Дисертації з теми "MECHANISM AND FRAGMENT"

This dissertation describes the benzylation of adenine under basic conditions, the unequivocal determination of the identity of the products of this reaction, an exploration of the effect of solvent on the reaction, a thorough computational study of the reaction mechanism and an investigation into the hydrogen-deuterium exchange reaction of the N-benzyladenine products and related compounds. The preferential sites of alkylation of adenine under basic conditions in DMSO were proven to be the N9 and N3 positions. X-ray crystal structures were obtained for both compounds. Formation of the N9-benzyladenine product is the most favoured in polar aprotic solvents, such as DMSO, and as the proportion of polar protic solvents, such as water, increases, so does the formation of the N3-benzyladenine product. Characteristic 1H NMR  chemical shifts of the purine ring protons and HMBC 1H-13C correlation NMR spectroscopy were useful tools to assign the 1H and 13C NMR spectra chemical shifts and confirm that the solution structures were the same as the isolated crystals. Simulating the SN2 mechanism for the N1-, N3-, N7- and N9-pathways computationally, employing DMSO as the simulated solvent, resulted in ambiguous results when considering the electronic energies of initial, TS and final products alone. However, a novel approach was developed (employing IQA-defined energy terms) to study fragment interactions along the reaction paths. It provided a full explanation of the reaction mechanism and yielded results which supported the N3/N9 positions of alkylation over the N1/N7 sites. The preference for the sites of alkylation occurs after the transition state, in which the N1/N7 reaction paths fail to proceed favourably to the end product, N1- and N7-benzyladenine, respectively. The N9-pathway dominates the N3-pathway at the product formation step, which corresponds to the N9- benzyladenine being the major product, as shown in Figure 1, and the N3-benzyladenine being the minor product from the benzylation of adenine. The faster rate of deuteration at the C8 position of N9-benzyladenine as compared to the deuteration rates at the C2 and the C8 of N3-benzyladenine, have shown support for a sp3 mediated mechanism and a carbene mediated mechanism of deuteration based on the “push” and “pull” mechanisms proposed for the C8 proton transfer of ATP in kinase enzymes. The deuteration of the C8 proton of 2,6-dichloropurine derivatives supports the existence of the carbene mediated mechanism since these compounds lack the amine moiety necessary for the sp3 mediated mechanism. These results demonstrate how experimentation and computation have led to greater insights into the reactivity of adenine and its derivatives. This strategy provides a useful platform for future research into adenine reaction mechanisms and the role adenine plays in kinase catalysis.
Dissertation (MSc)--University of Pretoria, 2015.
National Research Foundation (NRF)
Chemistry
MSc
Unrestricted

Multi-cue object tracking is a challenging eld of computer vision. In particular, the challenges originate from environmental variations such as occlusion, similar background and illumination variations or due to variations in target's appearance such as pose variations, deformation, fast motion, scale and rotational changes. In order to address these variations, a lot of appearance model have been proposed but developing a robust appearance model by fusing multi-cue information is tedious and demands further investigation and research. It is essential to develop a multicue object tracking solution with adaptive fusion of cues which can handle various tracking challenges. The goal of this thesis is to propose robust multi-cue object tracking frameworks by exploiting the complementary features and their adaptive fusion in order to enhance tracker's performance and accuracy during tracking failures. A real-time tracker using particle lter under stochastic framework has been developed for target estimation. The inherent problems of particle lter namely, sample degeneracy and impoverishment have been addressed by proposing a resampling method based upon meta-heuristic optimization. In addition, an outlier detection mechanism is designed to reduce the computational complexity of the developed tracker. A robust tracking architecture has been proposed under deterministic framework. Fragment-based tracker with a discriminative classi er has been designed that can enhance tracker's performance during dynamic variations. Periodic and temporal viii update strategy is employed to make tracker adaptive to changing environment. Extensive experimental analysis has been performed to prove the e ectiveness of the developed tracking solution. Multi-stage tracker based on adaptive fusion of multi-cue has been developed for multi-cue object tracking. The rst stage of target rough localization improves the accuracy of tracker during precise localization. In the appearance model complementary cues are considered to handle illumination variations and occlusion. Classi er mechanism and fragment based appearance model are proposed to improve the tracker's accuracy during background clutters and fast motion. Experimental validation on multiple datasets validates the performance and accuracy of the proposed tracker.

Biology
Ph.D.
The spacer region of pRb2/p130 was reported to be able to inhibit the kinase activity of Cdk2. The region responsible for the inhibitory effect was further narrowed down to a 39-amino-acid sequence, which was named as Spa310. In this dissertation, the anti-cancer functions and mechanisms of Spa310 were studied. The synthesized Spa310 peptide was able to inhibit the kinase activities of Cdk2/Cyclin E/A complexes. In vitro kinase assays showed the inhibition occurred in a dose-dependent manner. The half maximal inhibition concentration of the Spa310 in the kinase assay was 1.67mM. In addition, it has been shown that Spa310 peptide is able to inhibit the kinase activities of both Cdk2/Cyclin E and Cdk2/Cyclin A. Intra-cellular distribution study using fluorescein-labeled Spa310 peptide showed that Spa310 was able to localize to the nuclei of A549 cancer cells. Some data indicated the endoplasmic reticulum might play a role in transporting Spa310 peptide from cytoplasm to the nucleus. At high concentration, the treatment of Spa310 peptide was able to arrest cells at the G0/G1 phase of the cell cycle and reduce the growth of xenografted tumors in nude mice. Further studies indicated Spa310 peptide is not a specific inhibitor for Cdk2/Cyclin E/A. It is also able to inhibit the kinase activities of Cdk1/Cyclin B, Cdk4/Cyclin D and Cdk9/Cyclin T/K. Result of a binding assay using GST-Spa310 and in vitro transcribed/translated Cdk2 did not support a direct binding between Spa310 and Cdk2. Additionally, GST-Spa310 was unable to bind to the in vitro transcribed/translated Cyclin E. At first, co-immunoprecipitation experiments indicated a weak binding between Spa310 peptide and Cdk2. However, later this weak binding was proven to be unspecific and only occurred when the concentration of Spa310 peptide was high. Thus, the hypothesized mechanism of the inhibitory effect of Spa310 was not supported. After noticing three classic Cdk phosphorylation sites present in Spa310, it was proven that Spa310 is a substrate for Cdk1, 2, 4 and 9. Results of kinase assays supported the inhibitory effect of Spa310 on the different Cyclin-dependent kinases was resulted from a substrate-competitive mechanism. Although the data generated from this study does not support Spa310 is a potent peptide inhibitor for the Cdks, knowledge gained from and the approach used in this research can be applied to design and develop more potent and specific Cdk2 peptide inhibitors, which have their potentials to work as powerful anti-cancer reagents.
Temple University--Theses

Polarization is a term that is often excluded from almost all virtual screening. Polarizability helps explain interactions between nonpolar atoms and electrically charged species. When studying fragments in FBDD these minor interactions could have large effect in changing how well a ligand will bind to its target. After including the polarizability terms in docking a validation set of ligands (Favia et al., 2011) with GLIDE, it improved the results the amount good docked poses (< 2 Å RMSD) by up to 12%. However some ligands were bound in incorrect poses. Further investigation was carried out with MD to observe if given enough time ligands bound in an incorrect pose would return to the binding site. In the first stages of investigating MD we ascertained if we could use GPUs to simulate larger systems and faster. After some performing some MD simulations in GROMACs we found that GPUs were an improved option and thus continued the simulation work with ACEMD which allowed multiple GPUs in tandem. After running the MD simulations for 200ns with atomic charges generated from the polarization the results we found were quite interesting. Some ligands would be trapped in their binding site but would fluctuate quite readily such as 2GVV. Some ligands showed that despite low RMSD they would be ejected from the binding site. In some cases the ligands would then attempt to return to their binding site. Ligands such as in 2CIX would show binding based on the breathing movement of the protein. Some ligands such as 1F5F or 1F8E bound tightly to their binding site during the MD, these ligands also enjoyed improved docking polarization with 0.1 – 1.0 Å improvement. These could be carried forward to become good candidates for experimental testing. Polarization is shown to have an overall positive effect improving binding data and if implemented with simple methods would have little opportunity cost to be added to modern FBDD methods.

