Chez Arabidopsis thaliana, Les gènes d'ARN 5S sont regroupés en blocs situés dans l'hétérochromatine péricentromérique des chromosomes 3,4 et 5. La transcription des gènes d'ARN 5S est régulée par des facteurs épigénétiques altérant notamment leur structure chromatinienne. Une étude a été menée durant les premières étapes du développement post-germinatif pour identifier les évènements et des facteurs conduisant à l'élaboration de telles structures. Nous avons pu observer une décompaction de l'ADNr 5S immédiatement suivie d'une recondensation. Ces phénomènes impliquent respectivement ROS 1 et l'ARN polymérase IV. L'étude des formes Pol IVa et Pol IVb nous indique que Pol IVb, en plus de son activité partenaire de Pol IVa, possède une action spécifique dédiée au locus d'ADNr 5S du chromosome 4. Cette nouvelle activité de Pol IVb, qui est indispensable au silencing et à la compaction de ce locus, semble indépendante de la voie classique de méthylation de l'ADN dépendante des ARN.

Horizontal gene transfer (HGT) is now indisputably the predominant driving force, if not the sole force, behind speciation and the evolution of novelty in bacteria. Of all mechanisms of horizontal gene transfer (HGT), conjugation, the contact-dependent plasmid-mediated transfer of DNA from a bacterial donor to a recipient cell, is probably the most universal. First observed between bacteria, conjugation also mediates gene transfer from bacteria to yeast, plant and even animal cells. The range of environments in which bacteria naturally exchange DNA has not been extensively explored. The interior of the animal cell represents a novel and potentially medically relevant environment for gene transfer. Since most antibiotics are ineffective inside mammalian cells, our cells may be a niche for the evolution of resistance and virulence in invasive pathogens. Invading bacteria accumulate in vacuoles inside human cells, protected from antibiotics. Herein, I demonstrate the ability of intracellular Salmonella typhimurium to meet and exchange plasmid DNA by conjugation within animal cells, revealing the animal intracellular milieu as a permissive environment for gene exchange. This finding evokes a model for the simultaneous dissemination of virulence and antibiotic resistance within a niche protected from both antibiotics and the immune system and extends the variety of environments in which bacteria are known to exchange genes. Unlike conjugation between bacteria, conjugation between bacteria and eukaryotic cells requires the import of transferred DNA into the nucleus before the transferred genes can be expressed and inherited. Plant-cell nuclear transformation by the conjugation system of the Agrobacterium tumefaciens Ti plasmid is believed to be mediated by nuclear localization sequences (NLSs) carried within the proteins that accompany the T-DNA during transfer. Whether NLSs are equally important for transmission of other conjugative plasmids to eukaryotic cells is unknown. Herein, I demonstrate nuclear localization potential within the putative conjugative escort protein TraI of the IncPa plasmid RP4. In contrast, MobA, the putative escort protein from the IncQ plasmid RSF1010, lacked any clear nuclear localization potential. It is therefore likely that specific nuclear localization signals within conjugative proteins are not essential for nuclear transformation per se, although they may assist in efficient plasmid transmission.

Neisseria meningitidis is an obligate human pathogen causing over 1000 cases of meningococcal disease within the U.K., 10 % of which result in long-term disability or fatality. 10-70 % of the population carry N. meningitidis in their nasopharynx, the natural reservoir of this bacterium, as a commensal. The host-pathogen interactions of this species are complex and a greater understanding of the molecular mechanisms involved in pathogenesis and immune evasion is required. Three aspects of N. meningitidis pathogenesis were explored in this study. One mechanism of immune evasion which promotes serum resistance of N. meningitidis is recuitment of complement factor H through domains 6 and 7 (fH₆₇) by factor H binding protein (fHbp). In this study, mouse fH₆₇ was recombinantly expressed and purified. fHbp did not bind mouse fH₆₇ at physiologically relevant protein concentrations. The structure of mouse fH₆₇ was solved, showing differences in domain orientation and surface chemistry compared to the human version of this protein, potentially accounting for the host specificity of this interaction. Type IV pili (T4P) are crucial adhesins of N. meningitidis, the fibre of which is composed of thousands of copies of PilE. A method was developed to recombinantly produce large quantities of this protein from a variety of meningococcal strains and the structure was solved of one PilE protein. Subsequent analysis was performed with the PilE proteins investigating their interaction with the putative pilus receptor CD46 and human epithelia as well as their immunogenicity. A method was also established to produce PilC, the proposed tip-assocoated adhesin of T4P. ZapE has recently been identified as an important protein in pathogen colonisation, functioning as an ATPase linked to Z-ring formation in bacterial cell fission. Both N. meningitidis and E. coli ZapE were recombinantly produced. The domain boundaries were mapped and ATPase activity was confirmed. No interaction was seen with FtsZ but DNA binding and modulation was observed by shift assays, the exact function of which remains to be elucidated in future studies.

