No description available.

International Telemetering Conference Proceedings / October 26-29, 1998 / Town & Country Resort Hotel and Convention Center, San Diego, California / This paper describes a highly integrated and low cost GPS Translator/Telemetry system for use on missile platforms – the Digital GPS Translator (DGT), a component part of the Translated GPS Range System (TGRS). The DGT provides translated GPS tracking capability combined with transmission of telemetry at rates of up to 10 Mbps with optional encoding and/or encryption. This integrated approach to GPS tracking and telemetry results in a significant reduction in hardware size and cost compared to a segregated approach. The TGRS includes a ground-processing unit that provides real time processing of both the GPS and telemetry portions of the DGT transmission.

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.

Esta pesquisa abordou a questão do desenvolvimento social e econômico do Parque Estadual Turístico do Alto Ribeira (PETAR), localizado no sul do estado de São Paulo, a partir de um estudo geográfico realizado sob a luz da tríade analítica GTP: Geossistema, Território e Paisagem. Partindo dessa análise geográfica, foi possível identificar como as potencialidades e fragilidades do meio físico influenciaram na territorialização da região onde hoje se encontra o parque, e como a paisagem cultural é construída sobre esta realidade. Com isso foi possível contrapor a interpretação dos dados levantados ao Plano de Manejo proposto para o PETAR em 2010, que se encontra, desde aquele ano, sob análise no Conselho Estadual do Meio Ambiente do estado de São Paulo. O documento apresenta contradições internas no que tange à exploração dos recursos minerais. Com a aprovação da Lei Estadual 16.260/2016, que concede 25 unidades de conservação para a exploração da iniciativa privada, é necessário que não restem pontos obscuros no Plano de Manejo, para que fique claro quais são os recursos que poderão ou não serem explorados. Há indícios de que neste processo os interesses econômicos se sobrepuseram tanto ao objetivo de preservação da área como ao do desenvolvimento socioeconômico. Com essa problemática em foco, discutimos como a Geografia e as ciências, de maneira geral, podem ser úteis ao processo civilizatório ao estabelecer critérios éticos de forma objetiva, apontando assim, as causas dos problemas regionais relacionados à territorialização, as contradições políticas deste processo e de que forma seria possível promover o desenvolvimento socioambiental na região. Um dos principais problemas que levantamos foi a questão das restrições às atividades econômicas impostas pela legislação ambiental. Tendo o turismo como única fonte de sustento possível, a região se tornou altamente dependente dos programas de transferência de renda que, nos últimos anos, provaram ser cruciais para a redução da pobreza extrema e evolução dos índices de desenvolvimento humano. Uma vez que uma flexibilização muito grande das restrições ambientais seria desaconselhável e, levando em consideração que a área do parque é ocupada, historicamente, por populações tradicionais, como comunidades quilombolas, concluímos que seria razoável demandar do estado alguma compensação financeira. Esta medida evitaria que recaíssem, exclusivamente sobre a população local, os custos de uma conservação ambiental que é de interesse do conjunto da sociedade. / This research addressed the issue of the social and economic development of the Upper Ribeira State Tourist Park (PETAR), in the state of São Paulo, Brazil, from a geographic study conducted under the light of the analytical triad GTL: Geosystem, Territory and Landscape. Based on this geographical analysis, it was possible to identify how the potentialities and fragilities of the physical environment influenced the territorialization of the region where the park is today, and how the cultural landscape is built on this reality. Thus, it was possible to counter the interpretation of the data collected with the proposed Management Plan for PETAR in 2010, which has been under analysis by the State Environmental Council of the state of São Paulo since that year. The document presents internal contradictions regarding the exploitation of mineral resources. With the approval of State Law 16.260 / 2016, which grants 25 conservation units for the exploitation of the private initiative, it is necessary that there are no obscure points in the Management Plan so it is clear which resources may or may not be exploited. There are indications that, in this process, economic interests overshadowed both the objective of preserving the area and that of socioeconomic development. With this problematic in focus, we discuss how Geography and the sciences, in general, can be useful to the civilizational process by establishing objective ethical criteria, thus pointing to the causes of regional problems related to territorialization, the political contradictions of this process and how it would be possible to promote socio-environmental development in the region. One of the main problems raised is the issue of the restrictions on economic activities imposed by the environmental legislation. With tourism as the only possible source of income, the region has become highly dependent on income transfer programs which, in recent years, have proved to be crucial for the reduction of extreme poverty and the improvement of the human development indexes in the area. Since a significant relaxation of environmental restrictions would be inadvisable, and considering that the area of the park has historically been occupied by traditional populations such as quilombola communities, we have concluded that it would be reasonable to demand financial compensation from the state. This would prevent the local population from paying, alone, for the costs of an environmental conservation, that is in the best interest of society, as a whole.