C1q initie la voie classique du complement grace a sa region globulaire (gr). C3de correspond a la partie de c3b qui se fixe sur les cellules cibles. Nous avons cristallise ces proteines et enregistre un jeu de donnees a 3,2 a pour la gr, a 3,7 a pour c3de. Des problemes de reproductibilite et de taille des cristaux nous ont empeche de poursuivre l'etude. La subtilisine de bacillus lentus (savinase#t#m) est utilisee comme additif dans les detergents des lessives. Des inhibiteurs competitifs et reversibles (acides boroniques) sont ajoutes dans ces solutions pour eviter que la savinase#t#m ne degrade les autres enzymes presentes. Nous avons resolu la structure de 4 complexes a 2 a puis 1,7 a par remplacement moleculaire a partir d'une nouvelle forme cristalline. Les acides boroniques interagissent avec la proteine par une liaison covalente entre leur atome de bore et l'oxygene gamma de la serine active, et par des contacts hydrophobes entre leur groupement r et la poche de specificite s1. L'acide benzo furane 2 boronique (bf2ba) est l'inhibiteur le plus efficace parmi les 4 etudies. La reactivite entre le bore et la serine active depend de la nature du groupement r, du ph, de la concentration en inhibiteur. A partir du modele savinase#t#m/bf2ba, nous proposons des inhibiteurs potentiellement plus efficaces

The spontaneous formation of aggregates presents an obstacle in the developability, manufacturability and long term storage of numerous therapeutic proteins. These aggregates are a manifestation of the drug molecule's physical instability, potentially leading to reduced biological activity, differences in solution properties or increased immunogenic potential. Currently our understanding of the mechanisms behind aggregation and the role of a protein's extrinsic environment upon the molecule's aggregation propensity remains limited. In this study we investigated the aggregation behavior of a previously unstudied commercial biopharmaceutical, an α-TNF Fab' antibody fragment. Initially we mapped the differences in aggregation kinetics and product morphologies accompanying changes to the protein's aqueous environment, finding that the aggregation pathway adopted by the molecule depended upon the pH of the system. We then employed a combination of intrinsic and colloidal stability measurements to probe the causes of the behavioral differences seen. Together, these biophysical properties quantitatively captured the relative differences in aggregation rates observed. Furthermore, we were able to infer the existence of various partially folded intermediates whose populations govern the types and rates of aggregates formed. Finally, we sought to identify whether observable changes in the native state conformation precluded aggregate formation through low resolution structural analysis using small angle X-ray scattering. We showed that a drop in pH generated a conformationally expanded state which appeared to coincide with the titration of a key histidine residue within the protein's structure. We predict that an equivalent mechanism will lead to aggregation at low pH of other human derived antigen binding fragments.

Doctor of Philosophy
Department of Mechanical and Nuclear Engineering
Youqi Wang
Ballistic performance of textile fabric is affected by numerous elements, such as fabric architecture, material property, and projectile characteristics. Near fiber-level microstructures of soft body armor composed of multi-layer Kevlar KM-2 fabrics are generated for numerical simulation. The modified digital element approach (DEA) is applied to determine the ballistic limit of textile fabrics against fragment simulating projectiles (FSP). Different from other numerical models, the DEA takes a considerable amount of fiber-level detail into consideration and models the fabric at filament-level. In this approach, fabric is an assembly of yarns weaved and relaxed into pre-arranged pattern; yarn is simulated as a bundle of digital fibers. When the number of digital fibers per yarn reaches the number of actual fibers per yarn, fiber-level simulation is achieved. The DEA model successfully simulates real scale multi-layer fabric impacted by spherical projectile and accurately predicted fabric displacement and failure mechanism. It was assumed that the digital fiber is fully flexible and its bending rigidity is negligible. Shear force was thus neglected. However, for projectiles with sharp edge(s), such as FSP, due to resultant shear force, fabric failure starts where it interacts with projectile edge. As a result, the numerical results derived from the previous DEA overestimated the impact strength of fabrics against projectiles with shape edges. Therefore, shear force and fiber bending rigidity must be considered. In the modified DEA approach, numerical tests are employed to determine the effective bending rigidity of digital fiber. A combined tension-shear failure model is then incorporated into the DEA in order to calculate the shear force applied to fibers. The 3-D microscope is applied to measure the radius of FSP along the edge. The surface of the FSP is meshed into triangle elements. A unique algorithm is developed and employed to search contacts between textile fabric and projectile of arbitrary shape. In this research, first, an overview of ballistic impact analysis is discussed; the previous DEA model used in simulating ballistic impact and penetration process is presented. Second, the modified DEA approach used in simulating arbitrary shape projectile perforation process is established and verified. The method of searching and calculating contacts between textile fabric and solid body projectile is explained. The convergence and accuracy of digital element mesh are investigated statistically using tension-shear failure model. Third, fabric shear force and fiber bending rigidity are investigated using tension-shear failure model. The effective digital fiber area moment of inertia is numerically determined. Fourth, standard ballistic tests of real scale multi-layer Kevlar KM2 fabrics are simulated using FSP. Numerical results are compared to high-resolution experimental test data. The modified DEA is validated.

PhD
The aims of this study were to test the efficacy of immunoglobulin and its Fab and Fc fragment in the treatment of experimental autoimmune neuritis (EAN) in Lewis rats, to investigate which portion of immunoglobulin is operative in the effect of IVIg, and to clarify the possible mechanisms by which immunoglobulin exerts its action in the treatment of rats EAN. EAN was induced by immunization with whole bovine peripheral nerve myelin. The immunized rats were randomized into groups, assessed clinically, electrophysiologically, and histologically, and intravenously injected with normal saline, albumin, human IVIg preparation, purified Fab or Fc fragments. The treatment efficacy was compared between normal saline and albumin groups, albumin and IVIg groups, albumin and Fab groups, albumin and Fc groups, Fab and Fc groups, Fab and IVIg groups, and Fc and IVIg groups. Methods of myelin isolation, antibody purification, and Western blot techniques were also applied. The results revealed that treatment with Fc fragment and IVIg at the onset of signs of disease effectively prevented further progression of disease, shortened disease duration, and facilitating recovery from illness as shown in clinical, electrophysiological and histological parameters. In the study which the efficacy of albumin and IVIg was compared, 5 out of 17 rats (29%) in the albumin group and 12 out of 17 (71%) in the IVIg group completely recovered from the clinical disease by day 30. The animals receiving IVIg treatment exhibited lower clinical scores, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades. In the study which the efficacy of albumin, Fab fragment, Fc fragment, and IVIg was compared, 0 out of 8 (0%) in the albumin group, 1 out of 8 (13%) in the Fab group, 4 out of 8 (50%) in the Fc group, and 6 out of 9 (67%) rats in the IgG group completely recovered from the clinical disease by day 30. The animals receiving Fc fragment and IVIg treatment exhibited lower clinical scores, less prominent weight loss, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades.

