Les récepteurs couplés aux protéines G constituent la plus grande famille de protéines membranaires et interviennent dans de nombreux processus physiologiques. La compréhension de l’interaction ligand-récepteur d’un point de vue mécanistique mais également thérapeutique est cruciale. Appartenant à la famille des récepteurs couplés aux protéines G, les récepteurs de la vasopressine et de l’ocytocine ont été choisis comme modèle d’étude. Ces hormones jouent un rôle important dans la modulation de l’attachement et de l’affect chez les mammifères. Afin d’accélérer la découverte de ligands ocytocinergiques et d’explorer les mécanismes fondamentaux de leurs interactions, nous avons conçu les premiers ligands fluorescents non peptidiques des récepteurs de la vasopressine V1a et de l’ocytocine. Ces ligands ont été utilisés pour développer des tests de liaisons par TR-FRET et démontrer la dimérisation des récepteurs de la vasopressine V1a et V2 sur cellules. Des études autour de petites plates-formes dérivées d’aza-dicétopipérazine ont permis d’accéder à un nouvel antagoniste non peptidique du récepteur de l’ocytocine. L’optimisation de dérivés benzodiazépines ocytocinergiques par des études de relations structure-activité a permis d’identifier les meilleurs agonistes non peptidiques du récepteur de l’ocytocine à ce jour. Une étude in vivo chez la souris et chez le singe est amorcée pour apporter dans un futur, une solution thérapeutique aux problèmes d’interaction sociale en général et d’autisme en particulier. / G protein coupled receptors are the largest membrane protein family and play an important role in a large number ofphysiological processes. The comprehension of the ligand-receptor interaction from a mechanistic point of view but alsofor therapeutic use is crucial. Belonging to the G protein coupled receptors, the oxytocin and vasopressin receptors havebeen used as a model system. These two hormones play an important role in the modulation of attachment and affectin mammals. To accelerate the discovery of new ligands for oxytocin and vasopressin receptors and to explore thefundamental role of their interactions, we designed the first non-peptide fluorescent ligands for oxytocin and vasopressin V1a receptors. These ligands have been used to develop new binding tests based on TR-FRET technology and to prove the V1a and V2 receptor dimerisation. In parallel, we developed a new non-peptide oxytocin antagonist around an aza-diketopiperazine platform. . Optimization of benzodiazepine derivatives enables us to identify the best non peptideoxytocin agonists to date. In vivo studies in mice and monkeys are initiated to bring in the future a therapeuticsolution to social interaction problems in general and autism in particular

L’Opacification Capsulaire Postérieure (OCP) est la fibrose de la capsule développée sur la lentille intraoculaire implantée (LIO) suite à la dé-différenciation de cellules épithéliales cristalliniennes (LECs) subissant une transition épithélio-mésenchymateuse (EMT). La littérature a montré que l'incidence de l’OCP est multifactorielle, dont l'âge ou la maladie du patient, la technique de chirurgie, le design et le matériau de la LIO. La comparaison des LIOs en acryliques hydrophiles et hydrophobes montre que les premières ont une OCP plus sévère, médiée par la transition EMT. En outre, il est également démontré que l'adhérence des LECs est favorisée sur des matériaux hydrophobes par rapport à ceux hydrophiles. Une stratégie biomimétique destinée à promouvoir l’adhérence des LECs sans dé-différenciation en vue de réduire le risque de développement de l’OCP est proposée. Dans cette étude, les peptides RGD, ainsi que les méthodes de greffage et de quantification sur un polymère acrylique hydrophile ont