Researchers have identified several methods for treating acute myocardial infarction (AMI) patients affected by ischemia and reperfusion injury. Some of these therapies include thrombolysis, balloon angioplasty, and coronary arterial bypass graft (CAGB). This lab has previously demonstrated that transplantation of mitochondria into the ischemic zone of a rabbit heart during reperfusion significantly improved recovery as compared to current techniques. In order for this therapy to be translated into the clinic a rapid isolation method for producing highly pure and functional mitochondria will be required. Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 minutes to complete. Herein, a method for rapid isolation of mitochondria from mammalian tissue biopsies is described. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters which eliminates repetitive centrifugation steps. Mitochondrial isolation time is less than 30 minutes compared to 60-100 minutes using alternative methods. This isolation protocol yields approximately 2 x 10^10 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Depuis leur découverte au milieu des années quatre-vingt, les noyaux à halo n'ont cessé d'intriguer et de fasciner. Cette découverte a été rendue possible par le développement de faisceaux d'ions radioactifs au cours des années septante. Cette performance a ouvert le champ de l'exploration des propriétés des noyaux atomiques aux frontières de la stabilité. Les noyaux à halo présentent une taille beaucoup plus grande que prévue par l'hypothèse communément adoptée du noyau sphérique et seraient constitués d'un coeur, qui a les propriétés d'un noyau normal, auquel un ou deux neutrons qui forment le halo seraient faiblement liés. Cette propriété bouleverse complètement l'image traditionnelle du noyau atomique, celle d'un mélange quasiment homogène de protons et de neutrons. Les noyaux à halo ont tendance à facilement se dissocier lorsqu'ils entrent en collision avec un noyau cible. Leurs réactions de dissociation constituent donc un formidable outil expérimental pour étudier leurs propriétés. Cependant l'analyse de telles réactions nécessite une description précise du processus de collision. A cette fin, nous avons développé un nouveau modèle de réaction: l'approximation eikonale dynamique. Il s'agit d'une méthode purement quantique qui combine les avantages des approximations eikonale traditionnelle et semi-classique. Elle prend en compte aussi bien les effets dynamiques du mouvement interne du projectile que les interférences quantiques entre les trajectoires. Elle conduit à la résolution d'une équation de Schrödinger approchée similaire à celle de l'approximation semi-classique avec trajectoires rectilignes. Nous appliquons l'approximation eikonale dynamique à l'étude de réactions impliquant trois noyaux à halo différents : le $^{11}$Be, le $^{19}$C et le $^{8}$B. Pour les trois systèmes étudiés, nous confrontons nos résultats théoriques avec les données expérimentales disponibles. Nous constatons un très bon accord tant sur l'allure que sur l'ordre de grandeur des différentes sections efficaces. Ceci est valable aussi bien pour les collisions sur cible lourde que sur cible légère. Les motifs d'interférence présents dans les distributions angulaires sont également bien reproduits par notre modèle, y compris pour la diffusion élastique. Nous analysons la section efficace de dissociation expérimentale du $^{19}$C dans le but de déterminer la présence d'une résonance dans le spectre continu de ce noyau. Nous constatons que plusieurs options restent plausibles et que d'autres mesures sont nécessaires (sur cible légère, par exemple) pour confirmer nos hypothèses. La dissociation coulombienne du $^{8}$B fait l'objet de nombreuses études expérimentales dans le but d'obtenir des informations sur la réaction inverse qu'est la capture radiative $^{7}$Be(p,$gamma$)$^{8}$B. En analysant cinq expériences pour lesquelles différentes observables ont été mesurées, nous examinons la validité des hypothèses qui permettent de faire un lien direct entre dissociation et capture radiative. Nous observons que l'extraction d'informations sur la capture radiative à partir de données de dissociation semble plus compliqué qu'initiallement prévu. Cependant les différentes mesures permettent de valider un modèle de structure du $^{8}$B qui peut servir au calcul de la section efficace de capture radiative. L'approximation eikonale dynamique constitue donc un outil performant qui permet d'analyser toutes les observables liées à la dissociation élastique d'un noyau à halo sur une cible lourde ou légère à des énergies incidentes de quelques dizaines de MeV par nucléon.