The aims of this study were to test the efficacy of immunoglobulin and its Fab and Fc fragment in the treatment of experimental autoimmune neuritis (EAN) in Lewis rats, to investigate which portion of immunoglobulin is operative in the effect of IVIg, and to clarify the possible mechanisms by which immunoglobulin exerts its action in the treatment of rats EAN. EAN was induced by immunization with whole bovine peripheral nerve myelin. The immunized rats were randomized into groups, assessed clinically, electrophysiologically, and histologically, and intravenously injected with normal saline, albumin, human IVIg preparation, purified Fab or Fc fragments. The treatment efficacy was compared between normal saline and albumin groups, albumin and IVIg groups, albumin and Fab groups, albumin and Fc groups, Fab and Fc groups, Fab and IVIg groups, and Fc and IVIg groups. Methods of myelin isolation, antibody purification, and Western blot techniques were also applied. The results revealed that treatment with Fc fragment and IVIg at the onset of signs of disease effectively prevented further progression of disease, shortened disease duration, and facilitating recovery from illness as shown in clinical, electrophysiological and histological parameters. In the study which the efficacy of albumin and IVIg was compared, 5 out of 17 rats (29%) in the albumin group and 12 out of 17 (71%) in the IVIg group completely recovered from the clinical disease by day 30. The animals receiving IVIg treatment exhibited lower clinical scores, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades. In the study which the efficacy of albumin, Fab fragment, Fc fragment, and IVIg was compared, 0 out of 8 (0%) in the albumin group, 1 out of 8 (13%) in the Fab group, 4 out of 8 (50%) in the Fc group, and 6 out of 9 (67%) rats in the IgG group completely recovered from the clinical disease by day 30. The animals receiving Fc fragment and IVIg treatment exhibited lower clinical scores, less prominent weight loss, less prolongation of S wave latencies, better maintained S wave amplitudes, less reduction of distal motor NCVs, better maintained distal and proximal CMAP amplitudes, and lower histological grades.

This dissertation presents extensive structural studies of gas-phase peptide fragment ions, with a specific focus on b₂⁺ ions. Fragment ion structures can provide important insights into peptide fragmentation mechanisms. Based on the structures formed, information about the preference of competing b ion formation pathways can be obtained. b₂⁺ ion structures are of interest because of their large relative abundances in MS/MS spectra, which are difficult to predict. Prior to this work, only a few b₂⁺ ion structures were determined; these systems featured only aliphatic residues and all formed oxazolones. The work presented herein examines the influence of basic, acidic, and backbone-attached sidechains on peptide fragmentation mechanisms, as revealed by the resulting b₂⁺ fragment ion structure(s) formed. Specifically, the structures of several histidine, aspartic acid, and proline-containing b₂⁺ ions are determined by using action IRMPD spectroscopy, fragment ion HDX, and DFT calculations. The structures of a series of histidine analogue-containing b₂⁺ ions reveal that the location and availability of the pi-nitrogen is essential for diketopiperazine formation. The histidine sidechain bulk or strain interferes with the complete trans-cis isomerization required for diketopiperazine formation, so the oxazolone structure is also present. Xxx- Pro b₂⁺ ions favor oxazolone formation with aliphatic N-terminal residues. HP favors the diketopiperazine, combining the histidine effect and the proline cis conformation propensity. For Xxx-Asp b₂⁺ ions, aspartic acid significantly influences b₂⁺ ion structure only with an N-terminal histidine or lysine; both HD and KD form a mixture of oxazolone, anhydride, and diketopiperazine structures, presenting the first spectroscopic evidence for the anhydride b₂⁺ion structure. The HA and AH b₂⁺ ions feature the same structures, but HP and PH do not, showing that residue position matters. Additionally, while relative intensities and HDX rates featured some fluctuation, peptide precursor composition differences did not alter the mixture of b₂⁺ ion structures formed for a given b₂⁺ ion. To complement existing gas-phase structural methods, the utility of a new technique, QCID-HDX-IRMPD, was applied to m/z 552.28 from YAGFL-OH. Both the standard b₅⁺ fragment ion and an isobaric non-C-terminal water loss ion are present. Without separation of these isomers, MS/MS spectral interpretation would be complicated.

Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1 to 100 ng/ml) and cultured at 5% and 21% oxygen tension for 48 hours. In separate experiments, constructs were subjected to dynamic compression (15%) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5% and 21% oxygen tension using an ex-vivo bioreactor for 48 hours. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (p < 0.01) with values greater at 5% oxygen tension than 21%. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21%. The stimulation of GAG synthesis by dynamic compression was greater at 21% than 5% oxygen tension and this response was reduced with TNFα or reversed with L-NIO. The present findings revealed that TNFα has dose-dependent catabolic activities and increased production of inflammatory mediators at low oxygen tension. Dynamic compression or the NOS inhibitor downregulated the inflammatory effects induced by TNFα, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα induced pathways.

Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance.
Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions.
Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed.
Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.

The shapes of political pressure and the lobby appear as important tools for the private interestmanagement in the contemporary democracies This article displays a plot of actions of lobby at the Peruvian parliament and the role of building networks at the political arena in which the collision with other interests is avoid Thus, the lobby appears to be effective on issues that are out of the agenda of the public-media debate, issues of high specialization, or those that are not of interest of the party blocs (but represent a huge volume of legislative initiatives)
Las formas de presión política y el lobby aparecen como herramientas importantes para la gestión de intereses privados en las democracias contemporáneas Este artículo muestra una parcela de las acciones del lobby en el parlamento peruano y la función de construcción de redes en una arena política en la que se evita la colisión con otros intereses Así, el lobby aparece con eficacia en los temas que están fuera de la agenda del debate público-mediático, temas de alta especialización, o aquellos que no revisten interés de las bancadas (pero que representan un enorme volumen de iniciativas legislativas)
As formas da pressão política e o lobby aparecem como ferramentas importantes para a gestão dos interesses privados nas democracias contemporâneas Este artigo mostra uma parcela de ações do lobby no parlamento peruano e a função da construção das redes num cenário político, na que evita a colisão com outros interesses Assim, o lobby aparece com eficácia nos temas que estão fora da agenda do debate público midiático, tópicos de maior especialização, ou aqueles que não têm interesse das bancadas (mas representam um volumem muito grande de iniciativas legislativas)

