Methods of measurement of bone density

Předkládaná bakalářská práce se zabývá měřením hustoty kosti a důsledky jejích změn na biomechanické vlastnosti kostní tkáně. První část je věnována popisu metod kostní densitometrie, zejména metod radiologických, které jsou v současnosti nejrozšířenější. V druhé části je měřena hustota spongiózní kosti v Hounsfieldových jednotkách z CT snímků (femur prasete, zdravý a nemocný femur člověka a bederní obratel) v programu ROI Analysis. V třetí části práce je analyzován vliv změn hustoty kosti na deformačně-napěťové veličiny spongiózní kosti. K posouzení byl využit 2D výpočtový model kyčle řešený pomocí MKP.This bachelor's thesis focuses on methods for measuring bone density and consequences of changes in bone density on biomechanical properties of bone tissue. In first part are described methods of bone densitometry, especially radiological methods, which are currently most widely used. Second part focuses on measuring bone density from CT images (hog femur bone, healthy and damaged human femur bone and lumbar vertebra) using software ROI Analysis. Third part discusses influence of changes in bone density on strain-stress quantities of trabecular bone. For the assessment was created 2D computational model of hip using FEM.

Strain and stress analysis of the femur with distraction intramedullary nail

Diplomová práce se zabývá stanovením a analýzou deformace a napětí femuru s aplikovaným distrakčním intramedulárním hřebem pro léčbu nestejné délky končetin metodou distrakční osteogeneze. Práce je zaměřena především na stavy po ukončení distrakce s následnou konsolidací svalku. Problém stanovení deformace a napětí soustavy je řešen výpočtovým modelováním s využitím MKP. Součástí práce je detailní popis tvorby modelu jednotlivých prvků soustavy, k získání trojrozměrné geometrie kosti byly použity CT snímky. Výpočtový model je řešen pro 4 různé geometrie svalku a měněny jsou materiálové charakteristiky svalku. Dále je také analyzován vliv použití 3 a 2 distálních šroubů na deformaci a napětí.This master's thesis is focused on determination and analysis of stress and strain in femur with distraction intramedullary nail for treating leg length discrepancy with the method of distraction osteogenesis. Thesis is mainly focused on states after distraction when the callus consolidates. Problem of determining stress and strain is solved by computational modeling using FEM. Detailed description of modeling is included in this thesis, complicated 3D geometry of bone was acquired from segmentation of CT images. Computational model is solve with 4 different types of callus geometry and also material properties of callus are varied. The influence on stress and strain when the middle distal screw is not applied is also analyzed.

EVALUATION OF THE APPLICATION OF A THERMAL INSULATION SYSTEM: INSITU COMPARISSON OF SEASONAL AND DAILY CLIMATIC FLUCTUATIONS

The current outdated state of many institutional and administrative buildings in the EU region poses a significant burden from the energy sustainability point of view. According to the contemporary EU requirements on the energy efficiency of buildings maintenance, an evaluation of performed improvements is essential for the assessment of expended investments. This paper describes the effect of building envelope reconstruction works consisting in the installation of a thermal insulation system. Here, a long-term continuous monitoring is used for the extensive assessment of the seasonal and daily temperature and relative humidity fluctuations. The obtained results include temperature and relative humidity profiles in the wall cross-section as a response to the changing exterior climatic conditions. The analysis of measured data reveals substantial improvements in thermal stability of the analyzed wall during temperature peaks. While the indoor temperatures exceeding 28 °C are recorded during summer before application of the thermal insulation layer, the thermal stability of the indoor environment is distinctly upgraded after performed improvements. Based on the complex long-term monitoring, a relevant experience is gained for the future work on energy sustainability and fulfilment of the EU directives

HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat

<p>Abstract</p> <p>Background</p> <p>Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance.</p> <p>Results</p> <p>Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all <it>in vitro </it>selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases.</p> <p>Conclusions</p> <p>These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.</p

Current and Novel Inhibitors of HIV Protease

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed

Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure. © 2015 Ingr et al.INSERM; Grant Agency of the Czech Republic [P208-12-G016

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1 beta and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3(L351P) knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

BACKGROUND: HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. METHODS AND FINDINGS: We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. CONCLUSIONS: HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy