Letteratura scientifica selezionata sul tema "Bones – Molecular aspects"

Metabolic disorders caused by diabetes affect bone remodeling, alter the structure and reduce the strength of bone tissue, leading to the development of diabetic osteopathy. However, between diabetes mellitus (DM) type 1 and 2 there are noticeable differences in the effect on the bone structure, which is obviously due to the different cellular and molecular mechanisms of these processes. The density of bone tissue with DM typel decreases, which leads to an increase in the risk of fractures by 7 times. With DM type 2, bone mineral density is moderately elevated, which is expected to lead to a decrease in the incidence of osteoporotic fractures, but in fact, this index is approximately doubled. Pathophysiological mechanisms underlying osteoporotic changes in diabetes mellitus are complex and included hyperglycemia, oxidative stress and accumulation of advanced glycation endproducts that alter the properties of collagen, increase fatty infiltration of the bone marrow, release inflammatory factors and adipokines from visceral adipose tissue and potentially change the function osteoblasts. Additional factors are, some antidiabetic drugs that directly affect the metabolism of bones and minerals (such as thiazolidinediones), as well as an increased tendency to fall due to micro- and macroangiopathies, all contribute to an increased risk of low-fracture fractures in patients with diabetes mellitus.

Hypophosphatemic rickets (phosphate-diabetes) is a group of diseases associated with a defect in the reabsorption of phosphates in the proximal tubules, manifested by phosphaturia, hypophosphatemia and rickets deformities of the skeleton bones. Phosphate-diabetes has different genetic variants that determine the nature and severity of clinical manifestations. X-linked dominant hypophosphatemic rickets occurs most often (in 50-90% of cases). For the diagnosis, along with clinical characteristics, an important role is assigned to the study of partial renal functions, with the determination of clearance, excreted fraction of calcium and phosphates, as well as other indicators of calcium-phosphorus metabolism. Molecular genetic research helps to determine the form of the disease. Therapy for hypophosphatemic rickets should be differentiated depending on the type of disease. The timely appointment of an adequate pathogenetic therapy helps to slow down the formation of rickety deformities of the skeleton, positive growth dynamics, and an increase in physical activity.

Medullary thyroid carcinoma (MTC), arising from the parafollicular C cells of the thyroid, accounts for 1–2% of thyroid cancers. MTC is frequently aggressive and metastasizes to cervical and mediastinal lymph nodes, lungs, liver, and bones. Although a number of new imaging modalities for directing the management of oncologic patients evolved over the last two decades, the clinical application of these novel techniques is limited in MTC. In this article, we review the biology and molecular aspects of MTC as an important background for the use of current imaging modalities and approaches for this tumor. We discuss the modern and currently available imaging techniques—advanced magnetic resonance imaging (MRI)-based techniques such as whole-body MRI, dynamic contrast-enhanced (DCE) technique, diffusion-weighted imaging (DWI), positron emission tomography/computed tomography (PET/CT) with 18F-FDOPA and 18F-FDG, and integrated positron emission tomography/magnetic resonance (PET/MR) hybrid imaging—for primary as well as metastatic MTC tumor, including its metastatic spread to lymph nodes and the most common sites of distant metastases: lungs, liver, and bones.

The dorsal and ventral aspects of the turtle shell, the carapace and the plastron, are developmentally different entities. The carapace contains axial endochondral skeletal elements and exoskeletal dermal bones. The exoskeletal plastron is found in all extant and extinct species of crown turtles found to date and is synaptomorphic of the order Testudines. However, paleontological reconstructed transition forms lack a fully developed carapace and show a progression of bony elements ancestral to the plastron. To understand the evolutionary development of the plastron, it is essential to know how it has formed. Here we studied the molecular development and patterning of plastron bones in a cryptodire turtleTrachemys scripta. We show that plastron development begins at developmental stage 15 when osteochondrogenic mesenchyme forms condensates for each plastron bone at the lateral edges of the ventral mesenchyme. These condensations commit to an osteogenic identity and suppress chondrogenesis. Their development overlaps with that of sternal cartilage development in chicks and mice. Thus, we suggest that in turtles, the sternal morphogenesis is prevented in the ventral mesenchyme by the concomitant induction of osteogenesis and the suppression of chondrogenesis. The osteogenic subroutines later direct the growth and patterning of plastron bones in an autonomous manner. The initiation of plastron bone development coincides with that of carapacial ridge formation, suggesting that the development of dorsal and ventral shells are coordinated from the start and that adopting an osteogenesis-inducing and chondrogenesis-suppressing cell fate in the ventral mesenchyme has permitted turtles to develop their order-specific ventral morphology.

