Orientador: Carlos Francisco Sampaio Bonafé / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:21:51Z (GMT). No. of bitstreams: 1 Landini_GustavoFraga_D.pdf: 2852152 bytes, checksum: a366fb6b71cf198fd01af8b55114c8a9 (MD5) Previous issue date: 2007 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

Les récepteurs métabotropes du glutamate (mGluR) sont des récepteurs couplés aux protéines G qui régulent la transmission synaptique. Ce sont des cibles de choix pour le traitement de maladies neurologiques et psychiatriques telles que la maladie de Parkinson et la schizophrénie.J'ai développé une stratégie d'étude de l'assemblage multimérique des protéines membranaires dans des cellules vivantes, à l'aide de techniques de marquage orthogonal et de FRET en temps-résolu. De façon inattendue, j'ai montré que certaines sous-unités de mGluR, en plus de former des récepteurs homodimériques, peuvent former des récepteurs hétérodimériques fonctionnels. D'autre part, j'ai appliqué ces techniques à l'étude du mécanisme d'activation des mGluR et de leur régulation allostérique. J'ai démontré qu'un mouvement relatif des domaines extracellulaires au sein du dimère était responsable de l'action du glutamate.Ce travail a permis de mieux comprendre le fonctionnement des mGluR, et permet la conception de nouveaux tests de criblage. / Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate synaptic transmission. They are relevant therapeutic targets for neurological and psychiatric disorders, such as Parkinson disease and schizophrenia.I developed a strategy to study the multimeric assembly of membrane proteins in living cells, through a combination of orthogonal labeling and time-resolved FRET. Unexpectedly, some subunits of mGluRs, in addition to forming homodimeric receptors, were found capable of forming functional heterodimeric receptors. Then, I applied these techniques to study the activation mechanism of mGluRs and their allosteric regulation. I demonstrated that a conformational change of the dimeric extracellular domain is responsible for the action of glutamate.In addition to increase our understandings of how mGluRs function, this work opens new avenues for the design of drug screening tests.

No description available.

This thesis is devoted to the study of orientifolds and dynamical supersymmetry breaking in configurations of D-branes on toric Calabi-Yau singularities, through the lens of dimer models. We first review the basic ingredients of string theory that led to the formulation of gauge/gravity dualities in terms of dimers. Then, we discuss the non-abelian anomaly cancellation conditions for the supersymmetric gauge theories arising on D-branes and provide necessary geometric criteria to determine whether an orientifold projection can be safely introduced. We also find a new realization of orientifold projection without fixed loci in dimer models and expand on its physical features. We argue that it exhausts the possibilities of orientifolding dimer models. In the subsequent part of the thesis, we investigate dynamical supersymmetry breaking vacua in the same class of models and their typical instability along N=2 Coulomb branches. This leads us to formulate a no-go theorem against their stability based on geometrical features of the singularity, and then to establish a precise way to circumvent it. We eventually find the first instance of stable dynamical supersymmetry breaking vacuum in string theory from D-branes on a toric Calabi-Yau singularity, the Octagon. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Inledning: Patienter med covid-19 har en ökad risk att drabbas av lungemboli. Symtomen som patienter med covid-19 har liknar många gånger symtom på lungemboli. Förhöjda D-dimer-värden ökar misstanken om lungemboli. Detta är dock svårt att tolka eftersom patienter med covid-19 kan ha förhöjda D-dimervärden på grund av infektionen. För att misstänka lungemboli behövs ett positivt utslag på D-dimer och beaktande av symtom och för att påvisa lungemboli behöver patienter genomgå en undersökning med datortomografi (DT). Syfte: Beskriva symtom och kliniska tecken hos patienter med covid-19 som genomgår en datortomografiundersökning med frågeställningen lungemboli. Genom syftet utformades frågeställningen: Ses någon skillnad i symtom och kliniska tecken hos patienter med covid-19 som får diagnosen lungemboli och de som inte får diagnosen lungemboli? Metod: Studien har genomförts som en allmän litteraturöversikt och innefattar elva vetenskapliga artiklar med kvantitativ metod. Resultat: Ingen signifikant skillnad kunde ses i symtom, fysiska mätvärden, ålder och kön hos patienter med covid-19 som diagnostiserades med lungemboli jämfört med de som inte diagnostiserades med lungemboli, men däremot sågs en signifikant skillnad i D-dimernivåerna. Alla patienter med covid-19 hade förhöjda D-dimernivåer, och de patienterna med lungemboli hade högre värden. Slutsats: Det gick inte att urskilja något D-dimergränsvärde som med säkerhet kan utesluta lungemboli hos patienter med covid-19. Symtom och fysiska mätvärden hos patienter med covid-19 som drabbats eller inte drabbats av lungemboli är lika varandra. Förhöjda D-dimer- nivåer hos patienter med covid-19 är vanligt. Detta gör att många patienter med covid-19 remitteras till DT med frågeställningen lungemboli. Röntgensjuksköterskan ska kunna avgöra om en undersökning är berättigad för att en patient inte ska bli utsatt av en onödig stråldos, vilket är komplicerat i detta fall.