The focus of this dissertation is the introduction of surface-induced dissociation (SID) into a commercially available quadrupole time-of-flight mass spectrometer as an alternative ion fragmentation method. The performance of the SID device was characterized and its applications were demonstrated by dissociating peptides, proteins, inorganic salt clusters and non-covalent protein complexes. The SID setup allowed direct comparison of SID with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of Q-TOF instrumentation, including extended mass range, high sensitivity and resolution. With the SID setup installed, no significant reduction of the ion transmission was evident. SID fragmentation patterns of peptides are, in general, similar to CID, with slight differences in the relative intensities of immonium ions, backbone cleavage b- versus y- type ions, and y- versus y-NH3 ions. This suggests enhanced accessibility to high energy/secondary fragmentation channels with SID. SID studies on cesium iodide clusters (CsI) also revealed that SID deposits more internal energy.The utility of mass spectrometric methods to probe the gas phase cyclization process was studied with [D-Ala2]-Leucine Enkephalin amide. This peptide showed prominent formation of the [M-NH3]+ ion which is believed to be the linear b5 ion with a C-terminal oxazolone structure. Other fragments in the spectra indicate that the linear b5 ion undergoes cyclization, subsequent ring opening and further dissociation to rearranged fragments that cannot be explained by the initial sequence. The similarities between the cyclic and b5-ion from the linear peptide indicated the formation of a heterogeneous ion population and this is further supported by gas-phase H/D exchange experiments. An ion funnel interface to improve ion transmission at high pressures was tested in a custom built quadrupole-surface-quadrupole instrument. The ion transmission efficiency for selected bio-molecules such as YGGFLR, insulin chain-B, ubiquitin and cytochrome c showed to approach almost 90%, with the funnel interface installed. The ion transmission efficiency was effected by several factors including: the size of the analyte, the DC gradient, the RF frequency, and the RF amplitude. The higher fragmentation efficiencies for SID in the presence of the funnel interface indicated higher internal energy deposition for the funnel interface.

No description available.

This thesis explores the use of mass spectrometry to define the strengths, and understand solution phase speciation of phenolic oxime-based solvent extractants of the types used in the hydrometallurgical recovery of copper. Chapter 1 reviews briefly the current extraction technology for copper and focuses on hydrometallurgy and the use of phenolic oximes such as 5-nonylsalicylaldoxime. The modification of the latter to improve extraction efficiency is discussed, focussing on the introduction of X-substituents in the 3 position of the benzene ring. Modern mass spectrometry techniques are also discussed with a focus on their application to inorganic systems and their use in achieving the aims of this thesis, as defined above. The work described in chapter 2 involves the development of collision induced dissociation (CID) techniques to determine the relative gas phase stabilities of copper complex anions of the type [Cu(L)(L-H)]-, where LH is a 5-alkyl-3-X-2- hydroxybenzaldehyde oximes and X a range of substituents. The importance of interligand interactions in the outer-coordination sphere and their influence on gas phase anion stability, as defined by CID, is reported. The work described in Chapter 2 on CID is extended in chapter 3 and looks at the effect of charge, of ligand type, LH, and of the nature of the metal on the stability of ionic forms of [M(L)2] complexes, where LH is extended to include 5-alkyl-2- hydroxyphenylethanone oximes. The effects of substitution at the azomethine carbon atom and at the 3-position of the benzene ring and of variation of the nature of the metal on the ion dissociation mechanisms are shown to have a major influence on ion stability under CID conditions. In chapter 4 density functional theory calculations have been used to investigate the influences of substitution at the azomethine carbon atom and at the 3-position of the benzene ring and of variation of the nature of the metal on the gas phase structures of the neutral complexes, [M(L)2]. Gas phase deprotonation and dimerisation enthalpies of the ligands, LH, and enthalpies of formation of [M(L)2] complexes have been calculated and correlates with experimentally determined ligand extraction strength. The ligand type has been extended to include 3-X-2-hydroxybenzaldehyde hydrazones, which have previously been shown to have lower distribution coefficients for copper than the analogous 3-X-substituted oximes. The calculated gas phase formation enthalpies for [M(L)2] show a strong correlation with the strengths as extractants LH, measured as their pH0.5 values for metal uptake. Chapter 5 considers whether mass spectrometry can be used to define the solution equilibria when two different oxime-based ligands, LXH and LYH, compete for Cu(II) in a single phase solution. It has been established that shifts in the relative peak intensities of deprotonated ions derived from the Cu(II) complexes, [Cu(LX)2], [Cu(LY)2] and [Cu(LX)(LY)] reflect changes in the solution composition. The work described in chapter 6 extends the study of solution phase speciation using mass spectrometry. When the Cu(II) and proton concentrations of solutions were varied distinct changes in the resulting electrospray mass spectra were observed and the resulting species were identified using CID and high resolution mass spectrometry. A novel, [Cu3(L-H)3-μ3-O/OH]- species is determined to be a major component of solutions where Cu(II) concentrations are equal to/greater than the LH concentration. Various 3-X-2-hydroxybenzaldehyde oximes (X = CH2NR2) were synthesised. The incorporation of a protonatable arm in the 3-position enabled trinuclear complexes, [Cu3(L-H)3-μ3-OH], to be isolated and fully characterized, including two X-ray determined crystal structures.