Made available in DSpace on 2015-04-14T13:32:56Z (GMT). No. of bitstreams: 1 437701.pdf: 658664 bytes, checksum: 3842d70518efb4bc4633c3998b3a5859 (MD5) Previous issue date: 2012-03-28 / Sepsis is a systemic inflammatory disorder, and its progression to septic shock is a serious clinical problem associated with a high mortality rate. Despite significant advances in critical care, treatments do not reverse the systemic inflammatory response and its consequences. Gram-negative sepsis is initiated by exposure to a component of gram-negative bacterial membrane, lipopolysaccharide (LPS), and induces overproduction of host inflammatory cytokines, including tumor necrosis factor (TNF- α), interleukin-1 (IL-1) and interleukin-6 (IL-6) from immunocytes such as monocytes. Recent studies have shown that similar to LPS, the Respiratory syncytial virus - leading cause of severe lower respiratory tract infections in infants and young children requires the toll-like receptor 4 (TLR4) for signaling. Studies have shown that the respiratory syncytial virus attachment glycoprotein (RSV G) modulates cytokine and chemokine production in monocytes, inhibiting the inflammatory response elicited by LPS stimulation. In this report, we use murine monocytes from different knockout mice to show that RSV G can inhibit the release of cytokines in the immune response against lipoplysaccharide induced sepsis. We demonstrate the modulation of IL-1β, IL-6, IL-10 and TNF-α by RSV G in monocytes LPS stimulated. Importantly, we also considered the effect of RSV G subunits (peptides) and future directions for the clinical use of the glycoprotein on LPS-mediated inflammation. / Sepse ? uma desordem inflamat?ria sist?mica e sua progress?o para o choque s?ptico caracteriza um s?rio problema cl?nico, apresentando altas taxas de mortalidade. Apesar dos significativos avan?os no tratamento intensivo, os mesmos n?o s?o capazes de reverter a resposta inflamat?ria sist?mica, assim como suas conseq??ncias. A sepse por bact?rias gramnegativas ? desencadeada pela exposi??o a um componente da membrana destas bact?rias, conhecido como lipopolissacar?deo (LPS), o que leva a uma super produ??o de citocinas inflamat?rias no hospedeiro, incluindo o fator de necrose tumoral (TNF-α), a interleucina-1 (IL-1) e a interleucina-6 (IL-6), por sua vez provindas de c?lulas do sistema imune, como os mon?citos. Estudos recentes demonstraram que, de forma similar ao LPS, o v?rus sincicial respirat?rio (RSV) principal causa de infec??o respirat?ria baixa em crian?as e rec?m natos - utiliza o receptor toll-like 4 (TLR4) para sinaliza??o celular. Estudos demonstraram que a glicoprote?na de liga??o do v?rus sincicial respirat?rio (RSV G) age como imunomoduladora na produ??o de citocinas e quimiocinas por mon?citos, inibindo a resposta inflamat?ria estimulada por LPS. Neste artigo, n?s utilizamos mon?citos mon?citos murinos de diferentes camundongos knockout para demonstrar que RSV G pode inibir a produ??o de citocinas na resposta imune contra lipopolissacar?deos em um modelo de sepse. Demonstra-se tamb?m a modula??o das citocinas IL-1β, IL-6, IL-10 e TNF-α por RSV G em mon?citos estimulados por LPS. O artigo ainda considera de forma destacada o efeito de subunidades (pept?deos) de RSV G, bem como destaca perspectivas para o futuro uso cl?nico de glicoprote?nas na modula??o da resposta inflamat?ria mediada por LPS.

