Thèse--Faculté de droit de Paris. / "Principaux ouvrages à consulter": p. [5]-6.

The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity. / Thesis / Master of Science (MS)

The weakly electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. The EOD pulse is mainly shaped by sodium currents. There are two sodium channel α subunits orthologs of the mammalian Nav1.4 expressed in the EO of Sternopygus. Previous studies identified a novel splice variant of the Nav1.4b (Nav1.4bL), in which an extra 51-amino acid occurs in the N terminal end. Nav1.4bL currents inactivate and recover from inactivation significantly faster than Nav1.4bS. The voltage-dependence of steady-state inactivation of smNav1.4bL shifts to hyperpolarized potential. Structural analysis predicts an α-helix in the middle of the extended N terminus. Removal of a proline right after the α-helix significantly slows down current decay but has no effect on channel recovery from inactivation, suggesting inactivation and recovery have independent mechanism. Mutagenesis analysis of the extended N terminus showed that the short helical region, especially the positive charges in the helix, is an important determinant for channel voltage-dependence of steady-state inactivation. However, other residues outside the helical region are required for regulation of fast inactivation and recovery form inactivation. Functional and structural analysis provides evidence for the importance of the C terminus in fish Nav1.4b channel properties. Chimera in which the C terminus of smNav1.4bS was substituted by the human Nav1.4 C terminus, shows an 11 mV positive shift in voltage-dependence of activation and a -16 mV negative shift in inactivation. Deletion of the distal half of smNav1.4bS negatively shifted voltage-dependence of inactivation and significantly accelerated channel recovery from inactivation. In the deletion mutant, the regulation by the N segment is missing. Substitution of the C terminus mutant retains wild type channel inactivation and recovery properties and can be regulated by N segment again. My study provides evidence that the extended N terminus of smNav1.4bL binds the distal part of C terminal tail to modulate channel inactivation properties. This is the first time to show the distal C terminus is involved in channel recovery from inactivation. Studies in the fish sodium channel properties provide useful information to understand function and structure of voltage-gated sodium channels. / text

The key control step in E. coli chemotaxis is regulation of CheA kinase activity by a set of four transmembrane chemoreceptors. The receptor dimers can form trimeric complexes (trimers of dimers), and these trimers can be joined by a bridge thought to consist of a CheW monomer, a CheA dimer, and a second CheW monomer. It has been proposed that trimers of receptor dimers may be joined by CheW-CheA dimer-CheW links to form an extended hexagonal lattice that may be the structural basis of the chemoreceptor patches seen in E. coli. The receptor/CheA/CheW ternary complex is a membrane-spanning allosteric enzyme whose activity is regulated by protein interactions. The study presented in this dissertation investigated intermolecular and intramolecular interactions that affect the chemotactic signal processing. I have examined functional interactions between the serine receptor Tsr and the aspartate receptor Tar using a receptor coupled in vitro phosphorylation assay. The results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis. A mutational analysis of the extreme C-terminus (last fifty residues) of Tar is also presented. The results implicate the receptor C-terminus in maintenance of baseline receptor activity and in attractant-induced transmembrane signaling. They also suggest how adaptive methylation might counteract the effects of attractant binding.

No description available.

Runx1/AML1 transcription factor expression in hematopoietic cell lineages is differentially regulated via usage of two distinct promoters. The 5' UTR and a 19 amino acid encoding sequence transcribed from the distal promoter is inserted via alternative splicing into the 5' end of the mRNA transcript, replacing the 5' UTR and a 5 amino acid encoding sequence usually transcribed from the proximal promoter. Expression of proximal Runx1 in 32Dcl.3 cells delays G-CSF induced neutrophil terminal differentiation by increasing viability compared to distal Runx1. We utilized Runx1 Nterminal deletion and point mutants of three evolutionarily conserved residues to describe dual N-terminal isoform motifs that promote two distinct differentiation phenotypes as regulatory elements in hematopoietic cell differentiation. Runx1 isoforms were evaluated in established hematopoietic in vitro and ex vivo differentiation systems. Deletion of amino acids 3’-14’ (Δ3-14) or 3’-19’ (Δ3-19) of the distal Runx1 N-terminus delayed terminal differentiation of the 32Dcl.3 myeloid cell line, indicating a regulatory motif in distal Runx1 abrogates the delay of terminal differentiation induced by proximal Runx1. Deletion of amino acids 3’-8’ (Δ3-8) or mutation of amino acids serine 3’, serine 5’ and phenylalanine 7’ of the distal Runx1 N-terminus reduce Runx1 expression in the 32Dcl.3 cell line. The N-terminus motif, runt domain and nuclear matrix-targeting sequence of Runx1 modulated Ets1 activity on the KIR3DL1 bidirectional promoter element. The transcription factor YY1 promotes both forward and reverse activation of the KIR3DL1 bidirectional promoter element dominantly in the presence of Runx1, and additively with Ets1. Distinct Runx1 proximal and distal N-termini induced phenotypes were observed in myeloid and thymocyte differentiation, but not with the KIR3DL1 luciferase assay system. This work identifies a previously unknown N-terminal regulatory motif that acts with spatio-temporal and gene target specificity to add another level of control over Runx1 activity during hematopoiesis.