Biological material is a common type of evidence found at a crime scene, and body fluid identification is an essential process in crime scene investigation. One of the most common types of body fluids found is blood. After a stain has been presumptively identified as blood through the use of a colorimetric chemical test, additional testing may be necessary to better characterize the stain. SERATEC PMB Test is a relatively new lateral flow immunochromatographic assay that targets human hemoglobin and D-dimer simultaneously in order to distinguish peripheral blood and menstrual blood at the same time. Elevated levels of D-dimer, a fibrin degradation product, are found in menstrual blood, thrombosis formation and as part of the postmortem process. A previous study investigated levels of D-dimer in menstrual, peripheral and postmortem blood using the SERATEC PMB Test. In this study, all postmortem blood samples showed positive results for both hemoglobin and D-dimer; all peripheral bloodstain samples from living individuals showed positive results for hemoglobin detection, and negative results for D-dimer detection; and most menstrual bloodstain samples showed positive D-dimer results. The results suggest that this assay could be considered a presumptive test for both postmortem blood and menstrual blood. However, as D-dimer concentrations vary between individuals, additional testing is necessary to conclusively distinguish postmortem blood, menstrual blood and peripheral blood from living individuals with especially high D-dimer levels.

Very short lifetimes of excited states of isolated bases in nucleic acids, on the picosec- ond order, are believed to contribute to photostability of the genetic code. When embedded in DNA this behavior becomes more complex, mainly due to their inter- actions via stacking and hydrogen bonding. The DNA photophysiscs is not fully understood yet. It depends e.g. on the conformation and the character of excited states. The studies on smaller systems can help to improve the understanding of these phenomena. The aim of this work was to examine the dynamics of the excited states of the n → π∗ character of the complex of N-methylformamide dimer and two waters. The study was performed using non-adiabatic dynamics simulations with on-the-fly Surface Hopping algorithm based on the potential energy surfaces and non-adiabatic couplings obtained with multi-reference approach. The results show that after the vertical excitation into delocalized S2 state the system relaxes into S1 state within several tens femtoseconds. For majority of the population, the charac- ter of the state then oscillates between localized and delocalized during the whole course of the dynamics. Comparison with calculations with the waters removed in- dicates that the delocalization is caused by waters serving as a bridge between the two chromophores. 1

