Thermostructural Numerical Analysis of the Thrust Chamber of a Liquid Propellant Rocket Engine.

The numerical simulation of rocket engine thrust chambers is very challenging as several damaging phenomena, such as plasticity, low-cycle-fatigue (LCF) and creep occur during its service life. The possibility of simulating the thermostructural behavior of the engine, by means of non-linear finite element analyses, allows the engineers to guarantee the structural safety of the structure. This document reports the numerical simulations developed with the aim of predicting the thermostructural behaviour and the service life of the thrust chamber of a liquid-propellant rocket engine. The work represents a step ahead of previous researches by the authors, with particular reference to the addition of the Smith-Watson-Topper (SWT) fatigue criterion, and to the implementation of a sub-modelling technique, for a more accurate assessment of the most critical section of the component. It was found that the equivalent plastic strains in the most critical nodes obtained through the sub-modelling technique were about 20% lower than those calculated without sub-modelling. Consistently with experimental tests from literature conducted on similar geometries, the most critical areas resulted to be on the internal surface of the chamber. The analyses demonstrated that the LCF damaging contribution was significant, with a life prediction for the thrust chamber of about 3400 cycles.

Stress Relaxation Behavior of Additively Manufactured Polylactic Acid (PLA).

In this work, the stress relaxation behavior of 3D printed PLA was experimentally investigated and analytically modeled. First, a quasi-static tensile characterization of additively manufactured samples was conducted by considering the effect of printing parameters like the material infill orientation and the outer wall presence. The effect of two thermal conditioning treatments on the material tensile properties was also investigated. Successively, stress relaxation tests were conducted, on both treated and unconditioned specimens, undergoing three different strains levels. Analytical predictive models of the viscous behavior of additive manufactured material were compared, highlighting and discussing the effects of considered printing parameters.

Model-based evaluation of the microhemodynamic effects of PEGylated HBOC molecules in the rat brain cortex: a laser speckle imaging study.

Hemoglobin-based oxygen carriers (HBOCs) were developed with the aim of substituting transfusions in emergency events. However, they exhibit adverse events, such as nitric oxide (NO) scavenging, vasoactivity, enhanced platelet aggregation, presently hampering their clinical application. The impact of two prototypical PEGylated HBOCs, Euro-PEG-Hb and PEG-HbO2, endowed by different oxygen affinities and hydrodynamic volumes, was assessed on the cerebrocortical parenchymal microhemodynamics, and extravasation through the blood-brain-barrier (BBB) by laser speckle contrast imaging (LSCI) method and near-infrared (NIR) imaging, respectively. By evaluating voxel-wise cerebrocortical red blood cell velocity, non-invasively for its mean kernel-wise value ( v ¯ RBC ), and model-derived kernel-wise predictions for microregional tissue hematocrit, THt, and fractional change in hematocrit-corrected vascular resistance, R', as measures of potential adverse effects (enhanced platelet aggregation and vasoactivity, respectively) we found i) no significant difference between tested HBOCs in the systemic and microregional parameters, and in the relative spatial dispersion of THt, and R' as additional measures of HBOC-related adverse effects, and ii) no extravasation through BBB by Euro-PEG-Hb. We conclude that Euro-PEG-Hb does not exhibit adverse effects in the brain microcirculation that could be directly attributed to NO scavenging.

PEGylation promotes hemoglobin tetramer dissociation.

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

Modulation of the NO/cGMP pathway reduces the vasoconstriction induced by acellular and PEGylated haemoglobin.

Activation of the NO/cGMP pathway modulates smooth muscle cells relaxation and hence vasoconstriction, a major hindrance for the use of cell-free haemoglobin (Hb) as blood substitute, despite conjugation with 5-kDa maleimide poly(ethylene)-glycol (PEG) reduces vasoconstriction in vivo. We aimed at assessing how a recently developed PEGylated-Hb (Deoxy-PEGHb) and manipulation of the NO/cGMP pathway enable modulation of vasoconstriction in isolated rat hearts. Hearts were Langendorff-perfused with oxygenated Krebs-Henseleit (15 ml/min) while monitoring the coronary pressure (CPP) after injection (1 min) of 50 nM norepinephrine followed by a 1 microM Hb or Deoxy-PEGHb bolus, without altering the flow. Deoxy-PEGHb induced less vasoconstriction than Hb. Although the presence of PEG could contribute to vasoconstriction, Deoxy-PEGHb did not contain appreciable amounts of free PEG. Whereas reducing endothelial NO release by 0.2 mM L-NAME increased vasoconstriction, abolishing NO scavenging by Hb using its cyanomet derivative almost completely blunted it. Furthermore, maintaining intracellular cyclic GMP by inhibiting phosphodiesterase-5 with 0.02 mM sildenafil enabled control of Hb-induced vasoconstriction. We conclude that, although PEG-Hb represents a possible approach to limit Hb-induced vasoconstriction, manipulating the NO/cGMP pathway may provide a powerful way to circumvent this problem.

Identification of the sites of deoxyhaemoglobin PEGylation.

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].

The hemoglobin cyanomet ligation analogue and carbon monoxide induce similar allosteric mechanisms.

Current thermodynamic models of protein cooperativity predicting sigmoidal ligand equilibrium curves differ in the assumptions regarding the structural/functional properties of the intermediate ligation states. Quantitative information on the intermediates cannot be extracted from the equilibrium curves, but must be obtained from direct studies of the intermediates. Since the intermediates are intrinsically unstable species, ligation analogues with reduced mobility are indispensable tools for cooperativity studies provided that the tertiary/quaternary changes triggered by the ligation analogue are similar to those observed using the physiological ligands. We demonstrate that the valency exchange reactions occurring in mixtures of deoxy and cyanomethemoglobin yield non-random distributions of deoxy/cyanomet intermediates that resemble those observed in the equilibrium with carbon monoxide. Previous and new data using the analogue, in agreement with the studies of the CO intermediates, indicate that the mechanism of hemoglobin cooperativity is neither purely concerted nor sequential nor combinatorial, but contains some elements of each of these models.

Allosteric proteins: lessons to be learned from the hemoglobin intermediates.

Allosteric proteins, such as hemoglobin, are assemblies of functional units, which undergo quaternary structural transitions in response to concentration changes of a specific ligand. Functional properties of hemoglobin ligation intermediates indicate that the tertiary structural changes induced by the ligand do not promote an equilibrium of quaternary structures.