No description available.

Cardiovascular diseases affect one third of the U.S. population and are the number one cause of death globally. Acute myocardial infarction is one of the most catastrophic cardiovascular diseases that permanently alters patient's lives. Small molecule drugs, surgery, medical devices and lifestyle changes are the current treatment methods that only address symptoms and fail to cure cardiovascular disorders. Small therapeutic peptides are emerging methods to treat diseases ranging from cancer to auto-immune disorders. Due to their nature, they are non-toxic, non-immunogenic, biocompatible and highly target specific. However, because of their small size and lack of tertiary structure, they have a very short half-life. Alpha-carboxyl terminus 11 peptide (αCT11) is a 9 amino acid long small peptide that has shown to promote left ventricular function recovery when mouse hearts are perfused with the peptide prior to an ischemia-reperfusion injury. This study investigates the in vitro pharmacokinetics of αCT11 in rat plasma in the presence of protease and phosphatase inhibitor cocktails to provide a method to delay its degradation and to understand the degradation pattern of the peptide in vitro. The effect of time, temperature, presence of inhibitors and sex are investigated. Results have shown that while sex does not have a significant effect on αCT11 degradation, time and temperature significantly promote its degradation. Utilization of inhibitors also leads to a pronounced delay in αCT11 degradation, as the amount of αCT11 remaining in plasma increases from almost undetectable to 15-16% upon introduction of inhibitors. These results indicate that αCT11 degradation can be delayed significantly when inhibitor cocktails are used, bringing αCT11 closer to being used in a clinical setting to address ischemia-reperfusion injuries. / Master of Science / Cardiovascular diseases affect millions of people worldwide and they are the number one cause of death globally. Current treatments for cardiovascular diseases mainly focus on alleviating symptoms as they arise and delaying the disease progression using small molecule drugs and lifestyle changes, which unfortunately are unable to cure the diseases permanently. Peptide treatment is a novel method to address various traditionally incurable diseases, such as auto-immune disorders and cancer. These therapeutic peptides are highly target specific, typically non-toxic and highly biocompatible since they are designed based on native proteins. Even though small therapeutic peptides have numerous benefits, a major drawback is that they have a very short half-life in plasma. Alpha-carboxyl terminus 11 peptide (αCT11) is a small peptide derived from alpha-carboxyl terminus 1 peptide (αCT1), which is in phase 2 clinical trials for chronic wound healing. It has been shown that αCT11 has cardioprotective effects when the heart is perfused with the peptide before an ischemia-reperfusion injury, such as a heart attack. This study investigates the in vitro pharmacokinetic properties of αCT11 in rat plasma with respect to time, temperature and sex with the aim to provide an effective method to allow αCT11 to remain in plasma for a longer period of time. As a method to delay αCT11 degradation due to plasma enzymes, enzyme inhibitors are used, which delayed the αCT11 breakdown significantly. The results have also shown that time and temperature are the main factors affecting αCT11 degradation in rat plasma in vitro while sex is not a significant factor. These results indicate that this small peptide can be protected in plasma with the use of inhibitors. This discovery can be a stepping stone to use αCT11 in clinical settings to help treat cardiovascular diseases.

Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.

The aim of this thesis is to understand the conservation and transformation problems of railway heritage and to prepare a conservation and transformation principles for Iskenderun railway area. Primarily, it is tried to understand the historical process and the current situation of conservation of railway areas. In addition, in this study theoretical and historical framework of transformation of railway areas is developed in the light of international charters and documents. Transformation examples from Turkey and Europe were searched with analyzing the railway system and transformation approaches of the countries. In order to prepare a conservation and transformation principles for Iskenderun terminus area, considering the direct relation of the railway site with the port site, a wide border including the port for the study area is chosen and a detailed analyzes were done for whole area. For this study, the major concern is establishing strong principles in different scales. It is important to guide each conservation and transformation projects in Turkey. For rooted solutions, regulations have to start in the transformation strategies and railway policy.