Nous développons une méthode de couplage entre un fluide compressible non-visqueux et une structure tridimensionnelle mobile. Nous considérons d'abord une structure rigide, puis déformable, et enfin avec fragmentation. Le couplage repose sur une méthode conservative de type frontières immergées en combinaison avec une méthode de Volumes Finis pour le fluide et une méthode d'Éléments Discrets pour la structure. La méthode de couplage assure la conservation de la masse, de la quantité de mouvement et de l'énergie totale du système. Elle présente également des propriétés de consistance, comme l'absence d'effets de rugosité artificielle sur une paroi rigide. La méthode de couplage est explicite en temps dans le cas d'une structure rigide, et semi-implicite dans le cas d'une structure déformable. La méthode semi-implicite en temps évite que des déformations tangentielles de la structure ne se transmettent au fluide, et la résolution itérative jouit d'une convergence géométrique sous une condition CFL non restrictive sur le pas de temps. Nous présentons des résultats numériques montrant la robustesse de la méthode dans le cas d'une sphère rigide mise en mouvement par une onde de choc, une poutre encastrée fléchie par une onde de choc et un cylindre se fragmentant sous l'action d'une explosion interne
We develop a coupling method between an inviscid compressible fluid and a three dimensional mobile structure. We consider first a rigid structure, then a deformable, and finally a fragmenting one. The coupling hinges on a Conservative Immersed Boundary method combined with a Finite Volume method for the fluid and a Discrete Element method for the structure. The method yields conservation of mass, momentum, and energy of the system. The method also exhibits consistency properties, such as the absence of numerical roughness on a rigid wall. The method is explicit in time in the case of a rigid structure, and semi-implicit when the structure is deformable. The time semi-implicit method avoids that tangential deformations of the structure impact the fluid, and the method converges geometrically with a non-restrictive CFL condition on the time step. We present numerical results showing the robustness of the method in the case of a rigid sphere lifted by a shock wave, a clamped beam flexed by a shock wave, and a cylinder undergoing fragmentation owing to an intern explosion

L'expérience INDRA@GSI permet d'étudier les mécanismes de la réaction 12C+197Au en cinématique directe par l'utilisation du multidétecteur 4p INDRA et de faisceaux de 12C aux énergies relativistes. La source du spectateur de la cible est determinée pour les protons et les fragments légers, séparement de la source de cascade émettant des particules légeres et d'une source de haute energie émettant des fragments approximativement dans le centre de masse. Les spectres de protons en énergie cinétique sont comparés à des combinaisons du modèle de Cascade Intra-Nucléaire de Liège avec des modèles statistiques. Le scénario privilegié associe un processus de cascade avec une multifragmentation statistique. Les températures de pente des fragments determinées par une combinaison de fonctions de Maxwell-Boltzmann mettent en évidence une dépendance en énergie de faisceau, la centralité de la réaction étant donnée par la multiplicité de particules chargées. Les pions, detectés pour la première fois avec INDRA, et les protons rapides présentent une correlation avec le paramètre d'impact mais non avec la production de fragments.

Le fuselage des avions de nouvelle génération fera de plus en plus appel aux matériaux composites à matrice organique qui présentent des propriétés spécifiques particulièrement intéressantes. Or les structures aéronautiques sont exposées à de nombreuses exigences et en particulier à celle induite par l'impact de "petits fragments" à haute énergie. Le non percement du fuselage nécessite donc d'adjoindre une fonction de blindage à sa fonction habituelle de tenue mécanique. Par rapport à cette problématique, une approche avec intégration de fonction a été adoptée et a mené au développement d'un matériau composite multifonctionnel dans le cadre de ce travail de recherche. L'étude du comportement sous impact à basse vitesse (essais Charpy) et à haute vitesse (essais au canon à gaz) de composites à matrice organique aéronautiques courants et plus spécifiques au blindage a d'abord été réalisée. Cette étude a permis d'établir le lien entre les constituants des matériaux et les différents modes d'absorption de l'énergie d'impact. Plusieurs paramètres ont été discriminés : nature de la matrice (thermodurcissable - thermoplastique), nature de la fibre (minérale - organique), architecture du renfort (UD - tissée - tricotée), taux de porosité intra-mèche, apport d'éléments spécifiques aux inter-plis. Plusieurs concepts de multimatériaux sont ensuite proposés. Ils ont été définis à partir de combinaisons des différents comportements observés sur matériaux élémentaires et sont basés sur différents scénarii d'endommagement. Ils ont été testés à haute vitesse d'impact. Les observations révèlent une aptitude particulière de la fibre de polypara-phénylène-2,6-benzobisoxazole (PBO) à absorber une grande quantité d'énergie par déformation inélastique, notamment lorsqu'elle est faiblement imprégnée
Next generation aircraft fuselage will increasingly use polymer matrix composites that exhibit interesting specific properties. Aeronautical structures are exposed to many requirements and amongst them to that induced by the impact of high energy "small fragments". In order to avoid fuselage break through, an armour function has to be added to its usual mechanical function. With respect to this issue, an approach aiming the integration of such function was adopted and led to the development of a multifunctional composite material within this research work. The study of the behaviour under low speed (Charpy tests) and high speed (gas gun tests) impact of common and more specific organic matrix composites dedicated to armour was first performed. This study allowed establishing the link between the material components and the various modes of the impact energy absorption. Several parameters were discriminated: matrix type (thermosetting - thermoplastic), fibre type (mineral - organic), reinforcement architecture (UD - woven - knitted), intra-mesh porosity level, addition of specific inter-ply elements. Several concepts of multimaterials were then proposed. They were defined from combinations of various behaviours observed in the basic materials and are based on different damaging scenarios. They were tested under high speed impact. The observations show a particular aptitude of the polyparaphenylene-2,6-benzobisoxazole (PBO) fibre to absorb a large amount of energy by inelastic deformation, especially when it is poorly impregnated

Reactivite des complexes fe::(3)(co)::(9)(cch::(3))(coc::(2)h::(5)) ou fe::(3)(co)::(10)(cch::(3))(h). Les reactions de couplage des groupes alkylidynes avec les algues sont faciles. L'action de co a mis en evidence le couplage reversible dans des conditions douces de 2 fragments alkylidynes. En general, la presence du coordinat hydruro rend les reactions plus complexes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The research called Snapshots Fragments; a study of Bergsonian cinematographic mechanism in the Marcel Duchamp´s painting Nude Descending a Staircase, is based in arts and reflections in which paint is performed a study of the cinematic mechanism concept from the philosopher, Henri Bergson. The research starts from the questions about wich intersection point can exist between an art work and a philosophical concept, that were deeply relevant and fruitful in modernity with contemporary developments in both discussions: arts and philosophy. In other words, this work was done intending to find similarities and differences between Duchamp\'s painting and Bergson´s concept. The research was developed aiming to enable the dialogue establishment between art and philosophy, given through an element that would be the embrace of both: the movie.
A pesquisa intitulada Fragmentos instantâneos, um estudo do mecanismo cinematográfico bergsoniano na pintura Nu descendo uma escada de Marcel Duchamp, trata-se de uma pesquisa em fundamentos e reflexões em artes, no qual são realizados estudos da pintura Nu descendo uma escada , de Marcel Duchamp, e do conceito de mecanismo cinematográfico, do filósofo Henri Bergson. A pesquisa inicia-se a partir dos questionamentos sobre a possibilidade de haver um ponto de intersecção entre um trabalho em arte e um conceito filosófico, os quais foram profundamente relevantes na modernidade e profícuos de desdobramentos na contemporaneidade e, nas discussões tanto no campo das artes, quanto na filosofia. Em outras palavras este trabalho foi realizado objetivando-se verificar em quais aspectos há convergências e divergências entre a pintura de Duchamp e o conceito de Bergson. A pesquisa foi realizada visando possibilitar a instauração de um diálogo entre arte e filosofia, dado através de um elemento que seria o amplexo de ambos, o cinema.
Mestre em Artes