A computational model for mitochondrial function has been developed from oxygen concentration data measured in the Oroboros Oxygraph-2k and oxygen consumption rates measured in the Seahorse XF24 Analyzer. Measurements were acquired using embryonic-cultured cortical neurons and isolated mitochondria from CD1 mice. Based on the biological mechanism of mitochondrial activity, a computational model was developed using biochemical kinetic modelling. To modulate mitochondrial activity, dysfunctions were introduced by injecting the inhibiting reagents oligomycin, rotenone, and antimycin A, and the uncoupling reagent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) during measurements. To incorporate these changes, model equations were adapted and globally calibrated to experimental data using the genetic algorithm developed by Jason Fiege of the University of Manitoba by fitting oxygen concentration data. The model was coded in MATLAB R2014a along with the development of a graphical user interface for simulating mitochondrial bioenergetics in silico. / October 2016

Background: Chronic kidney disease (CKD) affects approximately 20 million Americans and is the cause of significant morbidity and mortality. Anemia, common in CKD, develops early in the disease process. It contributes to increased risk of cardiovascular disease, hospitalization, mortality, and diminishes health-related quality of life. Intravenous iron and Erythropoiesis Stimulating Agents (ESAs) are recommended for anemia management in CKD. The utilization patterns of IV iron and ESA, and their impact on hospital costs and length of stay merits investigation. Objectives: There were five general objectives of this investigation. The rate and extent of utilization of IV iron in anemic CKD patients was quantified across teaching hospitals in the US. Patient characteristics of those receiving IV iron and ESA and ESA alone were evaluated in detail. Predictors of IV iron and ESA use were determined. The impact of IV iron and ESA use was examined separately for total hospital costs and length of stay (LOS) while adjusting for confounding. Methods: This is a retrospective cohort analysis within the University Health System Consortium data warehouse. Eligible patients are those who were admitted to a hospital and received either IV iron and ESA or both at least once during the period of January 1, 2006, and December 31, 2008. Inclusion criteria include age > 18 years old with a primary or secondary diagnosis of CKD. The exposure of interest was IV iron and ESA therapy, and the outcome was the difference in total hospital costs and length of stay between patients only on ESA, and those on ESA and IV iron. A clustered binomial logistic regression using the GEE methodology was used to identify predictors of IV iron utilization. Propensity scores were used to control for confounding. A generalized estimating equations (GEE) model using a gamma distribution and log link was used to determine the adjusted hospital cost and length of stay for the IV iron and ESA and ESA alone therapy groups. Results: During the study period, 82,947 patients met all the inclusion and exclusion criteria. Of the 82,947 CKD patients on ESA therapy, only 8% (n = 6678) patients were on IV iron supplementation. Age, race, primary payer, admission status, severity of illness, dialysis status and physician specialty were identified as strong predictors of IV iron use in CKD patients. According to the multivariate model, the overall mean hospital cost for all 82,947 patients was $31,674. For patients using both IV iron and ESA (n=6678), mean costs were $34,756 compared to $31,404 for ESA users alone (n=76,269) – a difference of $3,352. The overall mean LOS for all patients was 9.75 days. For those using IV iron, the LOS was 10.71 days, and for those only using ESA, the LOS was 9.66 days– a difference of approximately 1 day. Conclusions: This inquiry is the first large multi-center investigation to quantify the impact of IV iron and ESA use on total hospital costs and LOS. Our investigation showed significant reduction in ESA doses with the use of IV iron supplementation, however, the overall prevalence of IV iron usage was low. Intravenous iron users were associated with a higher total hospital cost and longer length of stay than ESA users.

Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3'-PG (PO4CH2COOˉ) or 3'-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3'-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3'-PGs, in either the presence or absence of magnesium, to give a 3'-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3'-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3'- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl.

El presente informe, se ha estructurado con la finalidad de integrar temas conceptuales, técnicos y complementarios al diseño de las carreteras, que permita al profesional de Ingeniería Civil, la toma de decisiones bajo un lineamiento social en las construcciones de carretera. En la actualidad, para la construcción de carreteras transversales en el país, los métodos tradicionales son poco usados y por lo contrario son reemplazados por metodologías innovadoras de acuerdo a la demanda de diseños coherentes a la realidad de cada zona que atraviesa. El informe realizado, presenta las técnicas de construcción del mejoramiento y rehabilitación de la carretera Ayacucho - Abancay, tramo IV: Km 154+000 – Km 210+000 ubicada en el departamento de Apurímac, Provincia Chincheros y Distritos de Chincheros – Uripa, Se ha tenido en consideración las condiciones de los suelos, altitud, temperatura, precipitaciones, entre otras variables, donde propicia diseños por estratos, es decir por grupo de factores incidentes en una zona y que afectan a los diseños de las estructuras de pavimentos que pudieran ser causales de la degradación prematura.