été étudiés. La surface fonctionnalisée des LIOs favorisant l'adhérence des LECs via les récepteurs de type intégrine peut être utilisée pour reconstituer la structure capsule-LEC-LIO en sandwich, ce qui est considéré dans la littérature comme un moyen de limiter la formation de l‘OCP. Les résultats montrent que le biomatériau innovant améliore l'adhérence des LEC, et présente également les propriétés optiques (transmission de la lumière , banc optique) similaires et mécaniques (force haptique de compression, force d'injection de la LIO) comparables à la matière de départ. En outre, par rapport au matériau hydrophobe IOL, ce biomatériau bioactif présente des capacités similaires vis à vis de l’adhérence des LECs, le maintien de la morphologie, et l'expression de biomarqueurs de l’EMT. Les essais in vitro suggèrent que ce biomatériau a le potentiel de réduire certains facteurs de risque de développement de l’OCP. / Posterior Capsular Opacification (PCO) is the capsule fibrosis developed onto the implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LEC) undergoing Epithelial-Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including patient’s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs show the former has more severe PCO after EMT transition. Additionally, the LEC adhesion is favored onto the hydrophobic materials compared to the hydrophilic ones. A biomimetic strategy to promote LEC adhesion without de-differentiation to reduce PCO development risk is proposed. RGD peptides, as well as their grafting and quantification methods on a hydrophilic acrylic polymer were investigated. The surface functionalized IOL promoting LEC adhesion via integrin receptors can be used to reconstitute the capsule-LEC-IOL sandwich structure, which is considered to prevent PCO formation in literature. The results show the innovative biomaterial improves LEC adhesion, and also exhibits similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties comparing to the starting material. In addition, comparing to the hydrophobic IOL material, this bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression. The in vitro assays suggest this biomaterial has the potential to reduce some risk factors of PCO development.

Após a realização de uma sessão de treinamento (ST) é comum a ocorrência do fenômeno denominado dano muscular induzido pelo exercício (DMIE), que se caracteriza por prejuizos a estrutura da fibra muscular, com ruptura de alguns sarcômeros, desordem miofibrilar e alargamento das linhas Z. Ainda em consequência ao DMIE, surgem alguns sintomas que são utilizados como marcadores indiretos: dor muscular de início tardio (DMIT), redução na produção de força, aumento de enzimas e proteínas na corrente sanguínea e inchaço. O presente estudo examinou os efeitos da suplementação nutricional a fim de minimizar os efeitos deletérios do DMIE em 3 experimentos. No 1° estudo, 36 indivíduos inexperientes em treinamento de força (TF) foram suplementados com: placebo (PLA, n=12, 50mg·kg-1 de carboidrato); leucina (LEU) baixa dose (LBD, n=12, 50mg·kg-1 de LEU + 50mg·kg-1 de carboidrato) e LEU alta dose (LAD, n=12, 250mg·kg-1 de LEU + 50mg·kg-1 de carboidrato) por 6 dias antecedentes a sessão de treinamento (ST), e nos 3 dias seguintes. Foi observada redução significante, p<0.05, na dor muscular de início tardio (DMIT) do peitoral por palpação, e alongamento nos momentos 48h, e 72h após a ST no grupo LBD comparado ao PLA. A redução no teste de 1 repetição máxima (1RM) apresentou significância no grupo PLA em todos momentos após ST. O aumento na atividade da creatina quinase (CK) foi significante no grupo PLA comparado ao LAD em 24h, 48h e 72h após a ST, enquanto o aumento da concentração de mioglobina (Mb) foi significante no grupo