Enzymatic catalysis in the synthesis of new polymer architectures and materials

Over the last decade, progress in the research towards enzymatic ring-opening polymerization has lead to novel, biocatalytic, and cleaner processes for the synthesis of polymeric materials. So far, this research has predominantly been focused on how to utilize the enzyme’s selectivity to synthesize and modify polymers, which cannot easily be achieved via chemical routes. However, proper understanding of these processes has not been obtained yet. Additionally, already present polymers have been enzymatically synthesized for biomedical applications, without the use of a metal catalyst, e.g. poly(e-caprolactone). However, only little reports have been published on new materials, which were not readily accessible via traditional polymerization techniques. In this PhD research, the implementation of enzymes into polymer chemistry has been investigated, looking for an answer to the question: Can enzymes open new perspectives in polymer chemistry? Lipase was chosen as the enzyme for enzymatic ring-opening polymerization (e-ROP), as this is well-known in organic synthesis. The lipase that was used in this research is Candida antarctica Lipase B immobilized on an acrylic resin, which is commercially available under the name Novozym 435TM. The aim of this investigation is to (i) obtain insight into the critical parameters of e- ROP of lactones, and (ii) make new materials that are not (directly) accessible via chemical polymerization methods. In order to study the critical parameters of e-ROP of lactones, the present knowledge was investigated in more depth for e-caprolactone (e-CL) as monomer in the enzymatic synthesis of end-functionalized polymer using a functional initiator. It was found that only by optimizing reaction conditions such as temperature, presence of water, monomer concentration, and the type of initiator, well-defined polymeric structures could be obtained, thereby limiting the amount of polymeric side-products (i.e. polymer species that lack the specific end-group functionality). Moreover, the concentrations of end-functionalized polymer and the undesired side-products were quantified for the first time using Liquid Chromatography under Critical Conditions (LCCC). This technique has provided us new insights into the actual enzymatic process at different stages in the polymerization. Water appears to be the primary nucleophile during the initial stages of the reaction, even when all the reaction components are thoroughly dried. Depending on the type of functional initiator that is applied, this nucleophile is incorporated into the polymer, by both transesterification and initiation. Finally, cyclic polymer structures are formed during all stages of the reaction and their concentration depends strongly on the initial monomer concentration Subsequently, e-ROP was used in combination with controlled radical polymerization (atom transfer radical polymerization, ATRP) in order to investigate the compatibility of enzymes with other catalyst systems. All information that was previously obtained was used to synthesize a block copolymer (poly(CL-block-MMA)) by these two polymerization techniques. Ultimately, a cascade chemo-enzymatic polymerization was performed in which the two polymerization techniques were applied simultaneously from a bifunctional initiator to obtain a block copolymer. It was observed that enzymes are slowly deactivated in the presence of transition metals (i.e. copper- and nickel-based ATRP-catalysts), depending on the ligands used to coordinate these metals. Hence, cascade chemo-enzymatic synthesis is only feasible when the two catalysts are applied separately. In order to synthesize novel materials that are not (directly) accessible via chemical polymerization methods, a larger lactone (i.e. ¿-pentadecalactone, PDL) was polymerized using enzymatic ring-opening polymerization. Using chemical, metal-based catalysts, larger lactones cannot be polymerized to high molecular weight polyesters due to their low ringstrain, whereas enzymes have shown surprisingly high activity towards these monomers. The synthesis of this type of monomers opens a novel promising route to the production of biomedical materials. To test the mechanical properties of PPDL, the enzymatic synthesis was scaled up to obtain 30 g of polymer in one batch and optimized to obtain a polymer with a high molecular weight and a relatively narrow molecular weight distribution. The obtained PPDL was melt-spun into fibers, which after drawing show good properties for biomedical applications. In conclusion, it can be stated that enzymes do open new perspectives in polymer science. Careful analysis of the enzymatic process has revealed the critical parameters for proper enzymatic polymer synthesis. Enzymes can be used in combination with other catalysts and polymerization techniques to make polymer architectures that may not be directly available via chemical synthesis. Finally, a new range of monomers can be utilized specifically by enzymes to make novel, tailored biomedical materials

Microstructural topology effects on the onset of ductile failure in multi-phase materials - a systematic computational approach

Multi-phase materials are key for modern engineering applications. They are generally characterized by a high strength and ductility. Many of these materials fail by ductile fracture of the, generally softer, matrix phase. In this work we systematically study the influence of the arrangement of the phases by correlating the microstructure of a two-phase material to the onset of ductile failure. A single topological feature is identified in which critical levels of damage are consistently indicated. It consists of a small region of the matrix phase with particles of the hard phase on both sides in a direction that depends on the applied deformation. Due to this configuration, a large tensile hydrostatic stress and plastic strain is observed inside the matrix, indicating high damage. This topological feature has, to some extent, been recognized before for certain multi-phase materials. This study however provides insight in the mechanics involved, including the influence of the loading conditions and the arrangement of the phases in the material surrounding the feature. Furthermore, a parameter study is performed to explore the influence of volume fraction and hardness of the inclusion phase. For the same macroscopic hardening response, the ductility is predicted to increase if the volume fraction of the hard phase increases while at the same time its hardness decreases

Fracture initiation in multi-phase materials: a systematic three-dimensional approach using a FFT-based solver