El objetivo de esta tesis se centra en el estudio de la transferencia de electrones (ET), una de las reacciones más simples y cruciales en bioquímica. Para dichos procesos, obtener información directa de los factores que lo promueves, asi como del camino de transferencia electronica, no es una tarea trivial. Dicha información a un nivel de conocimiento detallado atómico y electrónico, sin embargo, es muy valiosa en términos de una mejor comprensión del ciclo enzimático, que podría conducir, por ejemplo, a un diseño más eficaz de inhibidores. El objetivo principal de esta tesis es el desarrollo de una metodología para el estudio cuantitativo de la ET en los sistemas biológicos. En este sentido, hemos desarrollado un nuevo método para obtener el camino de transferencia electrónico, llamado QM/MM e-­‐ Pathway, que se puede aplicar en sistemas complejos con ET de largo alcance. El método se basa en una búsqueda sucesiva de residuos importantes para la ET, utilizando la modificación de la región quantica en métodos mixtos QM/MM, y siguiendo la evolución de la densidad de espín dentro de la zona de transferencia. Hemos demostrado la utilidad y la aplicabilidad del algoritmo en el complejo P450cam/Pdx, identificando el papel clave de la Arg112 (en P450cam) y del Asp48 (en Pdx), ambos conocidos en la literatura. Además de obtener caminos de ET, hemos cuantificado su importancia en términos del acoplamiento electrónico entre el dador y aceptor para los diferentes caminos. En este sentido, se realizaron dos estudios de la influencia del solvente y de la temperatura en el acoplamiento electrónico para sistemas modelo oligopéptidos. Ambos estudios revelaron que los valores del acoplamiento electrónico fluctúan fuertemente a lo largo de las trayectorias de dinámica molecular obtenidas, y el mecanismo de transferencia de electrones se ve ampliamente afectado por el espacio conformacional del sistema. La combinación del QM/MM e-­‐pathway y de los cálculos de acoplamiento electronico fueron utilizados finalmente para investigar la ET en el complejo CCP/Cytc. Nuestros hallazgos indican el papel fundamental del Trp191 en localizar un estadio intermedio para la transferencia electronica, así como el camino ET principal que incluye Ala194, Ala193, Gly192 y Trp191. Ambos hallazgos fueron confirmados a través de la literatura. Los resultados obtenidos para el muestro de manios de ET, junto con su evaluación a través de cálculos de acoplamiento electrónico, sugieren un enfoque sencillo y prometedor para investigar ET de largo alcance en proteínas.
The focus of this PhD thesis lies on electron transfer (ET) processes, belonging to the simplest but most crucial reactions in biochemistry. Getting direct information of the forces driving the process and the actual electron pathway is not a trivial task. Such atomic and electronic detailed information, however, is very valuable in terms of a better understanding of the enzymatic cycle, which might lead, for example, to more efficient protein inhibitor design. The main objective of this thesis was the development of a methodology for the quantitative study of ET in biological systems. In this regard, we developed a novel approach to map long-­‐range electron transfer pathways, called QM/MM e-­‐Pathway. The method is based on a successive search for important ET residues in terms of modifying the QM region following the evolution of the spin density of the electron (hole) within a given transfer region. We proved the usefulness and applicability of the algorithm on the P450cam/Pdx complex, indicating the key role of Arg112 of P450cam and Asp48 of Pdx for its ET pathway, both being known to be important from the literature. Besides only identifying the ET pathways, we further quantified their importance in terms of electronic coupling of donor and acceptor incorporating the particular pathway residues. Within this regard, we performed two systematic evaluations of the underlying reasons for the influence of solvent and temperature onto electronic coupling in oligopeptide model systems. Both studies revealed that electronic coupling values strongly fluctuate throughout the molecular dynamics trajectories obtained, and the mechanism of electron transfer is affected by the conformational space the system is able to occupy. Combining both ET mapping and electronic coupling calculations, we finally investigated the electron transfer in the CcP/Cytc complex. Our findings indicate the key role of Trp191 being the bridge-­‐localized state of the ET as well as the main pathway consisting of Ala194, Ala193, Gly192 and Trp191 between CcP and Cytc. Both findings were confirmed through the literature. Moreover, our calculations on several snapshots state a nongated ET mechanism in this protein complex. The methodology developed along this thesis, mapping ET pathways together with their evaluation through electronic coupling calculations, suggests a straightforward and promising approach to investigate long-­‐range ET in proteins.

Les déchets plastiques s'accumulent depuis plusieurs décennies dans les océans où ils se fragmentent en particules appelés microplastiques lorsque leur taille est inférieure à 5 mm. Ces microplastiques sont retrouvés dans toutes les eaux du globe, dans les sédiments ainsi que dans de nombreux organismes marins. Le devenir physicochimique à long terme de ces particules et leur possible fragmentation en nanoplastiques sont complexes, encore peu documentés et nécessitent des études en laboratoire.Afin de comprendre les processus liés à la photodégradation et à la fragmentation des polymères dans l’environnement, mais également dans le but d’'appréhender l’évolution des fragments générés au cours de l’irradiation, un protocole de vieillissement accéléré en milieu abiotique a été mis en place sur des polymères modèles. Le suivi de l’oxydation et de la fragmentation des deux polymères étudiés,polyéthylène basse densité et polypropylène, a été mené à l’aide de techniques spectroscopiques (infrarouge, Raman), DSC, angles de contact, et microscopiques (lumière polarisée, MEB, AFM…).Ce travail a permis de mettre en évidence l’influence significative de l’environnement et de la morphologie initiale des polymères sur leurs cinétiques de vieillissement et leurs mécanismes de fissuration. Ainsi des distributions en nombres, tailles et formes de fragments très différentes ont été obtenues pour les deux polymères selon la présence d’eau. Enfin, après un long temps d’irradiation, des produits de dégradation ont pu être détectés mais la production significative de nanoplastiques n’a pas été démontrée. L'hypothèse d'une taille limite de fragmentation devrait être envisagée
Plastic wastes have been accumulating for several decades in the oceans where they break up into particles called microplastics when their size is less than 5 mm. These microplastics are found in all earth’s waters, in sediments and in many marine organisms. Their long-term physico-chemical fate and their possible fragmentation into nanoplastics are complex, still poorly documented and require laboratory studies.In order to understand the processes related to photodegradation and fragmentation of polymers, but also in order to understand the evolution of these fragments during irradiation, an accelerated aging protocol in abiotic conditions has been set up. The oxidation and fragmentation of two model polymers, low density polyethylene and polypropylene, were monitored using spectroscopic techniques (InfraRed, Raman), DSC, contact angles and microscopic technics (light microscopy, polarized light, SEM, AFM ...).This work has demonstrated a significant influence of the environment and the initial morphology of the polymers on their kinetics of aging and cracking mechanisms. This lead to significantly different distributions in numbers, sizes and shapes of the generated fragments. Moreover, after a long time of irradiaiton, other degradation products could be detected but the significant production of nanoplastics has not been demonstrated. The possibility of a size limit below which the fragmentation rate of plastics would strongly decrease should be considered