PLA comparado ao grupo LBD e LAD em 24h, 48h e 72h após a ST. O 2° estudo contou com a participação de 28 indivíduos com até 6 meses de experiência em TF. Os sujeitos foram suplementados com 3g de &beta;-hidroxi-&beta;-metilbutirato (HM) por 14 dias (H14, n=07); 7 dias (H07, n=07) e placebo por 14 dias (P14) ou 7 dias (P07, n=07) antecedentes a ST, e nos 3 dias seguintes. O aumento da DMIT por palpação e alongamento foi significante no grupo P14 comparado ao H14 em 24h (apenas alongamento), 48h e 72h após ST, ainda no momento 72h o grupo P07 era superior ao H07. A redução no teste de 1RM ocorreu nos 4 grupos imediatamente após, foi mantida em 24h após a ST nos grupos H14, H07 e P07, sem diferenças entre os grupos. O aumento na concentração de Mb foi significante no grupo P14 comparado ao grupo H14. No 3° estudo, 24 indivíduos experientes em TF foram suplementados com 7g de arginina (ARG, n=12) ou placebo (PLA, n=12, 7g carboidrato) 30 minutos pré-ST. O grupo PLA apresentou aumento significante na DMIT por palpação em 24h comparado ao grupo ARG. A redução no teste de 1RM alcançou significância apenas em 24h após a ST no grupo PLA, mas sem diferença entre os grupos. Os resultados do presente estudo permitem concluir que a suplementação nutricional implementada atenuou o comportamento de alguns marcadores indiretos DMIE, com maior efeito para a DMIT e parametros bioquímicos / After performing a training session (TS) is common the occurrence of the phenomenon called muscle damage induced by exercise (DMIE), which is characterized by damage to muscle fiber structure, breaking some sarcomeres, myofibrillar disorder and extension lines Z. As a consequence of DMIE, there are some symptoms that are measured as indirect markers: delayed onset muscle soreness (DOMS), reduction in strength production, increase of enzymes and proteins in the bloodstream, and swelling. The effect of nutritional interventions to minimize deleterious responses associated with exercise-induced muscle damage (EIMD) were investigated in 3 experiments. In study 1, 36 inexperienced subjects in resistance training (RT) were supplemented for 6 days prior to the training session (TS), and in the following 3 days with: placebo (PLA, n=12, 50mg·kg-1 of carbohydrate); leucine (LEU) low dose (LLD, n=12, 250mg·kg-1 LEU + 50mg·kg-1 + carbohydrate) and LEU high dose (LHD, n=12, 250mg·kg-1 LEU + 50mg·kg-1 + carbohydrate). There was a significant reduction (p <0.05) in delayed onset muscle soreness (DOMS), of the chest by palpation and stretching at 48h, after TS in the LLD group compared to PLA. A significant reduction in the one repetition maximum (1RM) test was observed in the PLA group at all times after TS. The increase in creatine kinase (CK) activity was significant in the PLA group compared to the LHD in 24h, 48h and 72h after TS, while the increase in myoglobin concentration (Mb) was significant in the PLA group compared to the LLD and LHD group in 24h, 48h, and 72h after TS. In study 2, 28 subjects with up to 6 months of RT experience were supplemented with 3g of &beta;-hydroxy-&beta;-methylbutyrate (HM&beta;) for 14 days (H14, n=7); for 7 days (H07, n=7), and placebo for 14 days (P14, n=7) or 7 days (P07, n=7) antecedent to ST, and in the next 3 days. The increase in DOMS by palpation and stretching was significant in the P14 group compared to H14 in 24h (stretching only), 48h and 72h after TS, yet at 72h the P07 group was higher than H07. The reduction in the 1RM test occurred in the 4 groups immediately after and maintained within 24h after TS in groups H14, H07 and P07, and there was no difference between groups. The increase in Mb concentration was significant in the P14 group compared to the H14 group. In study 3, 24 resistance-trained subjects were supplemented with 7g of arginine (ARG, n=12) or placebo (PLA, n=12, 7g of carbohydrate) 30 minutes pre- TS. The PLA group presented a significant increase in DOMS by palpation in 24h compared to the ARG group, and a significant reduction in the 1RM test only in 24h after ST in the PLA group, but without a significant difference between groups. The results of the present study suggest that the responses of indirect markers associated with EIMD were attenuated by nutritional interventions, with greater effect for DOMS and biochemical parameters