>Magister Scientiae - MSc / This study describes the synthesis and characterisation of Cu(II) and V(IV) complexes of tri- and quadridentate ligands L1 and L2 formed by condensation of ethylenediamine with acetylacetonate in 1:1 and 1:2 molar ratio, respectively. Encapsulation of these metal complexes in the nanocage of zoilite-Y generates new heterogeneous catalysts. These catalysts were synthesized employing the flexible ligand method encapsulation technique.The structures of these encapsulated complexes were established on the basis of various physico-chemical and spectroscopic studies. The results indicated that the complexes did not hinder or modify the framework or structure of the zeolite, confirming successful immobilization of Schiff-bases through the voids of zeolite Y.These encapsulated complexes were screened as heterogeneous catalysts for various oxidation reactions such as such as phenol, benzene, styrene and cyclohexene using a green oxidant (H2O2).For comparison, the corresponding neat complexes were screened as potential homogeneous catalysts for these oxidation reactions. The results proved that the corresponding homogeneous systems described here represent an efficient and inexpensive method for oxidation of phenol, benzene, styrene and cyclohexene, having advantages over heterogeneous catalysis are its high activity and selectivity and short reaction times. Its major problem is its industrial application regarding principally the separation of the catalyst from the products.The size of the substrate has a significant effect on the conversion by encapsulated complexes such as in styrene oxidation. Therefore, it was established that steric effects of the substrates play a critical role in the poor reactive nature of the encapsulated complexes.In general, the percentage conversion decreased upon encapsulation of complexes in zeolite Y. All catalysts studied proved to be potential catalysts for the various oxidation reactions.It has been shown in this study that encapsulation can effectively improve product selectivity but requires a longer reaction time in most cases for maximum activity.Furthermore,oxovanadium complexes were more reactive than copper-based catalysts in all oxidation reactions tested in this study.A reaction mechanism study revealed that the activity of the encapsulated and neat complexes occurs through either formation of peroxovanadium (V) or hydroperoxidecopper(II) intermediate species.The studies in this thesis, therefore, conclude that the Cu(II) and V(IV) complexes encapsulated in Y-zeolite are active heterogeneous catalysts for the selective oxidation of various substrates. Encapsulation of the metal complexes in the super cages (-cages) of the zeolite matrix has the advantages of solid heterogeneous catalysts of easy separation and handling, ruggedness, thermostability, reusability (regeneration of the deactivated catalysts) as well as share many advantageous features of homogeneous catalysts.

Iron deficiency anemia (IDA) is a common extra-intestinal manifestation of inflammatory bowel disease (IBD). It is particularly common in the pediatric population, with 40-60% of pediatric patients with IBD meeting criteria for anemia (Aljomah et al, 2018). A number of studies have examined the use of both IV and oral iron treatments to treat anemia in IBD, but few have examined the safety and efficacy of these treatments in children and how they impact a patient’s health-related quality of life. We conducted a prospective cohort study of 79 pediatric patients admitted to Boston Children’s Hospital for management of their IBD. 48 of these patients received IV iron, 13 received oral iron, and 12 were not treated with either. Treatment with IV iron resulted in a statistically significant increase in hemoglobin of 1.75g/dL+/-1.4g/dL (mean +/- SD) from admission to the time of their first post-discharge visit (p=0.0001). Prescription of oral iron at the time of discharge did not result in a significant increase in hemoglobin over the same interval (p=0.481). Though there was a positive trend, IV iron treatment did not result in a significant change in health-related quality of life (HRQOL) measurements as measured by the IMPACT-III questionnaire (p=0.06). Our study suggests that IDA is common in patients admitted for management of their IBD, IV iron is more efficacious in raising hemoglobin, and that larger studies will be needed to more fully demonstrate the impact of effective iron repletion on overall quality of life in these patients.

Die Dissertation beinhaltet die vollständige Edition des Kammerregisters Papst Martins IV. (Reg. Vat. 42, 1281 – 1285). Es ist das einzige Papstregister aus dem 13. Jahrhundert, das bisher nicht ediert worden war, und enthält 612 vollständige Briefe dieses Papstes. Ihr Inhalt ist aufschlussreich für die Geschichte des Kirchenstaates und das päpstliche Finanzwesen am Ende des 13. Jahrhunderts und eröffnet wertvolle Einsichten in die noch wenig erforschte päpstliche Kammer dieser Zeit. / My thesis concerns the complete edition of a register of Pope Martin IV’s chamber (Reg. Vat. 42, 1281 – 1285). It ist the only papal register of the 13th century which has not yet been edited and includes 612 complete letters of that pope. Their content ist of historical interest in that they reveal much about the Papal State and the papal finances during that part of the 13th century. They also give valuable insight into the camera apostolica at that time which has not been researched much previously.