Tsang, Chi Keung. / "November 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 103-116). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgement --- p.IV / Table of content --- p.V / List of Figures --- p.VIII / List of Tables --- p.XI / Abbreviations --- p.XII / Chapter Chapter 1: --- INTRODUCTION --- p.1 / Chapter 1.1. --- Hepatocellular carcinoma --- p.2 / Chapter 1.1.1. --- Etiological factors --- p.11 / Chapter 1.1.1.1. --- Chronic Hepatitis and Liver Cirrhosis --- p.13 / Chapter 1.1.1.2. --- HBV --- p.13 / Chapter 1.1.1.3. --- HCV --- p.17 / Chapter 1.1.1.4. --- Male gender --- p.20 / Chapter 1.1.1.5. --- Aflatoxin B1 exposure --- p.21 / Chapter 1.2. --- Genomic abnormalities in HCC --- p.23 / Chapter 1.3. --- GEF-H1 --- p.24 / Chapter 1.4. --- RhoA --- p.26 / Chapter 1.5. --- Epithelial-Mesenchymal Transition (EMT) --- p.29 / Chapter 1.6. --- Aims of Thesis --- p.31 / Chapter Chapter 2: --- MATERIALS AND METHODS --- p.32 / Chapter 2.1. --- Materials --- p.33 / Chapter 2.1.1. --- Chemicals and Reagents --- p.33 / Chapter 2.1.2. --- Buffers --- p.35 / Chapter 2.1.3. --- Cell Culture --- p.37 / Chapter 2.1.4. --- Nucleic Acids --- p.38 / Chapter 2.1.5. --- Enzymes --- p.39 / Chapter 2.1.6. --- Equipments --- p.40 / Chapter 2.1.7. --- Kits --- p.41 / Chapter 2.1.8. --- Antibodies --- p.42 / Chapter 2.1.9. --- Software and Web Resources --- p.43 / Chapter 2.2. --- Fluorescence In Situ Hybridization (FISH) --- p.44 / Chapter 2.2.1. --- Probe Preparation --- p.44 / Chapter 2.2.1.1. --- Human Bacterial Artificial Chromosome (BAC) probe preparation --- p.44 / Chapter 2.2.1.2. --- Nick translation --- p.44 / Chapter 2.2.2. --- Hybridization --- p.45 / Chapter 2.3. --- Genomic DNA extraction --- p.47 / Chapter 2.4. --- Copy number analysis --- p.48 / Chapter 2.5. --- Exon Sequencing analysis --- p.49 / Chapter 2.5.1. --- PCR amplification of GEF-H1 exons --- p.49 / Chapter 2.5.2. --- Cycle sequencing --- p.49 / Chapter 2.6. --- Ectopic expression of GEF-H1 in immortalized hepatocyte cell line --- p.52 / Chapter 2.6.1. --- Construction of GEF-H1 expressing vector --- p.52 / Chapter 2.6.2. --- Sub-cloning --- p.52 / Chapter 2.6.3. --- Transfection and clonal selection --- p.53 / Chapter 2.7. --- Gene Expression Analysis by Quantitative RT-PCR --- p.55 / Chapter 2.7.1. --- Total RNA extraction --- p.55 / Chapter 2.7.2. --- qRT-PCR analysis for gene expression --- p.55 / Chapter 2.8. --- Western blot --- p.58 / Chapter 2.9. --- Functional Analysis --- p.60 / Chapter 2.9.1. --- Cell viability (MTT) assay --- p.60 / Chapter 2.9.2. --- Cell proliferation assays (BrdU-incorporation) --- p.60 / Chapter 2.9.3. --- Mitomycin C treatment --- p.61 / Chapter 2.9.4. --- Migration and Invasion assays --- p.63 / Chapter 2.9.5. --- Wound healing assay --- p.65 / Chapter 2.9.6. --- Transient knock-down of RhoA --- p.65 / Chapter 2. --- 10. Immuno-fluorescent imaging --- p.66 / Chapter 2. --- 11. In vivo tumorigenic study of GEF-H1 by subcutaneous injection --- p.68 / Chapter 2. --- 12. Statistical analysis --- p.69 / Chapter Chapter 3: --- RESULTS --- p.70 / Chapter 3.1. --- Verifying copy number gain of GEF-H1 in high GEF-H1 expressing HCC --- p.71 / Chapter 3.2. --- Verifying if there is any GEF-H1 exon point mutation in HCC --- p.75 / Chapter 3.3. --- Functional roles of GEF-H1 in HCC --- p.77 / Chapter 3.4. --- GEF-Hl-induced functions were RhoA independent --- p.83 / Chapter 3.5. --- GEF-H1 Induction of Epithelial-mesenchymal transition in HCC --- p.88 / Chapter 3.6. --- GEF-H1 induced tumorigenicity of MIHA cells --- p.95 / Chapter Chapter 4: --- DISCUSSIONS --- p.96 / Chapter 4.1. --- GEF-H1 in HCC and other cancers --- p.97 / Chapter 4.2. --- GEF-H1 promotes cell motility --- p.98 / Chapter 4.3. --- GEF-H1 induced tumorigenicity --- p.100 / Chapter Chapter 5: --- CONCLUSIONS AND PROPOSED FUTURE INVESTIGATIONS --- p.101 / Chapter Chapter 6: --- REFERENCES --- p.103