This study’s aim was to explore face, content, concurrent validity and psychometric properties of the Swedish Child Dissociative Checklist (CDC) as well as and to provide guidelines towards validation. The CDC is used in Sweden when dissociation is highly probable, and validation could motivate wider use. Qualitative data was gathered from psychologists-in-training (N = 4) clinical psychologists (N = 5) and parents (N = 23) through a focus group, interviews and questionnaires, respectively. Quantitative data using the CDC and items from the Child Behaviour Checklist (CBCL) was obtained from parents. Results suggest good face, content and convergent validity. Factor analysis provides suggestions for future investigations into quantitative validation. Guidelines when validating is revising certain items, investigating the possibility of developing two versions of the CDC, utilizing an adequate sample size as well as assessment with a clinical group. / Studiens syfte var att undersöka face, innehålls- och konvergent validitet samt psykometriska egenskaper hos den svenska versionen av Child Dissociative Checklist (CDC), samt utforma riktlinjer för validering. I Sverige har CDC använts vid hög närvaro av dissociativa symptom, och validering kan motivera bredare användning. Kvalitativ data samlades in från psykologstudenter (N = 4), kliniska psykologer (N = 5) och föräldrar (N = 23) genom fokusgrupp, intervjuer respektive frågeformulär. Kvantitativa data erhölls från föräldrar med CDC och items från Child Behaviour Checklist (CBCL). Resultaten tyder på god face, innehålls- och konvergent validitet. Faktoranalys bidrar med förslag på framtida tillvägagångssätt för kvantitativ validering. Riktlinjer vid validering är revidering av vissa items, utforskning av möjligheten att utveckla två versioner av CDC, tillgång till ett stort sample samt jämförelse med en klinisk grupp.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

1. Abstract Charles University in Prague Faculty of Pharmacy in Hradec Kralove Department of Biophysics and Physical Chemistry Candidate: Miroslav Suchy Supervisor: Ing. Vladimir Kubicek, CSc. Title of Diploma Thesis: Physico-Chemical properties of drugs To test physico-chemical properties of new molecules is necessary during drug development. It could be helpful to understand or predict the pharmacokinetic parametres of a new drug in vivo/in vitro experiments. One of this parameters is a dissociation constant (pK). Dissociation constant is defined as " Number on pH scale, wherein is just fifty percent of molecule in a ionization condition". In real case this number can help us to know where in the gastro-intestinal tract (GIT) the drug will be absorbed. In GIT only molecules exhibiting pK from 3 to 11 could be absorbed. Out of this range it is not possible. In this work I would like to introduce the ways of experimental measurement of pK values. I was working with two methods to measure the pK values of water-soluble compounds. The spectrophotometric method and the potentiometric one. I had to find out, that potentiometric titration is primary method which gets us good and accurate results. Based on my measurement I evaluated the spectrophotometric method as the secondary method. Spectrophotometric method...

L’une des conséquences importantes de l’agression sexuelle est l’apparition de symptômes dissociatifs. La dissociation est un mécanisme de défense qui se manifeste pendant le traumatisme et qui permet à l’enfant de survivre émotionnellement à ce qu’il vit. L’objectif poursuivi dans ce mémoire est d’évaluer quelles variables, parmi différentes dimensions de facteurs (caractéristiques de l’agression sexuelle, facteurs personnels, parentaux, familiaux, autres formes de maltraitance et le dévoilement), influencent la présence de dissociation à un niveau clinique dans une population d’adolescentes ayant subi des agressions sexuelles à l’enfance et suivies par les services de la protection de la jeunesse. À la suite des analyses statistiques, trois variables ressortent comme étant significativement associées à la présence de dissociation d’intensité clinique : la durée de l’agression sexuelle, la méfiance de l’adolescente envers les autres et son sentiment d’être différente des autres. Ces trois variables expliqueraient 28,7 % de la variance de la présence ou de l’absence de dissociation à un niveau clinique. Les difficultés de l’adolescente à appréhender ses relations, sa perception d’elle-même et son identité devraient donc être les centrations des interventions réalisées auprès de la clientèle adolescente en agression sexuelle.