Neocarzinostatin chromophore: structural, mechanistic, and synthetic studies

Neocarzinostatin chromophore 1 is the active component of the antitumor antibiotic neocarzinostatin (NCS). The chromophore reacts with thiols to form a highly strained cumulene-enyne species which rapidly rearranges to a biradical intermediate which can abstract hydrogen atoms from DNA, leading to strand cleavage. DNA damage is the proposed source of biological activity for NCS. The structure of the methyl thioglycolate monoadduct 2 of NCS chromophore, including the absolute stereochemistry, was determined by NMR studies. The presence of the cumulene-enyne intermediate and the rearrangement to a biradical were supported by data from low temperature NMR investigations. Also included are synthetic approaches to NCS chromophore model compounds based on intramolecular addition of an acetylide to an aldehyde.

Late Transition Metals Supported by Aryl Ethers and Phenoxides Bearing Pendant Phosphines: Mechanistic Insights Relevant to Ether C-O Bond Cleavage

Terphenyl diphosphines bearing pendant ethers were prepared to provide mechanistic insight into the mechanism of activation of aryl C–O bonds with Group 9 and Group 10 transition metals. Chapters 2 and 3 of this dissertation describe the reactivity of compounds supported by the model phosphine and extension of this chemistry to heterogenous C–O bond activation.

Chapter 2 describes the synthesis and reactivity of aryl-methyl and aryl-aryl model systems. The metallation of these compounds with Ni, Pd, Pt, Co, Rh, and Ir is described. Intramolecular bond activation pathways are described. In the case of the aryl-methyl ether, aryl C–O bond activation was observed only for Ni, Rh, and Ir.

Chapter 3 outlines the reactivity of heterogenous Rh and Ir catalysts for aryl ether C–O bond cleavage. Using Rh/C and an organometallic Ir precursor, aryl ethers were treated with H2 and heat to afford products of hydrogenolysis and hydrogenation. Conditions were modified to optimize the yield of hydrogenolysis product. Hydrogenation could not be fully suppressed in these systems.

Appendix A describes initial investigations of bisphenoxyiminoquinoline dichromium compounds for selective C2H4 oligomerization to afford α-olefins. The synthesis of monometallic and bimetallic Cr complexes is described. These compounds are compared to literature examples and found to be less active and non-selective for production of α-olefins.

Appendix B describes the coordination chemistry of terphenyl diphosphines, terphenyl bisphosphinophenols, and biphenyl phosphinophenols proligands with molybdenum, cobalt, and nickel. Since their synthesis, terphenyl diphosphine molybdenum compounds have been reported to be good catalysts for the dehydrogenation of ammonia borane. Biphenyl phosphinophenols are demonstrated provide both phosphine and arene donors to transition metals while maintaining a sterically accessible coordination sphere. Such ligands may be promising in the context of the activation of other small molecules.

Appendix C contains relevant NMR spectra for the compounds presented in the preceding sections.

Self-Organized Criticality, Plasticity and Sensorimotor Coupling. Explorations with a Neurorobotic Model in a Behavioural Preference Task

During the last two decades, analysis of 1/f noise in cognitive science has led to a considerable progress in the way we understand the organization of our mental life. However, there is still a lack of specific models providing explanations of how 1/f noise is generated in coupled brain-body-environment systems, since existing models and experiments typically target either externally observable behaviour or isolated neuronal systems but do not address the interplay between neuronal mechanisms and sensorimotor dynamics. We present a conceptual model of a minimal neurorobotic agent solving a behavioural task that makes it possible to relate mechanistic (neurodynamic) and behavioural levels of description. The model consists of a simulated robot controlled by a network of Kuramoto oscillators with homeostatic plasticity and the ability to develop behavioural preferences mediated by sensorimotor patterns. With only three oscillators, this simple model displays self-organized criticality in the form of robust 1/f noise and a wide multifractal spectrum. We show that the emergence of self-organized criticality and 1/f noise in our model is the result of three simultaneous conditions: a) non-linear interaction dynamics capable of generating stable collective patterns, b) internal plastic mechanisms modulating the sensorimotor flows, and c) strong sensorimotor coupling with the environment that induces transient metastable neurodynamic regimes. We carry out a number of experiments to show that both synaptic plasticity and strong sensorimotor coupling play a necessary role, as constituents of self-organized criticality, in the generation of 1/f noise. The experiments also shown to be useful to test the robustness of 1/f scaling comparing the results of different techniques. We finally discuss the role of conceptual models as mediators between nomothetic and mechanistic models and how they can inform future experimental research where self-organized critically includes sensorimotor coupling among the essential interaction-dominant process giving rise to 1/f noise.