The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the “dynamic ligand binding” concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development. / Die vorliegende Studie beschäftigt sich mit der Synthese und der pharmakologischen Untersuchung von neu entwickelten dualsteren Liganden des muskarinischen Acetylcholinrezeptors, welcher zur Superfamilie der G-Proteine gehört. In derartigen bipharmakophoren Liganden sind die Vorteile des orthosteren Bindemodus und des räumlich davon getrennten allosteren Bindemodus vereint. Der orthostere Bindemodus bewirkt eine hohe Affinität zum Rezeptor, während der allostere Bindemodus Subtypselektivität vermittelt. Dadurch weisen diese Verbindungen weniger Nebenwirkungen auf. Dies eröffnet einen neuen Therapieansatz in der medikamentösen Behandlung von z.B. chronischen Schmerzen, Drogenentzug, Morbus Parkinson und Morbus Alzheimer. Die neu synthetisierten, dualsteren Verbindungen wurden pharmakologisch untersucht, um ein besseres Verständnis über das Bindungsverhalten und die Signalweiterleitung an muskarinischen Acetylcholinrezeptoren zu erhalten, besonders in Hinblick auf Partialagonismus. Die Entwicklung des Konzeptes der „dynamischen Ligandenbindung“ bietet neue Perspektiven in Hinblick auf das Bindungsverhalten und die Signalweiterleitung an G-Protein gekoppelten Rezeptoren. Somit werden GPCRs nicht mehr nur in ihrem aktiven oder inaktiven Zustand betrachtet. Vielmehr können dualstere Liganden sowohl einen dualsteren Bindemodus, welcher den aktiven Rezeptorzustand widerspiegelt, als auch einen rein allosteren Bindemodus, welcher durch einen inaktiven Rezeptorzustand charakterisiert ist, einnehmen, was schließlich zu Partialagonismus führt. Die Stärke des resultierenden Partialagonismus hängt vom Verhältnis zwischen aktiver und inaktiver Rezeptorbesetzung ab. Auf Basis dessen wurden orthostere/orthostere Hybridverbindungen, bestehend aus einem Antagonisten, Atropin oder Scopolamin, und einem Agonisten, Iperoxo oder Isoxazol, die über eine Alkylkette unterschiedlicher Länge miteinander verknüpft sind, synthetisiert, um mit deren Hilfe den Partialagonismus zu steuern (Abbildung 1). Abbildung 1: Strukturen der synthetisierten Iperoxo/Isoxazol-Atropin/Scopolamin-Hybride. Es wurden verschiedene quartäre sowie tertiäre Homodimere, welche entweder aus zwei Iperoxo-Einheiten oder aus zwei Acetylcholin-Einheiten bestehen, synthetisiert, um deren Ausmaß in Bezug auf Partialagonismus untersuchen zu können (Abbildung 2). Die beiden Agonisten wurden über unterschiedlich lange Alkylketten miteinander verknüpft. Bindungsstudien an CHO-hM2 Zellen der quartären Verbindungen zeigten, dass die Dimerisierung eines Agonisten zu einer verringerten Wirkstärke führt. Die Dimere von Iperoxo erreichten sowohl auf dem Gi- als auch auf dem Gs-Signalweg höhere Maximaleffekte als die Dimere von Acetylcholin. Neben der Wahl des orthosteren Bausteins (Wirkstärke des Agonisten) spielt auch die Länge der Alkylkette eine entscheidende Rolle für die Stärke des Partialagonismus. Abbildung 2: Strukturen der