Reaktion von Arginin mit Abbauprodukten 1,4-verknüpfter Disaccharide Im Verlauf der Reaktion von Arginin mit Abbauprodukten 1,4-glycosidisch verknüpfter Disaccharide entsteht ein Hauptderivatisierungsprodukt des Arginins, welches aus Inkubationsansätzen von Lactose mit N-(tert-Butoxycarbonyl)-L-arginin (Boc-Arg) bzw. N-a-Hippuryl-L-arginin (Hip-Arg) isoliert und als N-d-[5-(3-Hydroxypropyl)-4-oxo-imidazolon-2-yl]-L-ornithin (PIO) identifiziert werden konnte. PIO stellt ein spezifisches Reaktionsprodukt von Arginin mit Abbauprodukten 1,4-glycosidisch verknüpfter Disaccharide dar. Zum Nachweis des Precursors von PIO wurden die Bildung und der Abbau von 1,2-Dicarbonylverbindungen in Inkubationsansätzen von Lactose mit und ohne Hip-Arg nach der Hitzebehandlung mit o-Phenylendiamin untersucht. Es zeigte sich, dass ein als 1,2-Dicarbonylverbindung identifiziertes Abbauprodukt von Lactose nur in Abwesenheit von der Aminokomponente (Hip-Arg) als Hauptabbauprodukt bestimmbar war. Nach Isolierung dieser 1,2-Dicarbonylverbindung in Form ihres stabilen Chinoxalin-Derivates und der Strukturaufklärung ist es gelungen, dieses Hauptabbauprodukt der Lactose als (3'-Hydroxypropyl)-chinoxalin also das Chinoxalin der 3,4-Didesoxypentosulose (3,4-DDPs) zu identifizieren. Bestimmung von 1,2-Dicarbonylverbindungen in Lebensmitteln Glyoxal (GO), Methylglyoxal (MGO), 3-Desoxyglucosulose (3-DG) und 3-Desoxypentosulose (3-DPs) konnten nach Umsetzung mit o-Phenylendiamin erstmals in Milch- und Milchprodukten quantifiziert werden. Für Glyoxal wurden Gehalte von 0,06 bis 3,5 mg/ l und für Methylglyoxal von 0,2 bis 4,7 mg/ l bestimmt. 3-Desoxyglucosulose wurde mit Gehalten von 0,7 bis 3,5 mg/ l und 3-Desoxypentosulose von 0,1 bis 4,7 mg/ l bestimmt. Des Weiteren erfolgte die Bestimmung von Glyoxal, Methylglyoxal und 3-Desoxyglucosulose in käuflich erworbenen deutschen Honigen, in Honigen des Imkerverbandes Dresden und in neuseeländischen Honigen. Im Vergleich zu den Milchprodukten wurden deutlich höhere Gesamtgehalte an 1,2-Dicarbonylverbindungen (124 bis 1550 mg/ kg) bestimmt. Für 3-Desoxyglucosulose wurden 119 bis 1451 mg/ kg, für Glyoxal 0,2 bis 4,6 mg/ kg und für Methylglyoxal 0,5 bis 743 mg/ kg ermittelt. Ein Zusammenhang zwischen hohen Gehalten an 1,2-Dicarbonylverbindungen und der antibakteriellen Aktivität der Honige wurde untersucht. Hier stellten die neuseeländischen Manuka-Honige (Manuka: Leptospermum scoparium, Teebaum) den Schwerpunkt der Untersuchung dar. Für die untersuchten Manuka-Honige konnten ungewöhnlich hohe Gehalte an Methylglyoxal bestimmt werden (von 347 bis 743 mg/ kg). Von 12 verschiedenen Honigen deutscher und neuseeländischer Herkunft konnten nur Manuka-Honige als antibakteriell wirksam eingestuft werden. Bezogen auf den Gehalt an Methylglyoxal liegen die MIC-Werte für Staphylococcus aureus bei 1,5 mmol/ l für Manuka-Honig (35 % v/v), 1,4 mmol/ l für Manuka-Honig &amp;quot;active&amp;quot; (30 % v/v), 1,1 mmol/ l für Manuka-Honig UMF 10+ (25 % v/v) bzw. 1,8 mmol/ l für Manuka-Honig UMF 20+ (20 % v/v). Es zeigte sich, dass die antibakterielle Aktivität des Honigs unmittelbar auf den Methylglyoxal-Gehalt zurückführbar war.

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA.

La chaîne invariante (Ii) agit à titre de chaperon dans l’assemblage et le trafic des molécules du complexe majeur d’histocompatibilité de classe II (CMHII). Chez l’humain, les deux isoformes prédominantes, p33 et p35, diffèrent par la présence d’un motif di-arginine (RXR). Ce dernier permet la rétention de p35 au réticulum endoplasmique (RE) jusqu’à son masquage par une molécule de CMHII. La chaîne invariante forme des trimères auxquels s’associent successivement jusqu’à trois dimères αß de CMHII résultant en la formation de pentamères, heptamères et nonamères. Toutefois, la stœchiométrie exacte des complexes Ii-CMHII qui quittent le RE et le mécanisme permettant le masquage du motif RXR demeurent un sujet de débats. Dans un premier temps, nous avons examiné par une approche fonctionnelle la stœchiométrie des complexes formés autour de p33 et de p35. Nous avons observé que p35 engendre la formation de complexes nonamériques (αßIi)3 et permet l’incorporation de différents isotypes de CMHII autour d’un même trimère de p35 alors que p33 facilite la formation de pentamères (αß)1Ii3. Lors de l’étude du masquage du motif RxR par les CMHII, nous avons montré que son inactivation requiert une interaction directe (en cis) entre les sous-unités p35 et CMHII, résultant en une rétention des trimères de p35 insaturés au RE. Aussi, nous avons observé que contrairement aux complexes p33-CMHII, les complexes p35-CMHII sont retenus au RE lorsque coexprimés avec la protéine NleA de la bactérie Escherichia coli entérohémorragique. Comme l’expression de NleA interfère avec la formation des vésicules COPII responsable de l’export du RE, nous supposons que la sortie du RE des complexes p35-CMHII dépend des vésicules COPII alors que la sortie des complexes formés autour de l’isoforme p33 est indépendante de la formation de ces vésicules. La trimérisation d’Ii représente la toute première étape dans la formation des complexes Ii-CMHII. Deux domaines d’Ii permettent la formation de trimères; le domaine de trimérisation (TRIM) et le domaine transmembranaire (TM). Nous nous sommes intéressés à la nécessité de ces domaines dans la trimérisation de la chaîne et la formation subséquente de complexes avec les CMHII. Nous avons démontré que le domaine TRIM n’est pas essentiel à la trimérisation de la chaîne, à la formation de pentamères et de nonamères ainsi qu’au trafic adéquat de ces complexes Ii-CMHII dans la cellule. En absence des domaines TM d’Ii et des CMHII, nous avons observé la formation de complexes pseudo-nonamériques. Ceci suppose que la présence de ce domaine n’est pas un prérequis à la formation de nonamères. En conséquence, la présence d’un seul domaine de trimérisation de Ii est requise pour la formation de trimères et de complexes nonamériques. L’ensemble de nos résultats démontrent que la fonction de p35 n’est pas redondante à celle de p33. p35 influence de manière distincte le trafic des CMHII puisqu’il affecte la stœchiométrie des sous-unités incorporées aux complexes Ii- CMHII. / The invariant chain (Ii) assists in the folding and trafficking of MHC class II molecules (MHCII). Four different isoforms of the human Ii have been described (p33, p35, p41 and p43). The main isoforms, p33 and p35, differ by the presence of a di-arginine (RXR) endoplasmic reticulum (ER) retention motif in p35. This motif is inactivated upon binding of MHCII. In the ER, p33 and p35 assemble into trimers before associating with MHCII. The sequential binding of up to three MHCII αß dimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. However, the exact stoichiometry of the Ii-MHCII complex and the mechanism allowing shielding of the ER retention motif remain a matter of debate. To shed light on these issues, we chose a functional approach to examine the stoichiometry of complexes formed around the p33 and p35 isoforms. We showed that p35 promotes formation of nonameric complexes (αßIi)3 while formation of pentameric complexes (αß)1Ii3 was observed for p33. We then showed that formation of nonameric complexes can result in the inclusion of distinct MHCII isotypes around a single trimeric p35 scaffold. When answering the question wetter masking of the p35 RXR motif by MHCII results in the formation of nonamers, we showed that the actual inactivation of motif requires a direct cis-interaction between p35 and the MHCII, precluding ER egress