Solid-state NMR study of nitric oxide adsorption in MOFs. Amine functionalized Cu3btc2 MOFs shows chemisorption of NO as NONOates. NO also adsorbed in Cu open metal site(OMS). All of these information is characterized by 1H, 13C and 15N NMR studies. NO adsoprtion in Al based MOFs MIL-100(Al) is investigated to get details about direct detection of OMS site by 27Al NMR. First time detection of 15NO as dimer is acheived by 15N NMR studies.:Contents.............................................................................. v List of Figures...................................................................... vii Abbreviations............................................................................. ix 1 Motivation .............................................................................1 2 Introduction .............................................................................3 2.1 Nitric oxide (NO): A Potent Gasotransmitter . . . . . . . . . . . . . . . . . . 3 2.1.1 Biological action in human biology: . . . . . . . . . . . . . . . . . . . . 3 2.1.2 Structure and chemistry of NO . . . . . . . . . . . . . . . . . . . . . . 4 2.2 NO storage in porous materials . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.1 Physisorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.2 Chemisorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.3 Current NO storage materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.4 Metal-organic frameworks (MOFs) . . . . . . . . . . . . . . . . . . . . . . . . 8 2.4.1 Cu3btc2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.4.2 MIL-100(Al) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3 Experimental techniques .............................................................................15 3.1 Nuclear spin interactions in solid-state NMR . . . . . . . . . . . . . . . . . . . 15 3.2 NMR Techniques and Pulse Sequences . . . . . . . . . . . . . . . . . . . . . . 19 3.3 NMR sample tube preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4 Gas adsorption procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4 Overview and enclosed papers 29 References .............................................................................121 5 Contribution .............................................................................137 / Gas storage in solids is becoming more important as a technology, with applications ranging in fields such as energy, the environment, and more importantly in biology and medicine. Porous solid storage materials are also increasingly important to advancements in science, as seen through their use in emergent gas-delivery technologies that include storage of the signaling molecule, nitric oxide (NO). The deficiencies of NO biosynthesis have been interconnected to a number of diseases, such as cardiovascular dysfunction, thrombosis and cancer. To date, one of the promising NO delivery materials are the metal-organic frameworks (MOFs), a new class of porous materials, which can store significant quantities of NO and then deliver it to specific sites in the body. MOFs contain open metal sites (OMS) that can physisorbed NO. Furthermore, amine functionalized MOFs can store NO covalently as N,N -diazeniumdiolates (NONOate). The thesis at hand is a collection of the publications written and co-authored by the author. The following thesis will investigate NO adsorption of one of the most highly studied carboxylate-based MOFs, Cu3btc2, and its amine derivatives, and MIL-100(Al) [Materials Institute Lavoisier] by magic angle spinning (MAS) NMR. However, NMR observation of Cu3btc2 is quite difficult, because it behaves as a paramagnet at room temperature. This paramagnetic behavior originates from the presence of antiferromagnetically coupled Cu-Cu ions, which result in an S=1 electronic state at higher temperatures (above 90 K). In that case, a significant insight into the understanding of NO interaction and the changing of electronic properties of NO loaded Cu3btc2 and the formation of NONOate in Cu3(NH2btc)2, which is known as University of Hamburg materials (UHM-30), has been obtained by MAS NMR. In paper (A) the effect of NO adsorption on the Cu3btc2 and UHM-30 has been followed by adsorbing different amounts of NO/Cu via the gas phase. The relevant NMR parameters, e.g., chemical shift, hyperfine coupling and 1H T1 of NO loaded MOFs displayed the change of electron density at the Cu site because of NO adsorption as well as indirect suggestion of NONOate formation. Further studies are carried out on the secondary amine functionalized MOFs, Cu3(NHRbtc)2, as they opened up the greater potential for NONOate formation in the MOFs. The structural characterization of four different Cu3(NHRbtc)2 is carried out by MAS NMR in (B) which revealed better incorporation of the btc ligand compared to NHRbtc in MOFs. In (C) NO loaded UHM-37 is extensively investigated by MAS NMR in order to understand the sorption priority, e.g., chemisorption or physiosorption. The multinuclear approach together with the fact that the MOFs contain antiferromagnetically coupled Cu-Cu pairs and NO being paramagnetic shows significant effects on spectra that allow for the deduction of adsorption effects in these MOFs. In the amine-functionalized UHM-37, first chemisorption of NO takes place to form NONOates. When this reaction is completed, additional adsorption at the OMS takes place. This observation is also in accordance with observed 13C shift changes upon NO adsorption. With 15N-labeled NO, we were able to directly determine signals of NONOate formation in UHM-37. To the best of our knowledge, this is the first report on 15N NMR data of NONOates in porous systems. In (D), NO interaction of another type of carboxylate MOF, MIL-100(Al) is investigated by 1H, 13C and 27Al MAS NMR. 27Al NMR data show that half of all Al sites are free for gas adsorption and that additional Al(OH)3 is present inside the pores, which is well-documented by 27Al 1H HETCOR spectra. 1H T1 of NO loaded MIL-100(Al) decreases with NO loading representing uniform distribution of NO in the MOF. In addition, the MIL-100(Al) five-coordinated Al site intensity is decreasing with increasing NO loading, while six-coordinated site intensity is increasing and a maximum of 1 NO per Al trimer can be adsorbed. This indicates rather weak NO adsorption. The magnetic properties of NO make it quite interesting for NMR measurements. Therefore, isotopically leveled bulk 15NO is studied for the first time by NMR in (E). The manuscript is accepted for publication and is included in this thesis. 15N NMR spectra have been obtained in the liquid and the solid state. The dynamic equilibrium ranges between (NO)2 and NO is characterized in gas - liquid transition temperature of NO. The variation of 15N chemical shift, line width and 15N T1 of NO with temperature represents the fast dynamic equilibrium. SQUID measurements are carried out on the same sample for further confirmation of the NMR results.:Contents.............................................................................. v List of Figures...................................................................... vii Abbreviations............................................................................. ix 1 Motivation .............................................................................1 2 Introduction .............................................................................3 2.1 Nitric oxide (NO): A Potent Gasotransmitter . . . . . . . . . . . . . . . . . . 3 2.1.1 Biological action in human biology: . . . . . . . . . . . . . . . . . . . . 3 2.1.2 Structure and chemistry of NO . . . . . . . . . . . . . . . . . . . . . . 4 2.2 NO storage in porous materials . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.1 Physisorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.2 Chemisorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.3 Current NO storage materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.4 Metal-organic frameworks (MOFs) . . . . . . . . . . . . . . . . . . . . . . . . 8 2.4.1 Cu3btc2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.4.2 MIL-100(Al) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3 Experimental techniques .............................................................................15 3.1 Nuclear spin interactions in solid-state NMR . . . . . . . . . . . . . . . . . . . 15 3.2 NMR Techniques and Pulse Sequences . . . . . . . . . . . . . . . . . . . . . . 19 3.3 NMR sample tube preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.4 Gas adsorption procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4 Overview and enclosed papers 29 References .............................................................................121 5 Contribution .............................................................................137

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonucleasesensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65-70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.