This paper studies a two-phase material with a microstructure composed of a hard brittle reinforcement phase embedded in a soft ductile matrix. It addresses the full three-dimensional nature of the microstructure and macroscopic deformation. A large ensemble of periodic microstructures is used, whereby the individual grains of the two phases are modeled using equi-sized cubes. A particular solution strategy relying on the Fast Fourier Transform is adopted, which has a high computational efficiency both in terms of speed and memory footprint, thus enabling a statistically meaningful analysis. This solution method naturally accompanies the regular microstructural model, as the Fast Fourier Transform relies on a regular grid. Using the many considered microstructures as an ensemble, the average arrangement of phases around fracture initiation sites is objectively identified by the correlation between microstructure and fracture initiation -- in three dimensions. The results show that fracture initiates where regions of the hard phase are interrupted by bands of the soft phase that are aligned with the direction of maximum shear. In such regions, the hard phase is arranged such that the area of the phase boundary perpendicular to the principal strain direction is maximum, leading to high hydrostatic tensile stresses, while not interrupting the shear bands that form in the soft phase. The local incompatibility that is present around the shear bands is responsible for a high plastic strain. By comparing the response to a two-dimensional microstructure it is observed that the response is qualitatively similar (both macroscopically and microscopically). One important difference is that the local strain partitioning between the two phases is over-predicted by the two-dimensional microstructure, leading to an overestimation of damage

Kinetics of the avian influenza-specific humoral responses in lung are indicative of local antibody production

The role and kinetics of respiratory immunoglobulins in AIV infection has not been investigated. In this study we determined the numbers of both total antibody secreting cells (ASC) and virus-specific ASC in lung, spleen, blood and bone marrow (BM) following low-pathogenic AIV infection. Antiviral humoral immune responses were induced both locally in the lung and systemically in the spleen. Responses in the lung and BM preceded responses in the spleen and in blood, with virus-specific IgY ASC already detected in lung and BM from 1 week post-primary inoculation, indicating that respiratory immune responses are not induced in the spleen, but locally in the lung. ASC present in the blood of the lungs and co-isolated during lymphocyte isolation from the lungs have no major impact on the ASC detected in the lungs based on statistical correlatio

Neutrophil Gelatinase-Associated Lipocalin as a Diagnostic Marker for Acute Kidney Injury in Oliguric Critically Ill Patients: A Post-Hoc Analysis

__Background:__ Oliguria occurs frequently in critically ill patients, challenging clinicians to distinguish functional adaptation from serum-creatinine-defined acute kidney injury (AKIsCr). We investigated neutrophil gelatinase-associated lipocalin (NGAL)'s ability to differentiate between these 2 conditions. __Methods:__ This is a post-hoc analysis of a prospective cohort of adult critically ill patients. Patients without oliguria within the first 6 h of admission were excluded. Plasma and urinary NGAL were measured at 4 h after admission. AKIsCr was defined using the AKI network criteria with pre-admission serum creatinine or lowest serum creatinine value during the admission as the baseline value. Hazard ratios for AKIsCr occurrence within 72 h were calculated using Cox regression and adjusted for risk factors such as sepsis, pre-admission serum creatinine, and urinary output. Positive predictive values (PPV) and negative predictive values (NPV) were calculated for the optimal cutoffs for NGAL. __Results:__ Oliguria occurred in 176 patients, and 61 (35%) patients developed AKIsCr. NGAL was a predictor for AKIsCr in univariate and multivariate analysis. When NGAL was added to a multivariate model including sepsis, pre-admission serum creatinine and lowest hourly urine output, it outperformed the latter model (plasma p = 0.001; urinary p = 0.048). Cutoff values for AKIsCr were 280 ng/ml for plasma (PPV 80%; NPV 79%), and 250 ng/ml for urinary NGAL (PPV 58%; NPV 78%). __Conclusions:__ NGAL can be used to distinguish oliguria due to the functional adaptation from AKIsCr, directing resources to patients more likely to develop AKIsCr

Seed quality of high protein corn lines in low input and conventional farming systems

Seed quality is a major issue for crop establishment especially in low input farming systems, where varieties often grow under more stressful conditions than in conventional farming systems. Corn (Zea mays L.) seed for organic (low input) production will eventually need to be grown organically, thus research is needed to ensure excellent seed quality in organic corn seed production. The objective of this study was to compare seed quality and composition differences between a group of high protein corn genotypes grown under low input and conventional farming systems, and to compare the relative seed quality of these genotypes to two well known inbreds, B73 or Mo17. Twenty high protein breeding genotypes were planted during two growing seasons in conventional and organic nurseries near Ames, Iowa, to produce seeds for laboratory tests. The germination, saturated cold, accelerated aging, and soak test percentages of seeds produced organically were 5 to 11% lower than for seeds produced conventionally. Protein, measured by near-infrared reflectance, was unaffected by the production location, but the oil content of seeds produced organically was significantly higher (between 0.2 and 0.3% higher) than in the conventional system. Location by genotype interactions for most tests were non significant both years, indicating that genotypes selected for high seed quality in a conventional system will also have high seed quality when grown in a low input, organic system

Stick-slip synchronization in stack of elastically coupled frictional interfaces

We perform physical and numerical experiments to study the stick-slip response of a stack of slabs in contact through dry frictional interfaces driven in quasistatic shear. The ratio between the drive's stiffness and the slab's shear stiffness controls the presence or absence of slip synchronization. A sufficiently high stiffness ratio leads to synchronization, comprising periodic slip events in which all interfaces slip simultaneously. A lower stiffness ratio leads to asynchronous slips and, experimentally, to the stick-slip amplitude being broadly distributed as the number of layers in the stack increases. We interpret this broadening in light of the combined effect of surface disorder, complex loading paths of the asynchronous slips, and creep. Consequently, the ageing rate can be readily extracted from the stick-slip cycle. The extracted aging rate is found to be of the same order of magnitude as existing experimental results on a similar material. Finally, we discuss the emergence of slow slips and an increase in creep-rate variations when more slabs are added to the stack