synthetisierten quart./tert. Iperoxo/Acetylcholin-Homodimere. Um weitere Partialagonisten zu entwickeln, wurden Chinolon-basierte Verbindungen, die mit dem Superagonisten Iperoxo oder mit dem endogenen Liganden Acetylcholin über eine Alkylkette mit unterschiedlicher Länge verknüpft sind, synthetisiert (Abbildung 3). FRET-Messungen bestätigen, dass es sich bei den Hybriden um M1-subtypselektive Substanzen handelt und das FRET-Signal von der Länge der Zwischenkette abhängig ist. Das stärkste positive FRET-Signal wurde mit der Verbindung Chinolon-C6-Iper erzielt, welches durch positive Kooperativität zwischen den beiden Liganden, Alloster und Orthoster, zustande kommt. Im Gegensatz zu den kurzkettigen Hybriden beobachtete man bei den langkettigen Hybriden ein inverses FRET-Signal, welches auf einen anderen Bindemodus zum Rezeptor hindeutet, z.B. könnte es sich um eine rein allostere Bindung handeln. Außerdem wurde die flexible Alkylkette durch einen starren Linker ersetzt, welches im Hybrid Chinolon-rigide-Iperoxo verwirklicht ist (Abbildung 3). FRET-Messungen dieser starren Hybridverbindung am M1-Rezeptor zeigten eine verzögerte FRET-Kinetik, welche vermutlich auf Wechselwirkungen zwischen dem starren Linker und dem aromatischen Deckel, der sich zwischen der orthosteren und der allosteren Bindestelle befindet, zurückzuführen ist. Es wird vermutet, dass das starre Hybrid bitopisch in den Rezeptor bindet. Um diese Annahme bestätigen zu können, müssten Mutationsstudien durchgeführt werden. Abbildung 3: M1-selektive Hybridverbindungen. Ein weiteres Ziel dieser Arbeit war das Wirkstoffdesign und die Synthese von neuen Hybridverbindungen, die als Agonisten am M1- und am M2-Rezeptor sowie als Inhibitoren der AChE als auch der BChE im Hinblick auf die Alzheimer`sche Krankheit wirken sollen. Verschiedenartige Hybridverbindungen, bestehend aus unterschiedlichen pharmakophoren Gruppen (katalytische, aktive Seite: Phthalimid, Naphthalimid, Tacrin; periphere, anionische Seite: Iperoxo, Isoxazol), die über eine Polymethylenkette unterschiedlicher Länge miteinander verknüpft sind, wurden synthetisiert. Tac-C10-Iper (Abbildung 4), bestehend aus Tacrin und dem Superagonisten Iperoxo, welche über eine C10 Polymethylenkette miteinander verknüpft sind, zeigte exzellente Anticholinesterase-Aktivitäten sowohl für die AChE (pIC50 = 9.81) als auch für die BChE (pIC50 = 8.75). Docking-Experimente lieferten ein Strukturmodell, welches die inhibitorische Aktivität in Bezug auf die AChE begründet. Zusätzlich zeigten die aus Tacrin bestehenden Hybride Affinität zum M1- als auch zum M2-Rezeptor. Solche Verbindungen, die mehr als ein Zielmolekül adressieren, sind für multifaktorielle Krankheiten, wie z.B. die Alzheimer`sche Krankheit, von Vorteil. Abbildung 4: Struktur der aktivsten Substanz in Bezug auf die Anticholinesterase-Aktivität. Zusammenfassend kann festgestellt werden, dass sowohl die Wahl des Pharmakophors, deren Verbindungsstelle als auch die Zusammensetzung, Länge und Flexibilität des Linkers eine große Rolle für die Aktivität der entwickelten bivalenten Verbindungen spielen. Kurzkettige Linker können im Gegensatz zu längeren Zwischenketten nicht beide Bindestellen gleichzeitig überbrücken. Andererseits können zu lange Zwischenketten unerwünschte sterische Wechselwirkungen hervorrufen. Weitere Untersuchungen in Bezug auf strukturelle Veränderungen der Hybridverbindungen, mit oder ohne quartäre Ammoniumgruppen, sind in Bezug auf die Arzneimittelentwicklung notwendig.