La reproduction expérimentale de transitoires thermiques accidentels de type Accident par Perte de Réfrigérant Primaire (APRP) en laboratoire a permis d’observer la fragmentation du combustible fortement irradié lorsque la gaine se déforme sous l’augmentation de la température. Ces fragments de petites tailles peuvent se relocaliser dans le ballon voire être éjectés hors du crayon cas de rupture de gaine. La zone High Burnup Structure (HBS) des combustibles fortement irradiés est la plus susceptible de se fragmenter et d’être relocalisée par sa position en périphérie de pastille. Pour expliquer ce phénomène, l’hypothèse retenue est que le transitoire provoque une surpression dans les bulles HBS ce qui mène à la décohésion des joints de grains et à la fragmentation. Cette thèse a pour but de développer un critère de fissuration mécanique du combustible pour mieux comprendre le comportement des bulles HBS lors des conditions thermiques APRP. Ce travail se base sur une méthode une méthode micromécanique en trois étapes : i) la représentation qui permet de caractériser la microstructure de la zone HBS (leurs dimensions, leur fraction volumique, et la pression interne). Deux sources d’informations seront utilisées : les observations expérimentales provenant de disques ou de pastilles de combustible irradiés à fort taux de combustion et d’outils numériques(avec Alcyone-Caracas [JSB+14])
Under Loss Of Coolant Accident(LOCA) transients conditions, the high irradiated fuel is fragmented in small sizes fragments who can be relocated in the balloon, or being ejected out of the fuel rod if the latter burst. This work focuses on the pellet rim, where bubbles density increases owing to a higher irradiation level. Usually the hypothesis used to explain fuel fragmentation during transient is grain cleavage induced by over pressurized fission gas bubbles, located at the grain boundary. The aim of this study is to define a macroscopic fragmentation model based on a micro mechanical approach to have a better understanding of the fuel mechanical behaviour at lower scale : size and volume fraction of fragments. This PhD introduces a stepwise micromechanical method based on three steps : i) firstly, we detail how to model the HBS microstructure including pressurized porosities, based on experimental or numerical data and define a representative volume element (RVE)

碩士
國立臺灣大學
生物環境系統工程學研究所
93
The main purpose of this study is to investigate the effects of rock fragments used as mulch cover on interrill soil erosion. The soil used in the soil erosion tests was collected from a mountainside at Sindian, Taipei County. Erosion Boxes and a rainfall simulator were specially designed and fabricated for this study. During the tests, the slope steepness of the erosion boxes was maintained at 20%. Mortar Balls were placed at the soil surface as the mulch cover. The rate and type of cover were changed, and two different rainfall intensities were simulated and applied in this study. The erosion tests were repeated on soil samples at the cover rates of 0%, 10%, 20%, 30%, 40% and 50% and types of completely exposed to the soil surface and partially embedded into the soil (i.e. the embedded ratio was 0% and 50%), and rainfall intensities of 40 and 90 mm/hr. It was found from the test results that while the rainfall intensity was low and the cover rate was less than 40%, the runoff rate decreased. While the rock fragments were completely exposed to the soil surface, the runoff erosion, splash erosion and total erosion rate were found less compared to that while the rock fragments were partially embedded into the soil. At the cover rate of 10%, it was found that while the rock fragments were partially embedded into the soil, the runoff erosion and total erosion rate were found less. Nevertheless, both the two types of cover could effectively reduced the erosion. It was also found that while the rock fragments were completely exposed to the soil surface, the rainfall intensity was low and the cover rate was greater then 40%, the reducing effect of soil erosion was the best. After regression analysis, an equation was proposed to relate soil erosion, cover rate, embedded ratio and rainfall intensity. The relationship were found very satisfactory.

Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire, ils sont fortement régulés par les modifications post-traductionnelles et peuvent se produire dans tous les compartiments subcellulaires. À ce jour, aucune méthode générale n’a été développée pour permettre une dissection fine de ces complexes macromoléculaires. L'objectif de cette thèse est d'établir et de démontrer une nouvelle stratégie pour disséquer les complexes protéines formés lors du cycle cellulaire de la levure Saccharomyces cerevisiae (S. cerevisiae). Dans cette thèse, je décris le développement et l'optimisation d'une stratégie simple de sélection basée sur un essai de complémentation de fragments protéiques en utilisant la cytosine déaminase de la levure comme sonde (PCA OyCD). En outre, je décris une série d'études de validation du PCA OyCD afin de l’utiliser pour disséquer les mécanismes d'activation des facteurs de transcription et des interactions protéine-protéines (IPPs) entre les régulateurs du cycle cellulaire. Une caractéristique clé du PCA OyCD est qu'il peut être utilisé pour détecter à la fois la formation et la dissociation des IPPs et émettre un signal détectable (la croissance des cellules) pour les deux types de sélections. J'ai appliqué le PCA OyCD pour disséquer les interactions entre SBF et MBF, deux facteurs de transcription clés régulant la transition de la phase G1 à la phase S. SBF et MBF sont deux facteurs de transcription hétérodimériques composés de deux sous-unités : une protéine qui peut lier directement l’ADN (Swi4 ou Mbp1, respectivement) et une protéine commune contenant un domain d’activation de la transcription appelée Swi6. J'ai appliqué le PCA OyCD afin de générer un mutant de Swi6 qui restreint ses activités transcriptionnelles à SBF, abolissant l’activité MBF. Nous avons isolé des souches portant des mutations dans le domaine C-terminal de Swi6, préalablement identifié comme responsable dans la formation de l’interaction avec Swi4 et Mbp1, et également important pour les activités de SBF et MBF. Nos résultats appuient un modèle où Swi6 subit un changement conformationnel lors de la liaison à Swi4 ou Mbp1. De plus, ce mutant de Swi6 a été utilisé pour disséquer le mécanisme de régulation de l’entrée de la cellule dans un nouveau cycle de division cellulaire appelé « START ». Nous avons constaté que le répresseur de SBF et MBF nommé Whi5 se lie directement au domaine C-terminal de Swi6. Finalement, j'ai appliqué le PCA OyCD afin de disséquer les complexes protéiques de la kinase cycline-dépendante de la levure nommé Cdk1. Cdk1 est la kinase essentielle qui régule la progression du cycle cellulaire et peut phosphoryler un grand nombre de substrats différents en s'associant à l'une des neuf protéines cycline régulatrice (Cln1-3, Clb1-6). Je décris une stratégie à haut débit, voir à une échelle génomique, visant à identifier les partenaires d'interaction de Cdk1 et d’y associer la cycline appropriée(s) requise(s) à l’observation d’une interaction en utilisant le PCA OyCD et des souches délétées pour chacune des cyclines. Mes résultats nous permettent d’identifier la phase(s) du cycle cellulaire où Cdk1 peut phosphoryler un substrat particulier et la fonction potentielle ou connue de Cdk1 pendant cette phase. Par exemple, nous avons identifié que l’interaction entre Cdk1 et la γ-tubuline (Tub4) est dépendante de Clb3. Ce résultat est conforme au rôle de Tub4 dans la nucléation et la croissance des faisceaux mitotiques émanant des centromères. Cette stratégie peut également être appliquée à l’étude d'autres IPPs qui sont contrôlées par des sous-unités régulatrices.
Proteins are the end-products of gene interpretative machinery. Proteins serve essential roles in defining the structure, integrity and dynamics of the cell and mediate most chemical transformations needed for everything from metabolic catalysis to signal transduction. We know that the central dogma of molecular biology, one gene = one mRNA = one protein is a gross simplification and that a protein may do different things depending on the form in which its mRNA was spliced, how and where it is post-translationally modified, what conformational state it may be in or finally, which other proteins it may interact with. Formation of protein complexes may, themselves, be governed by the states in which proteins are expressed in specific cells, cellular compartments or under specific conditions or dynamic phases such has growth or division. Protein complexes involved in mitotic cell cycle regulation are particularly challenging to dissect since they could only form during specific phases of the cell cycle, are highly regulated by post-translational modifications and can be found in any subcellular compartments. To date, no general methods have been developed to allow fine dissection of these protein complexes. The goal of this thesis was to establish and demonstrate a novel strategy for dissecting protein complexes regulating the budding yeast Saccharomyces cerevisiae (S. cerevisiae) mitotic cell cycle. In this thesis, I describe my development and optimization of a simple survival-selection Protein-fragment Complementation Assay using the prodrug-converting enzyme, yeast cytosine deaminase as reporter (OyCD PCA). I further describe, in a series of proof of principle studies, applications of the OyCD PCA to dissect the mechanism of transcriptional activation by key mitotic transcription factors and to dissect protein-protein interactions (PPIs) among regulators of the mitotic cell cycle. A key feature of the OyCD PCA is that it can be used to detect both formation and disruption of PPIs by virtue of having positive readouts for both assays. I applied the OyCD PCA in a strategy to dissect interactions between the key transcription factors of the G1/S phase: SBF and MBF. These two heterodimeric transcription factors are composed of, respectively, two distinct DNA-binding subunits named Swi4 and Mbp1 and a common transcription activation subunit called Swi6. I took advantage of the dual selection by OyCD PCA to engineer a specific mutant of Swi6 in order to demonstrate the rewiring of a transcriptional network. We isolated Swi6 with mutations found in its C-terminal domain previously identified for binding Swi4 and Mbp1 and important for SBF and MBF activities. Our results support a model where Swi6 undergoes a conformational change upon binding to Swi4 or Mbp1. In addition, this Swi6 mutant was used to dissect the regulatory mechanism that governs the entry of S. cerevisiae to a new round of cell division also known as START. We found that the SBF and MBF repressor Whi5 directly binds to the C-terminal domain of Swi6. Finally, I applied the OyCD PCA to dissect the yeast cyclin dependent kinase Cdk1-protein complexes. Cdk1 is the essential kinase that regulates cell cycle progression and can phosphorylate a large number of different substrates by teaming up with one of nine cyclin regulatory proteins (Cln1-3, Clb1-6). I describe a strategy to identify interaction partners of Cdk1 that can easily be scaled up for a genome-wide screen and associate the complexes with the appropriate cyclin(s) required for mediating the interaction using the OyCD PCA and deletion of the cyclin genes. My results allow us to postulate which phase(s) of the mitotic cell cycle Cdk1 may phosphorylate proteins and what function potential or known substrates of Cdk1 may take on during that phase(s). For example, we identified the interaction between Cdk1 and the γ-tubulin (Tub4) to be dependent upon Clb3, consistent with its role in mediating nucleation and growth of mitotic microtubule bundles on the spindle pole body. This strategy can also be applied to study other PPIs that are contingent upon accessory subunits.

