapies. Heterologous expression of proteins from different organisms has been used to recover some mitochondrial diseases. Biochemical and molecular studies in our laboratory demonstrated the presence of a mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) in Aspergillus fumigatus. Ndt1 protein performs cytosolic NAD+ transport to the mitochondria matrix, thus being an important protein to keep the redox balance in A. fumigatus. Thus, the aim of this work was to obtain a line of HEK293 mammalian cells with knockdown for the SLC25A13 gene, an in vitro CTLN2 model and the heterologous expression of the Ndt1 protein as a form of metabolism recovery. Cells with citrin knockdown showed an increase of cytosolic NADH/NAD+ ratio, reduction of glycolysis, reduction of urea concentration, and increase of ammonia concentration. Expression of Ndt1 protein was able to reduce cytosolic NADH/NAD+ ratio and recovered the glycolytic activity. However, Ndt1 protein was not able to increase the urea concentration and reduce of ammonia concentration caused by CTLN2. Thus, our results suggest that expression of Ndt1 protein in mammal cells recovers the mitochondrial metabolism and glycolytic activity in CTLN2 cells but does not improve urea cycle and reduce ammonia concentration.

L'intestin grêle à un rôle vital, il est impliqué dans de multiples fonctions : absorptive, endocrinienne, lymphoïde, barrière, et dans le métabolisme de l'arginine. L'exploration du grêle est difficile chez les patients graves hospitalisés en réanimation, alors même que ces patients sont à risque d'avoir une dysfonction intestinale aiguë du fait de la sensibilité du grêle à l'ischémie. La découverte et la validation de biomarqueurs entérocytaires, l'un de fonction, la citrullinémie, l'autre de lyse entérocytaire, l'intestinal fatty acid-binding protein (I-FABP), pourrait permettre de mieux évaluer l'intégrité et la fonctionnalité du grêle chez les patients graves. Les objectifs de cette thèse étaient de décrire la prévalence des anomalies entérocytaires chez des patients graves, de comprendre les mécanismes enjeu dans l'atteinte entérocytaire aigiie, d'évaluer la valeur pronostique de ces biomarqueurs, et d'évoquer des perspectives. Entre 2007 et 2013, nous avons mesuré la citrullinémie et/ou l'I-FABP chez des patients hospitalisés au CHU de Besançon ayant un risque théorique de souffrance entérocytaire : patients admis en réanimation, patients nécessitant un pontage aortocoronarien, patients nécessitant une anesthésie générale. Des anomalies de biomarqueurs entérocytaires étaient présentes chez plus de la moitié des patients hospitalisés en réanimation dès leur admission. Il s'agissait d'une citrullinémie basse et/ou d'une concentration plasmatique élevée d'I-FABP. La synthèse des résultats obtenus fait apparaître qu'une cytolyse entérocytaire précoce et courte est fréquente chez le patient admis en réanimation (élévation de la concentration plasmatique d'I-FABP), puis diminue avec la stabilisation clinique des patients. Ce pic initial d'I-FABP semble suivi d'une diminution de la citrullinémie au cours des 48 premières heures d'hospitalisation en réanimation. D'autre part, le sepsis, qu'il soit ou non d'origine digestive, est associé à une diminution de la citrullinémie, ce qui suggère une entéropathie septique indépendante du champ nosologique de la destruction entérocytaire ischémique. Le couple de biomarqueurs, l'un de lyse cellulaire (I-FABP), l'autre de fonction cellulaire (citrulline), semble permettre de caractériser des altérations entérocytaires aiguës du patient grave, en estimant les degrés de destruction ou de dysfonction entérocytaire, et leurs évolutions. Nos études ont mis en évidence une surmortalité chez les patients qui ont une citrullinémie basse et/ou une concentration plasmatique d'I-FABP élevée. La valeur pronostique de l'atteinte entérocytaire nécessite toutefois d'être confirmée par d'autre groupes. Si la compréhension des modifications des valeurs de ces biomarqueurs entérocytaires progresse, avec les limites d'interprétation de leurs variations, et si leur dosage devient plus rapide du fait d'une automatisation de la technique permettant un rendu rapide de résultat, ils pourraient devenir un outil de diagnostic et d'adaptation rapide de la prise en charge thérapeutique des patients graves. / The small bowel is a complex organ involved in numerous vital functions. The small bowel dysfunction is suspected to be the cornerstone of the development of multiple organ dysfunction syndrome in critically ill patients. The development of enterocyte biomarkers is promising for evaluating the small bowel dysfunction and damage in the most severe patients. Plasma citrulline concentration is a validated marker of small bowel function, reflecting the functional enterocyte mass. Plasma citrulline concentration is frequently decreased in critically ill patients, and a decreased plasma citrulline concentration at ICU admission is associated with elevated plasma CRP concentration, and with increased 28-day mortality. Intestinal fatty acid binding protein (I-FABP) is a small cytosolic protein that is released in extracellular space in case of enterocyte destruction. In critically ill patients plasma I-FABP concentration is increased in about one half of patients at ICU admission. Increased plasma I-FABP concentration is associated with shock state, and with elevated catecholamine dose. Both biomarkers are probably complementary and may help the clinician to identify patients presenting enterocyte damage. Further studies are needed to improve the interpretation of plasma citrulline concentration which is probably complex in critically ill patients.