Magíster en genética / Las paraplejias espásticas hereditarias (PEHs) corresponde a diversos desórdenes genéticos caracterizados por la espasticidad de miembros inferiores y una marcada debilidad muscular debido a una degeneración de los axones más largos en los seres humanos. Los genes más conocidos causantes de las PEHs (atlastina, espastina, VCP) están involucrados en el tráfico intracelular, localización y conformación de organelos membranosos. El gen atlastina (atl) codifica para una GTPasa que media la fusión homotípica en las membranas del retículo endoplásmico (RE). En el tratamiento de PEHs asociadas a mutaciones en atl no se han descrito blancos terapéuticos efectivos ya que inhibir cualquiera de las proteínas asociadas resulta en defectos en la morfogénesis del RE. En Drosophila melanogaster existen genes codificantes para moduladores de la expresión y actividad de atlastina. Como el gen es conservado tanto en humanos como en Drosophila, se utilizó este modelo biológico para identificar genes que modulen su actividad. En esta tesis se rastrearon genes del cromosoma 2 (aproximadamente un 40% del genoma de Drosophila) que actúen como modificadores dominantes del fenotipo asociado a la disminución de la expresión de atl en neuronas motoras. Nuestros resultados indican que existen genes que interactúan con atlastina, destacándose particularmente uno de ellos, en que la proteína codificada por este participa de la vía de señalización de BMP, que juega un rol fundamental en el correcto desarrollo de la unión neuromuscular. Esto es el inicio de una potencial investigación enfocada en encontrar más interactores de atlastina y los mecanismos moleculares que subyacen a estos descubrimientos. / The hereditary spastic paraplegias (HSPs) corresponds to a variety of genetic disorders, characterized by spasticity of the lower limbs and a marked muscle weakness due to degeneration of the longest axons in humans. The best known genes of the HSP (atlastin, spastin, VCP) are involved in intracellular trafficking, localization and formation of membranous organelles. The atlastin (atl) gene encodes a GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes. There have not been described effective therapeutic targets for the treatment of HSPs associated to atl mutations, because the inhibition of any associated proteins results in defects in morphogenesis ER. In Drosophila melanogaster there are genes coding for modulators of expression and activity of atlastin. As this gene is conserved in both humans as in Drosophila, this biological model is used to identify genes that modulate its activity. In this thesis we will screen genes of chromosome 2 (about 40% of the Drosophila genome) that acts as dominant modifiers of the phenotype associated with decreased expression of atl in motor neurons. Our results indicate that there are genes that interact with atlastin, particularly highlighting one of them, in which the protein encoded by this participates of the BMP signaling pathway, which plays a fundamental role in the proper development of the neuromuscular junction. This is the beginning of a potential research focused on finding more interactors of atlastin and the molecular mechanisms underlying these findings.

<p>The aim of this thesis was to configure an FPGA with high speed ports to capture data from a prototype 4 bit ΣΔ analogue-to-digital converter sending data at a rate of 2.4 Gbps in four channels and to develop a protocol for transferring the data to a PC for analysis. Data arriving in the four channels should be sorted into 4 bit words with one bit taken successively from each of the channels. A requirement on the data transfer was that the data in the four channels should arrive synchronously to the FPGA. A Virtex-5 FPGA on a LT110X platform was used with <em>Rocket^TMIO</em> GPT transceivers tightly integrated with the FPGA logic. Since the actual DUT (Device Under Test) was not in place during the work, the transceivers of the FPGA were used for both sending and receiving data. The transmission was shown to be successful for both eight and ten bit data widths. At this stage a small skew between the data in the four channels was observed. This was solved by storing the information in separate memories, one for each of the channels, to make possible to later form the 4 bit words in the PC (MatLab). The memories were two port FIFOs writing in data at 240 MHz (10 bit data width) or 300 MHz (8 bit data width) and read out at 50 MHz.</p> / <p>Syftet med examensarbetet var att konfigurera en FPGA med höghastighetsportar så att data från en prototyp av en 4 bitars ΣΔ analog-till-digital omvandlare kan samlas in med en hastighet av 2.4 Gbps i var och en av fyra kanaler och att utveckla ett protokoll för överföring av dessa data från FPGAn till en PC för analys. Insamlade data ska sorteras i 4 bitars ord med en bit successivt tagen från var och en av kanalerna. Ett krav på dataöverföringen var att data i de fyra kanalerna skulle anlända synkront till FPGAn. En Virtex-5 FPGA på en LT110X plattfrom användes med <em></em>GTP transceivrar tätt integrerade med FPGA logiken. Då utrustningen som skulle testas inte var tillgänglig under tiden arbetet utfördes användes FPGAns transceivrar till att både sända och ta emot data. Överföring av data med både 8 och 10 bitars datavidd uppnåddes framgångsrikt. Data i de fyra kanalerna visade sig dock inte anlända synkront till mottagaren. Detta problem löstes genom att lagra informationen i separata minnen, ett för varje kanal, överföra data från minnena till PCn och där med hjälp av MatLab sortera dem till 4 bitars ord. Som minnen användes tvåportars FIFOn där data skrivs in med en hastighet av 240 MHz (10 bitars datavidd) eller 300 MHZ (8 bitars datavidd) och läses ut med en hastighet av 50 MHz.</p>

The aim of this thesis was to configure an FPGA with high speed ports to capture data from a prototype 4 bit ΣΔ analogue-to-digital converter sending data at a rate of 2.4 Gbps in four channels and to develop a protocol for transferring the data to a PC for analysis. Data arriving in the four channels should be sorted into 4 bit words with one bit taken successively from each of the channels. A requirement on the data transfer was that the data in the four channels should arrive synchronously to the FPGA. A Virtex-5 FPGA on a LT110X platform was used with RocketTMIO GPT transceivers tightly integrated with the FPGA logic. Since the actual DUT (Device Under Test) was not in place during the work, the transceivers of the FPGA were used for both sending and receiving data. The transmission was shown to be successful for both eight and ten bit data widths. At this stage a small skew between the data in the four channels was observed. This was solved by storing the information in separate memories, one for each of the channels, to make possible to later form the 4 bit words in the PC (MatLab). The memories were two port FIFOs writing in data at 240 MHz (10 bit data width) or 300 MHz (8 bit data width) and read out at 50 MHz. / Syftet med examensarbetet var att konfigurera en FPGA med höghastighetsportar så att data från en prototyp av en 4 bitars ΣΔ analog-till-digital omvandlare kan samlas in med en hastighet av 2.4 Gbps i var och en av fyra kanaler och att utveckla ett protokoll för överföring av dessa data från FPGAn till en PC för analys. Insamlade data ska sorteras i 4 bitars ord med en bit successivt tagen från var och en av kanalerna. Ett krav på dataöverföringen var att data i de fyra kanalerna skulle anlända synkront till FPGAn. En Virtex-5 FPGA på en LT110X plattfrom användes med GTP transceivrar tätt integrerade med FPGA logiken. Då utrustningen som skulle testas inte var tillgänglig under tiden arbetet utfördes användes FPGAns transceivrar till att både sända och ta emot data. Överföring av data med både 8 och 10 bitars datavidd uppnåddes framgångsrikt. Data i de fyra kanalerna visade sig dock inte anlända synkront till mottagaren. Detta problem löstes genom att lagra informationen i separata minnen, ett för varje kanal, överföra data från minnena till PCn och där med hjälp av MatLab sortera dem till 4 bitars ord. Som minnen användes tvåportars FIFOn där data skrivs in med en hastighet av 240 MHz (10 bitars datavidd) eller 300 MHZ (8 bitars datavidd) och läses ut med en hastighet av 50 MHz.