In this thesis, the synthesis and reactivity of metal complexes containing phosphido (PR2−) and phosphenium (PR2+) ligands for the hydrophosphination of alkenes were investigated. The mechanisms of hydrophosphination mediated by these M-PR2 fragments were explored. Based on previous work in the Rosenberg group, Ru(η5-indenyl) complexes were explored and developed as catalysts for hydrophosphination. It was determined that Ru-phosphido complexes are key intermediates in the hydrophosphination of electron-deficient alkenes. A detailed study on the mechanisms of hydrophosphination catalyzed by the phosphido complexes Ru(η5-indenyl)(PPh2)(L)(PPh3) (4a, L = NCPh; b, L = PPh2H; c, L = CO) was performed. Evidence for product inhibition was found for this catalyst system using Reaction Progress Kinetic Analysis. Product inhibition is consistent with the observed catalyst resting state of a complex containing product phosphines and the determination that substitution of the product phosphine from Ru is rate-limiting. The ancillary ligands (L) of 4 were found to influence catalytic activity by enabling catalyst deactivation (L = NCPh) or off-cycle processes including alkene telomerization (L = CO). Proposed mechanisms for catalysis were devised based on these findings. These results are important mechanistic insights that will be useful for designing new catalysts for hydrophosphination. The unprecedented viability of metal phosphenium complexes as intermediates in hydrophosphination was also explored. Three Mo phosphenium complexes were synthesized via P-H bond hydride abstraction from coordinated secondary phosphines, PR2H. These complexes were found to mediate the stoichiometric hydrophosphination of alkenes and ketones. In particular, trans-[Mo(CO)3(PPh2H)2(PPh2)]+ (13) mediates the hydrophosphination of a wide scope of alkenes that includes ethylene, propene and 1-hexene, which are challenging substrates for metal-catalyzed hydrophosphination. Preliminary attempts were conducted to render this synthetic phosphenium-mediated hydrophosphination catalytic. These results provide evidence for the putative steps of a hydrophosphination cycle utilizing metal phosphenium complexes as intermediates. The phosphenium complexes trans-[Mo(CO)4(PR2H)(PR2)] (12a R = Tolp2, b R = Ph) were also investigated as Lewis acid catalysts for hydrosilylation. A tentatively-assigned η1-HSiEt3 adduct of 12a, [Mo(CO)4(PTolp2H)(PTolp2{HSiEt3})] (20a), was observed by low temperature 31P{1H} NMR and was studied computationally. Complex 12b is proposed to behave as a Lewis acid catalyst for hydrosilylation. An off-cycle equilibrium is proposed that results in the formation of EtSi+. This work is a unique example of P(III) Lewis acid catalysis, of which there are few examples in the literature.
Graduate
2020-07-29