Muscle signaling in exercise intolerance: Insights from the McArdle mouse model

We recently generated a knock-in mouse model (PYGM p.R50X/p.R50X) of McArdle disease (myophosphorylase deficiency). One mechanistic approach to unveil the molecular alterations caused by myophosphorylase deficiency, which is arguably the paradigm of 'exercise intolerance', is to compare the skeletal-muscle tissue of McArdle, heterozygous, and healthy (wild type (wt)) mice. We analyzed in quadriceps muscle of p.R50X/p.R50X (n=4), p.R50X/wt (n=6) and wt/wt mice (n=5) (all male, 8 wk-old) molecular markers of energy-sensing pathways, oxidative phosphorylation (OXPHOS) and autophagy/proteasome systems, oxidative damage and sarcoplamic reticulum (SR) Ca handling. We found a significant group effect for total AMPK (tAMPK) and ratio of phosphorylated (pAMPK)/tAMPK (P=0.012 and 0.033), with higher mean values in p.R50X/p.R50X mice vs. the other two groups. The absence of massive accumulation of ubiquitinated proteins, autophagosomes or lysosomes in p.R50X/p.R50X mice suggested no major alterations in autophagy/proteasome systems. Citrate synthase activity was lower in p.R50X/p.R50X mice vs. the other two groups (P=0.036) but no statistical effect existed for respiratory chain complexes. We found higher levels of 4-hydroxy-2-nonenal-modified proteins in p.R50X/p.R50X and p.R50X/wt mice compared with the wt/wt group (P=0.011). Sarco(endo)plasmic reticulum ATPase 1 (SERCA1) levels detected at 110kDa tended to be higher in p.R50X/p.R50X and p.R50X/wt mice compared with wt/wt animals (P=0.076), but their enzyme activity was normal. We also found an accumulation of phosphorylated SERCA1 in p.R50X/p.R50X animals. Myophosphorylase deficiency causes alterations in sensory energetic pathways together with some evidence of oxidative damage and alterations in Ca handling but with no major alterations in OXPHOS capacity or autophagy/ubiquitination pathways, which suggests that the muscle tissue of patients is likely to adapt overall favorably to exercise training interventions.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Mechanistic-based Design-Integrated Reliability Validation Framework for Mechanical Systems

Abstract: New product design challenges, related to customer needs, product usage and environments, face companies when they expand their product offerings to new markets; Some of the main challenges are: the lack of quantifiable information, product experience and field data. Designing reliable products under such challenges requires flexible reliability assessment processes that can capture the variables and parameters affecting the product overall reliability and allow different design scenarios to be assessed. These challenges also suggest a mechanistic (Physics of Failure-PoF) reliability approach would be a suitable framework to be used for reliability assessment. Mechanistic Reliability recognizes the primary factors affecting design reliability. This research views the designed entity as a “system of components required to deliver specific operations”; it addresses the above mentioned challenges by; Firstly: developing a design synthesis that allows a descriptive operations/ system components relationships to be realized; Secondly: developing component’s mathematical damage models that evaluate components Time to Failure (TTF) distributions given: 1) the descriptive design model, 2) customer usage knowledge and 3) design material properties; Lastly: developing a procedure that integrates components’ damage models to assess the mechanical system’s reliability over time. Analytical and numerical simulation models were developed to capture the relationships between operations and components, the mathematical damage models and the assessment of system’s reliability. The process was able to affect the design form during the conceptual design phase by providing stress goals to meet component’s reliability target. The process was able to numerically assess the reliability of a system based on component’s mechanistic TTF distributions, besides affecting the design of the component during the design embodiment phase. The process was used to assess the reliability of an internal combustion engine manifold during design phase; results were compared to reliability field data and found to produce conservative reliability results. The research focused on mechanical systems, affected by independent mechanical failure mechanisms that are influenced by the design process. Assembly and manufacturing stresses and defects’ influences are not a focus of this research.