of unsaturated p35 trimers. Interestingly, as opposed to p33-MHCII complexes, p35-MHCII complexes remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Expression of this bacterial protein is thought to interfere with the formation of COPII vesicles, leading to the conjecture that p35-MHCII and p33-MHCII complexes exit the ER in a COPII-dependant and COPII-independent manner, respectively. The trimerization of Ii represents the very first step in the formation of Ii-MHCII complex. Two domains of Ii, the trimerization domain (TRIM) and the transmembrane (TM) domain have been shown to trigger the trimerization of the chain. We focused our attention on the requirement of the two trimerization domains in Ii self-association and in the formation of pentameric and nonameric complexes. We showed that the TRIM domain of Ii is not essential for the chain’s trimerization, formation of pentamers and nonamers and for proper traffic with MHCII molecules. In absence of the Ii and MHCII TM domains, we observed the formation of a nonamer-like structure hereby suggesting that the presence of this domain is not a prerequisite for nomamer complex formation. Consequently, our results showed that either Ii trimerization domains are sufficient for Ii trimer formation and nonameric complex trafficking. Taken together, our results demonstrate that the function of the p35 isoform is not redundant, influencing distinctively MHCII trafficking as the subunit stoichiometry of oligomeric Ii/MHCII complexes is affected by p35.

<p>Pathogenic bacteria are evolving to drug resistant strains at alarming rates. The threat posed by drug resistant bacterial infections emphasize the need to establish new antimicrobial agents. Of immediate concern regarding the dangers of antibiotic resistance is the existence of intracellular bacteria, which find refuge from bactericidal devices by hiding within mammalian cells. Unfortunately, many therapeutics, such as vancomycin, do not possess membrane penetrating abilities to achieve efficacious eradication of bacteria at the subcellular level, allowing infections to persist. In an effort to target pathogens that thrive within mammalian cells, features of cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) were combined to develop a dual action antimicrobial CPP, cationic amphiphilic polyproline helices (CAPHs). CAPHs have proven to be an effective antimicrobial agent to combat an array of both Gram negative and Gram positive bacteria. </p> <p> </p> <p>Herein, to improve CAPHs activity, we have demonstrated how the incorporation of strategic modifications has resulted in increased cell uptake, alternative subcellular locations for CAPHs, and advanced antimicrobial potency. By simultaneously extending the helical length of CAPHs while incorporating different hydrophobic groups in place of the original isobutyl moiety that compose CAPHs we have created a <b>FL-P17-5R </b>series of peptides with five carbon aliphatic motifs: <b>Fl-P17-5B</b>, <b>Fl-P17-5C</b> and <b>Fl-P17-5L. </b>Through these modifications the peptides proved to be 2 to 5-fold more efficient in accumulating in macrophage cells than parent peptide Fl-P14LRR and where able to clear intracellular pathogenic bacteria, such as <i>Listeria</i>, from infected macrophages by 26 to 54%. </p> <p> </p> <p>In addition to making the <b>Fl-P17-5R</b> series of CAPHs to potentiate CAPHs activity, modifications to the cationic moiety of CAPHs were explored. By incorporating a new cationic monomer into the CAPHs sequence, a guanylated amino proline (GAP) residue, we produced <b>Fl-P14GAP</b>, a CAPHs peptide with an organized cationic charge display. This modification resulted in a 5-fold increase in cell uptake and a 2 to 16-fold decrease in minimum inhibitory concentration (MIC) values against strains of enteric and ESKAPE pathogens in comparison to Fl-P14LRR. <b>Fl-P14GAP</b> also executed superior clearance of intracellular pathogenic bacteria that resulted in the complete eradication of a drug resistant strain of <i>A. baumannii</i> from infected macrophage cells. Overall, our efforts with the <b>Fl-P17-5R</b> series of CAPHs and <b>Fl-P14GAP</b> have strengthened the therapeutic potential of CAPHs in the hopes of addressing the need for novel antibiotics with the propensity to eradicate intracellular pathogens.</p>

No description available.