Themitochondrialmalate aspartate shuttle system(MAS)maintains the cytosolicNAD+/NADH redox balance, thereby sustaining cytosolic redox-dependent pathways, such as glycolysis and serine biosynthesis. Human disease has been associated with defects in four MAS-proteins (encoded by MDH1, MDH2, GOT2, SLC25A12) sharing a neurological/epileptic phenotype, as well as citrin deficiency (SLC25A13) with a complex hepatopathic-neuropsychiatric phenotype. Ketogenic diets (KD) are high-fat/low-carbohydrate diets, which decrease glycolysis thus bypassing the mentioned defects. The same holds for mitochondrial pyruvate carrier (MPC) 1 deficiency, which also presents neurological deficits. We here describe 40 (18 previously unreported) subjects with MAS-/MPC1-defects (32 neurological phenotypes, eight citrin deficiency), describe and discuss their phenotypes and genotypes (presenting 12 novel variants), and the efficacy of KD. Of 13 MAS/MPC1- individuals with a neurological phenotype treated with KD, 11 experienced benefits—mainly a striking effect against seizures. Two individuals with citrin deficiency deceased before the correct diagnosis was established, presumably due to high-carbohydrate treatment. Six citrin-deficient individuals received a carbohydrate-restricted/fat-enriched diet and showed normalisation of laboratory values/hepatopathy as well as age-adequate thriving. We conclude that patients with MAS-/MPC1- defects are amenable to dietary intervention and that early (genetic) diagnosis is key for initiation of proper treatment and can even be lifesaving.

In the framework of this thesis was performed genotyping of 46 specimens of the breed Czech red cattle from the University farm in Czech Budejovice, which monitored the incidence of autosomal recessive genetic disorders, specifically bovine citrullinemia (BC) in exon 5 and deficiency of blood coagulation factor XI (FXI) in exon 9 and 12. Genotyping for BC was done using PCR/RFLP methods and for the disorder FXI in both exons genotypes were determined on the basis of different length of fragments using PCR technology and horizontal agarose electrophoresis. The presence of mutant allele was detected only in the locus for BC and that is in 7 heterozygous carriers, who produced three bands with a length of 185 bp fragments, 103 bp and 82 bp. The frequency of mutant allele and the frequency of heterozygous carriers to 7.6% and 15.2%. Results of the study show that the presence of mutant allele for BC in our tested panel of animals is relatively high. In the future it will be necessary to adopt measures that will lead to the elimination of this allele. Otherwise, its further dissemination would have a negative impact on the health of the population and there might occur complications in the regeneration of Czech red cattle, which is one of our farm animal genetic resources. The literature review deals with the problems of congenital disorders and discusses the importance of health heredity and understanding of the genomic information of cattle.