ATR-FTIR spectroscopy and spectroscopic imaging of solvent and permeant diffusion across model membranes

The uptake and diffusion of solvents across polymer membranes is important in controlled drug delivery, effects on drug uptake into, for example, infusion bags and containers, as well as transport across protective clothing. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has been used to monitor the effects of different solvents on the diffusion of a model compound, 4-cyanophenol (CNP) across silicone membrane and on the equilibrium concentration of CNP obtained in the membrane following diffusion. ATR-FTIR spectroscopic imaging of membrane diffusion was used to gain an understanding of when the boundary conditions applied to Fick's second law, used to model the diffusion of permeants across the silicone membrane do not hold. The imaging experiments indicated that when the solvent was not taken up appreciably into the membrane, the presence of discrete solvent pools between the ATR crystal and the silicone membrane can affect the diffusion profile of the permeant. This effect is more significant if the permeant has a high solubility in the solvent. In contrast, solvents that are taken up into the membrane to a greater extent, or those where the solubility of the permeant in the vehicle is relatively low, were found to show a good fit to the diffusion model. As such these systems allow the ATR-FTIR spectroscopic approach to give mechanistic insight into how the particular solvents enhance permeation. The solubility of CNP in the solvent and the uptake of the solvent into the membrane were found to be important influences on the equilibrium concentration of the permeant obtained in the membrane following diffusion. In general, solvents which were taken up to a significant extent into the membrane and which caused the membrane to swell increased the diffusion coefficient of the permeant in the membrane though other factors such as solvent viscosity may also be important.

A mechanistic explanation of the Sargasso Sea DMS summer paradox

In the Sargasso Sea, maximum dimethylsulfide (DMS) accumulation occurs in summer, concomitant with the minimum of chlorophyll and 2 months later than its precursor, dimethylsulfoniopropionate (DMSP). This phenomenon is often referred to as the DMS "summer paradox". It has been previously suggested that the main agent triggering this pattern is increasing irradiance leading to light stress-induced DMS release from phytoplankton cells. We have developed a new model describing DMS(P) dynamics in the water column and used it to investigate how and to what extent processes other than light induced DMS exudation from phytoplankton, may contribute to the DMS summer paradox. To do this, we have conceptually divided the DMS "summer paradox" into two components: (1) the temporal decoupling between chlorophyll and DMSP and (2) the temporal decoupling between DMSP and DMS. Our results suggest that it is possible to explain the above cited patterns by means of two different dynamics, respectively: (1) a succession of phytoplankton types in the surface water and (2) the bacterially mediated DMSP(d) to DMS conversion, seasonally varying as a function of nutrient limitation. This work differs from previous modelling studies in that the presented model suggests that phytoplankton light-stress induced processes may only partially explain the summer paradox, not being able to explain the decoupling between DMSP and DMS, which is possibly the more challenging aspect of this phenomenon. Our study, therefore, provides an "alternative" explanation to the summer paradox further underlining the major role that bacteria potentially play in DMS production and fate.

Parameterisation of bivalve functional traits for mechanistic eco-physiological dynamic energy budget (DEB) models

Mechanistic models such as those based on dynamic energy budget (DEB) theory are emergent ecomechanics tools to investigate the extent of fitness in organisms through changes in life history traits as explained by bioenergetic principles. The rapid growth in interest around this approach originates from the mechanistic characteristics of DEB, which are based on a number of rules dictating the use of mass and energy flow through organisms. One apparent bottleneck in DEB applications comes from the estimations of DEB parameters which are based on mathematical and statistical methods (covariation method). The parameterisation process begins with the knowledge of some functional traits of a target organism (e. g. embryo, sexual maturity and ultimate body size, feeding and assimilation rates, maintenance costs), identified from the literature or laboratory experiments. However, considering the prominent role of the mechanistic approach in ecology, the reduction of possible uncertainties is an important objective. We propose a revaluation of the laboratory procedures commonly used in ecological studies to estimate DEB parameters in marine bivalves. Our experimental organism was Brachidontes pharaonis. We supported our proposal with a validation exercise which compared life history traits as obtained by DEBs (implemented with parameters obtained using classical laboratory methods) with the actual set of species traits obtained in the field. Correspondence between the 2 approaches was very high (>95%) with respect to estimating both size and fitness. Our results demonstrate a good agreement between field data and model output for the effect of temperature and food density on age-size curve, maximum body size and total gamete production per life span. The mechanistic approach is a promising method of providing accurate predictions in a world that is under in creasing anthropogenic pressure.