Habitat loss and fragmentation are key drivers of global species loss. In fragmented landscapes species must persist in small, isolated and often degraded habitat patches where they can be subject to high risk of extinction due to deterministic and stochastic forces. Species respond to habitat fragmentation according to species-specific life-history traits, with habitat generalist, edge or mobile species being less impacted compared to specialists and less mobile species. The impact of habitat fragmentation on species and their consequent probability of persistence depends on a series of key, concatenated events occurring at different biological and spatial scales. The response of single individuals to landscape change can translate into effects at the level of populations; coexisting species can reciprocally influence their responses through the alteration of interspecific relationships; inter-population dynamics can also occur, involving the movement of individuals between populations in different habitat fragments and affecting the persistence of entire systems of populations. Given the complexity of factors involved, including direct and interacting responses, it is extremely difficult to understand the actual effects triggered by habitat fragmentation without a thorough knowledge of the underlying ecological mechanisms. The aim of this PhD project was to contribute to understanding the mechanisms underlying the response of species to habitat fragmentation. By following a holistic approach, I used a set of mechanistic field studies on four rodent species specifically designed to investigate the series of key events involved in the persistence of species in fragmented landscapes: 1) Population and individual scale responses of small mammals to patch size, isolation and quality. The aim of this section was to determine the relative effects of landscape structure (habitat amount and configuration) and patch quality (here measured as abundance of shrub resources) on individuals (survival and litter size) and populations (density and colonization/extinction dynamics). A large-scale demographic field study was conducted, encompassing 30 woodland sites nested within three landscapes and surveyed monthly for three years by means of a capture-mark-recapture protocol. Model species was an arboreal rodent, the hazel dormouse (Muscardinus avellanarius), known to be sensitive to habitat loss and fragmentation. Habitat quality influenced populations at different biological scales by concatenated effects: it enhanced individual survival, increased the chances of colonizing vacant patches and sustained higher population densities. It was therefore related to the performance of single populations and systems of populations through re-colonization dynamics. Habitat quality, however, did not influence local extinction probability, which was ultimately related to the extent of available habitat, likely due to the absolute size of populations: a high absolute number of individuals reduces the chances of population extinction. 2) The role of interspecific interactions in shaping small mammal communities in fragmented landscapes. The aim of this section was to evaluate the strength of interspecific interactions as a shaping force of animal communities in fragmented landscapes. A large-scale demographic field study was conducted to measure the degree of competitive interference between species. Model system was constituted by the community of forest-dwelling ground rodents of central Italy, including the species Apodemus sylvaticus, Apodemus flavicollis and Myodes glareolus. Populations, inhabiting 29 wood patches in a fragmented landscape, were surveyed for two years by means of a capture-mark-recapture protocol. I modeled species' distribution as a function of landscape (habitat cover and connectivity provided by hedgerows) and habitat variables (vegetation structure and food resources) to look for evidences of competitive spatial segregation. Then I tested for each species the effect of competitors on several biological parameters: survival, recruitment, reproduction, body mass, population density. Even though populations' relative distribution was consistent with a mechanism of competitive spatial segregation, with habitat specialists being favored by high-quality, well-connected fragments and generalists exploiting more isolated and degraded patches, results on demographic parameters did not fully confirm this result. The strongest competitive effects were exerted by A. sylvaticus on A. flavicollis, whereas a little degree of interference was found between Apodemus spp. and M. glareolus. Nevertheless, competitive effects were weak, acting on a few biological parameters and not translating into strong effects at the level of populations (density of individuals). These results suggest that populations were mainly distributed according to their ecological requirements; competitive exclusion of specialists from isolated and degraded fragments was actually acting but was likely to play a minor role in determining the observed pattern of distribution. 3) Perceptual range and movement ability of small mammals in fragmented landscapes. The aim of this section was to broaden our understanding of animal orientation and movements in the agricultural matrix, with a special attention on the use of plantation rows as navigation cues. Experiments consisted in releasing individuals of forest-dwelling small mammals (species A. flavicollis, A. sylvaticus, M. glareolus) in fields characterized by different types of matrices: a bare field, a grass field with random pattern of vegetation, and a wheat field at three different stages of growth. Animals (N=119) were marked with fluorescent powder and released at progressive distances from target wood fragments; in this type of experiments individuals are assumed to go directly toward the wood as soon as they perceive it. Animal tracks were then analyzed to determine perceptual ranges and movement abilities. Perceptual ranges were species-specific, with habitat specialists perceiving woods at smaller distances compared to generalists. The presence of vegetation in the fields (either grass or wheat) strongly reduced perceptual ranges of all species by obstructing individuals' view. Furthermore, wheat plantation rows drastically influenced animal movements, possibly facilitating or hampering the reaching of a wood. Individuals of all species, in fact, followed the direction of wheat rows at any stage of growth, even if they were not directed toward the target wood. This study is one of the few examples investigating in detail the demographic mechanisms of response of species to habitat fragmentation. The holistic approach allowed me to provide an overview on the process by which factors such as landscape features, habitat characteristics, and co-occurring species affect the performance of populations in fragmented landscapes. Interspecific interactions play a minor role in shaping the community of small mammals in the studied system. A major role, instead, is played by landscape characteristics (such as habitat cover, connectivity, matrix properties) and local features (such as food resources and habitat structure), in both cases depending on species-specific life-history traits. Increased individual performance (e.g. due to habitat quality) can help to increase the viability of systems of populations; at the same time animals are constrained by the physical structure of the landscape where they live, and individual-scale effects are not necessarily transferred to the level of population. Results suggest that in order to increase the viability of animal systems in fragmented landscapes there is the need to manage the quality of habitat, which proves to be a major determinant of animal populations' performance. Nevertheless, findings also strongly suggest not to ignore the overall landscape context where populations are embedded. In landscapes that have been extensively cleared, restoration aimed to increase the amount of habitat and management of outside-patch landscape elements (hedgerows, agricultural fields) might also be a critical step to ensure the persistence of animal communities.

Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

Habitat loss and fragmentation is a crisis affecting wildlife worldwide. In Tanzania, East Africa, a dramatic and recent (<80 years) expansion in human settlement and agriculture threatens to reduce gene flow among protected areas for many species of large mammals. Wildlife linkages can mitigate population isolation, but linkage designs lacking empirical justification may be controversial and ineffective. Connectivity conservation requires an understanding of how biogeographic factors shaped gene flow prior to habitat loss or fragmentation, however the history of interaction among populations is rarely known. The goal of my study was to provide context for connectivity conservation in central and southern Tanzania by identifying landscape features that have shaped gene flow for three ungulate species with different dispersal capabilities. I investigated historical patterns of connectivity for Maasai giraffe (Giraffa camelopardalis tippelskirchi), impala (Aepyceros melampus), and eland (Tragelaphus oryx) by estimating genetic structure among four to eight protected areas per species. Genetic structure changes very slowly among large populations and thus is likely to reflect historical processes instead of recent anthropogenic influences. I collected noninvasive DNA samples and generated microsatellite genotypes at 8 to 15 loci per species, then estimated genetic diversity metrics (allelic richness, AR, and expected heterozygosity, H[subscript E]) for each population (defined by reserve). I also calculated genetic distance (F[subscript ST] and Nei's unbiased genetic distance, D[subscript hat]) and an estimate of gene flow (Nm) between all population pairs for each species. To elucidate the possible causes of genetic structure between these populations, I tested for isolation by distance and isolation by resistance based on a suite of biogeographic factors hypothesized to affect gene flow. To do this, I created GIS-based resistance surfaces that assigned different costs of movement to landscape features. I created one or more resistance surfaces for each hypothesis of landscape effect. I used circuit theory to estimate the cumulative resistance between each pair of reserves for each weighting scheme, and then performed Mantel tests to calculate the correlation between these resistances and the observed population pairwise genetic distances (D[subscript hat]). I chose the optimal resistance model for each species as the model that was most highly correlated with observed genetic patterns. To verify that the correlation of resistance models with genetic distance was not an artefact of geographic distance, I performed partial Mantel tests to calculate correlation while controlling for the effect of geographic distance. Finally, I compared historical gene flow patterns to the distribution of contemporary human activity to predict areas that are at risk of a loss of connectivity. Indices of genetic diversity were moderate for all three species and comparable to previously reported values for other savannah ungulates. Diversity (both H[subscript E] and A[subscript R]) was highest in eland and lowest in giraffe for these populations, and was not consistently correlated with reserve size as has been reported for other species in East Africa. Although patterns in genetic distance were broadly similar across all three species there were also striking differences in connectivity, highlighting the importance of cross-species comparisons in connectivity conservation. At this scale, resistance models based on slope strongly predicted population structure for all three species; distance to water was also highly correlated with genetic distance in eland. For all three species, the greatest genetic distances occurred between populations separated by the Eastern Arc Mountains, suggesting that the topography of this area has long acted as a barrier to gene flow, but this effect is present in varying degrees for each species. I observed high levels of historical gene flow between centrally located populations (Ruaha National Park and Rungwa Game Reserve) and those in the southwest (Katavi National Park and Rukwa Game Reserve). Although human settlement in this area has been low relative to other areas, the connection between the Katavi/Rukwa and Ruaha ecosystems may be threatened by increased human activity and warrants conservation. High levels of historical gene flow were also seen between reserves in the northeast (Tarangire National Park, Swagaswaga Game Reserve) and the central and southwest populations. These connections appear highly threatened due to current land use practices, and may have already suffered a loss of gene flow. Field surveys in the lands surrounding the northeastern reserves are needed to quantify current levels of connectivity and determine whether corridors could be established to maintain or restore gene flow with other reserves.
Graduation date: 2012