Die myeloische Entwicklung wird durch die Familie der Transkriptionsfaktoren CCAAT/Enhancer-Binding-Protein (C/EBP) reguliert. Eine aberrante Expression oder Funktion von C/EBPs stört die normale myeloische Differenzierung und wird bei vielen Arten hämatopoetischer Malignome beobachtet. Mutationen von CEBPA führen zu einem veränderten Expressionsanteil der verkürzten Isoform C/EBPa p30 und werden bei etwa 15% der AML-Patienten (akute myeloische Leukämie) nachgewiesen. Obwohl die verkürzte Isoform C/EBPα p30 als Onkogen identifiziert wurde da sie die Proliferation myeloischer Vorläufer fördert, behält sie dennoch eine Differenzierungsfunktion. Unser Interesse gilt der Frage, wie diese beiden Funktionen von C/EBPα p30 reguliert werden. Die C/EBP-Familie gehört der Gruppe intrinsisch ungeordneter Proteine an, die zudem viele posttranslationale Modifikationen (PTMs) aufweisen. PTMs auf C/EBPα verändern seine biologische Funktionsweise stark. Frühere Forschungsarbeiten haben drei Argininreste am N-Terminus von C/EBPα p30 identifiziert, die aufgrund des Methylierungsstatus differentiell mit anderen Proteinen interagieren. In dieser Arbeit untersuchen wir den Einfluss der C/EBPα p30 Arginin-Methylierung auf seine pro-leukämische Aktivität sowie dessen Fähigkeit zur Neuausrichtung der hämatopoietischen Differenzierungslinie. Mit Hilfe von Aminosäuresubstitutionen fanden wir heraus, dass C/EBPα p30 Mutanten der Methylierungsmimesis oder Ladungsabschaffung die myeloische Differenzierung verstärkt, während Ladungserhalt-Mutanten die Erneuerung und Proliferation hämatopoetischer Stamm-/Vorläuferzellen unterstützt. Transkriptionelles Profiling von Zellen, die mutierte C/EBPα -p30-Varianten exprimieren, deutet auf potenzielle Ziele der methyliertem bzw. unmethyliertem C/EBPα p30 hin. Die Ergebnisse legen nahe, dass der Arginin-Methylierungsstatus das Leukämie- und Differenzierungs-Potenzial von C/EBPα p30 verändert und somit ein neues Ziel der Leukämietherapie darstellen könnten. / Myeloid development is regulated by the family of transcription factors CCAAT/enhancer-binding-protein (C/EBP). Aberrant expression or functioning of C/EBPs disturbs normal myeloid differentiation and is found in many types of hematopoietic malignancies. Mutations of CEBPA lead to imbalanced expression of the truncated isoform C/EBPα p30 and are found in approximately 15% of AML (acute myeloid leukemia) patients. Yet, how C/EBPα participates in leukemic progression remains to be discovered. More specifically, the truncated isoform C/EBPα p30, although being identified as an oncogenic isoform that promotes proliferation of myeloid progenitors, still retains differentiation function. The question of how both functions of C/EBPα p30 are regulated, is of our interest. C/EBP family also represents a group of intrinsically disordered proteins, which contain many post-translational modifications (PTMs). PTMs on C/EBPα greatly alter its functioning. Previous works have identified three arginine residues at the N-terminus of C/EBPα p30 that interact differently with others protein dependent on their methylation status. We hypothesize, that methylation of these arginine residues plays important roles in the biology of C/EBPα p30. In this study, we used a lymphoid-to-myeloid transdifferentiation (LMT) system to investigate the influence of arginine-methylation on C/EBPα-induced lineage switch and its pro-leukemic activity. Using amino acid substitution, we found that C/EBPα p30 mutants that resemble arginine-methylated p30 enhanced myeloid differentiation, while the charge-retention mutant, resembling arginine-unmethylated p30, supported renewability and proliferation of hematopoietic progenitors. Transcriptional profiling of cells expressing C/EBPα p30 variants suggested potential targets of either methylated or unmethylated p30. The results implied that arginine methylations alter C/EBPα p30’s leukemic potential and might comprise novel targets of leukemia therapy.