Some properties of the speciation model for food-web structure - Mechanisms for degree distributions and intervality

We present a mathematical analysis of the speciation model for food-web structure, which had in previous work been shown to yield a good description of empirical data of food-web topology. The degree distributions of the network are derived. Properties of the speciation model are compared to those of other models that successfully describe empirical data. It is argued that the speciation model unities the underlying ideas of previous theories. In particular, it offers a mechanistic explanation for the success of the niche model of Williams and Martinez and the frequent observation of intervality in empirical food webs. (c) 2005 Elsevier Ltd. All rights reserved.

A Monte Carlo model of DNA double-strand break clustering and rejoining kinetics for the analysis of pulsed-field gel electrophoresis data

In studies of radiation-induced DNA fragmentation and repair, analytical models may provide rapid and easy-to-use methods to test simple hypotheses regarding the breakage and rejoining mechanisms involved. The random breakage model, according to which lesions are distributed uniformly and independently of each other along the DNA, has been the model most used to describe spatial distribution of radiation-induced DNA damage. Recently several mechanistic approaches have been proposed that model clustered damage to DNA. In general, such approaches focus on the study of initial radiation-induced DNA damage and repair, without considering the effects of additional (unwanted and unavoidable) fragmentation that may take place during the experimental procedures. While most approaches, including measurement of total DNA mass below a specified value, allow for the occurrence of background experimental damage by means of simple subtractive procedures, a more detailed analysis of DNA fragmentation necessitates a more accurate treatment. We have developed a new, relatively simple model of DNA breakage and the resulting rejoining kinetics of broken fragments. Initial radiation-induced DNA damage is simulated using a clustered breakage approach, with three free parameters: the number of independently located clusters, each containing several DNA double-strand breaks (DSBs), the average number of DSBs within a cluster (multiplicity of the cluster), and the maximum allowed radius within which DSBs belonging to the same cluster are distributed. Random breakage is simulated as a special case of the DSB clustering procedure. When the model is applied to the analysis of DNA fragmentation as measured with pulsed-field gel electrophoresis (PFGE), the hypothesis that DSBs in proximity rejoin at a different rate from that of sparse isolated breaks can be tested, since the kinetics of rejoining of fragments of varying size may be followed by means of computer simulations. The problem of how to account for background damage from experimental handling is also carefully considered. We have shown that the conventional procedure of subtracting the background damage from the experimental data may lead to erroneous conclusions during the analysis of both initial fragmentation and DSB rejoining. Despite its relative simplicity, the method presented allows both the quantitative and qualitative description of radiation-induced DNA fragmentation and subsequent rejoining of double-stranded DNA fragments. (C) 2004 by Radiation Research Society.

Common Themes in Glycoconjugate Assembly Using the Biogenesis of O-Antigen Lipopolysaccharide as a Model System

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

An efficient and flexible synthesis of model lignin oligomers

The use of model compounds in the development of selective lignin depolymerisation processes has been limited by the lack of complexity of these models compared with lignin itself. In this paper we report a convergent and efficient synthetic method for the flexible, multi-gram preparation of model lignin hexamers and octamers containing three of the most common connectivity motifs found within native lignin, namely ß-O-4', 5-5' and ß-5', which will be used to further the mechanistic understanding of lignin depolymerisation processes.

Microneedle/nanoencapsulation-mediated transdermal delivery:Mechanistic insights

A systematic study was undertaken to gain more insight into the mechanism of transdermal delivery of nanoencapsulated model dyes across microneedle (MN)-treated skin, a complex process not yet explored. Rhodamine B (Rh B) and fluorescein isothiocyanate (FITC) as model hydrophilic and hydrophobic small/medium-size molecules, respectively, were encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and delivered through full thickness porcine skin pretreated with MN array. Permeation through MN-treated skin was affected by physicochemical characteristics of NPs and the encapsulated dyes. Dye flux was enhanced by smaller particle size, hydrophilicity, and negative zeta potential of NPs. Regarding encapsulated dyes, solubility at physiological pH and potential interaction with skin proteins proved to outweigh molecular weight as determinants of skin permeation. Data were verified using confocal laser scanning microscopy imaging. Findings coupled with the literature data are supportive of a mechanism involving influx of NPs, particularly of smaller size, deep into MN-created channels, generating depot dye-rich reservoirs. Molecular diffusion of the released dye across viable skin layers proceeds at a rate determined by its molecular characteristics. Data obtained provide mechanistic information of importance to the development of formulation strategies for more effective intradermal and transdermal MN-mediated delivery of nanoencapsulated therapeutic agents.

PTEN Phosphatase-Independent Maintenance of Glandular Morphology in a Predictive Colorectal Cancer Model System

Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na/H exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.