Parasiten durchlaufen in ihrem Lebenszyklus morphologisch verschiedene Stadien. Die Kontrolle des Übergangs zwischen den Stadien kann die Transmission in einen neuen Wirt begünstigen. Bei vielen Parasiten ist unbekannt, welche Faktoren die Progression des Lebenszyklus beeinflussen. Der Ablauf kann genetisch prädeterminiert sein (kanalisiert) oder von äußeren Einflüssen abhängen (phänotypische Plastizität). Hier wurde die Progression des Lebenszyklus zweier Darmparasiten in Mäusen untersucht. Die Oozysten von Eimeria falciformis wurden quantifiziert und die Transkriptome von Parasit und Wirt wurden in Mäusen unterschiedlicher Immunkompetenz analysiert. Wenngleich erwartet wurde, dass die Immunantwort einen Stressor für das Pathogen darstellt, hatte die Immunkompetenz des Wirts keine Auswirkungen auf den Zeitpunkt der Oozystenausscheidung und das Transkriptomprofil des Parasiten. E. falciformis konnte nicht von der Immunschwäche des Wirtes profitieren; ist also hinsichtlich der Immunantwort des Wirts genetisch kanalisiert. In G. duodenalis wurde untersucht, inwiefern die Progression des Lebenszyklus, d.h die Trophozoitenreplikation bzw. die Zystenausscheidung, von Arginin abhängt. Die Replikation der Trophozoiten war nicht von Arginin aus der Nahrung abhängig; die Ausscheidung infektiöser Zysten war unter argininarmen Bedingungen jedoch verringert. Dies lässt vermuten, dass der Ablauf des Lebenszyklus von G. duodenalis, insbesondere die Enzystierung, an die Argininzufuhr gekoppelt ist. Die Umstellung des Metabolismus von G. duodenalis hin zur Produktion eines wichtigen Zystenwandbestandteils wird hier als mechanistische Verbindung zwischen ATP-Erzeugung aus Arginin in Nichtsäugetieren (Arginindihydrolase-Stoffwechselweg), verringerter Glykolyse und der Zystenwandsynthese erörtert. Somit könnte Arginin als Stimulus für phänotypische Plastizität bei der Enzystierung von G. duodenalis dienen. / Eukaryotic parasites have life cycles with morphologically distinct stages. Accurate timing of the conversion from one stage into another can be beneficial for transmission into a new host. Often little is known about determinants for such life cycle progression or the genes involved. Timing can be genetically pre-determined (canalized) or depend on exposure to a stimulus (phenotypic plasticity). Here, life cycle progression of two unicellular intestinal parasites was investigated in mice. For Eimeria falciformis, oocyst stage parasites were quantified, and parasite and host transcriptomes analyzed in differently immune competent hosts. Host immune response stimuli are expected to induce stress on the pathogen, but different host immune competences did not change the timing of oocyst shedding or influence parasite transcriptome profiles. E. falciformis was unable to benefit from hosts with weakened immune responses. It is therefore an example of a genetically canalized parasite with regards to host immune stimulus. In Giardia duodenalis, dependence on arginine for life cycle progression was investigated. The in vivo relevance for parasite replication is unknown. Trophozoite stage replication and cyst shedding were assessed in hosts fed normal and arginine-free diets. G. duodenalis did not depend on dietary arginine for trophozoite replication, but infective cysts were reduced in number under arginine-poor conditions. Dependence on arginine for life stage switching suggests that G. duodenalis could time progression by encysting upon arginine exposure. G. duodenalis metabolic reprograming to generate a major cyst wall component is discussed as a strategy to mechanistically link 1) non-mammalian ATP generation (arginine dihydrolase pathway) from arginine with 2) decreased glycolytic flux and 3) cyst wall generation. Therefore, arginine may be an external stimulus for phenotypic plasticity of encystation in G. duodenalis.