A decline in the body’s motor functions has been linked to decreased muscle mass and function in the oral cavity and throat; however, aging of the junctions of the muscles and bones has also been identified as an associated factor. Basic and clinical studies on the muscles, tendons and bones, each considered independently, have been published. In recent years, however, research has focused on muscle attachment as the muscle–tendon–bone complex from various perspectives, and there is a growing body of knowledge on SRY-box9 (Sox9) and Mohawk(Mkx), which has been identified as a common controlling factor and a key element. Myostatin, a factor that inhibits muscle growth, has been identified as a potential key element in the mechanisms of lifetime structural maintenance of the muscle–tendon–bone complex. Findings in recent studies have also uncovered aspects of the mechanisms of motor organ complex morphostasis in the superaged society of today and will lay the groundwork for treatments to prevent motor function decline in older adults.

The histology of sauropod long bones often appears uniform and conservative along their evolutionary tree. One of the main aspects of their bone histology is to exhibit a fibrolamellar complex in the cortex of their long bones. Here, we report another bone tissue, the radial fibrolamellar bone (RFB), in the outer cortex of the humeri of a young adult cf. Isanosaurus (Early to Late Jurassic, Thailand) and an adult Spinophorosaurus nigerensis (Early to Middle Jurassic, Niger) that do not exhibit any pathological feature on the bone surface. Its location within the cortex is unexpected, because RFB is a rapidly deposited bone tissue that would rather be expected early in the ontogeny. A palaeopathological survey was conducted for these sampled specimens. Observed RFB occurrences are regarded as spiculated periosteal reactive bone, which is an aggressive form of periosteal reaction. A ‘hair-on-end’ pattern of neoplasmic origin (resembling a Ewing's sarcoma) is favoured for cf. Isanosaurus , while a sunburst pattern of viral or neoplasmic origin (resembling an avian osteopetrosis or haemangioma) is favoured for Spinophorosaurus . This study highlights the importance of bone histology in assessing the frequency and nature of palaeopathologies. This article is part of the theme issue ‘Vertebrate palaeophysiology’.

The operculum is a large flap consisting of several flat bones found on the side of the head of bony fish. During development, the opercular bones form within the second pharyngeal arch, which expands posteriorly and comes to cover the gill-bearing arches. With the evolution of the tetrapods and the assumption of a terrestrial lifestyle, it was believed that the operculum was lost. Here, we demonstrate that an embryonic operculum persists in amniotes and that its early development is homologous with that of teleosts. As in zebrafish, the second pharyngeal arch of the chick embryo grows disproportionately and comes to cover the posterior arches. We show that the developing second pharyngeal arch in both chick and zebrafish embryos express orthologous genes and require shh signalling for caudal expansion. In amniotes, however, the caudal edge of the expanded second arch fuses to the surface of the neck. We have detailed how this process occurs and also demonstrated a requirement for thyroid signalling here. Our results thus demonstrate the persistence of an embryonic opercular flap in amniotes, that its fusion mirrors aspects of amphibian metamorphosis and gives insights into the origin of branchial cleft anomalies in humans.

Rickets refers to a deficient mineralization of the growth plate cartilage, predominantly affecting longer bones. Despite the fact that preventive measures are available, it is still a common disease worldwide; nutritional rickets, due to vitamin D deficiency or dietary calcium inadequate intake, remains the most common form. Medical history, physical examination, radiologic features and biochemical tests are essential for diagnosis. Although recent studies suggest hypophosphatemia as the leading alteration, rickets is classically divided into two categories: calcipenic rickets and phosphopenic rickets. Knowledge of this categorization and of respective clinical and laboratory features is essential for rapid diagnosis and correct management. The aim of this review is to analyze the epidemiological, pathogenetic, clinical, and therapeutic aspects of the different forms of rickets, describing the novelties on this “long-lived” disease.

Solute transport is one of the important aspects involved in maintaining the physiological activity of tissues. The mechanical environment drives nutrition in and waste out in articular cartilage due to its avascularity, which plays a key role in the biological activity of articular cartilage. The human knee joint motion is a complex interaction between different bones including relative rolling and/or sliding movements. Rolling-compression process is a typical physiological load in knee joint motion. To investigate solute transport behavior in articular cartilage under rolling-compression load, fluorescence tracers with molecular weights of 40kDa and 0.43kDa were used respectively to mark the transport in fresh articular cartilage of mature pigs. Solute fluorescence intensity changing with time and depth of cartilage layer was measured under rolling-compression load and static state, respectively, and the distribution of corresponding relative concentration was calculated by the fluorescence microscope imaging method. The experiment results show that the solute relative concentration in articular cartilage under rolling-compression load increases significantly, even up to 62.4%, comparing with that under static state, and the changes of concentration vary in different layers and that small molecular weight solute is easier to transport than relatively large molecular weight solute in articular cartilage. Therefore, rolling-compression load can promote the solute transport in cartilage, and the mechanical loading may have application in functional cartilage tissue engineering.

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular.
The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.

Orientador: Sandra Cecilia Botelho Costa
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O Citomegalovírus Humano (HCMV) continua sendo uma causa significante de morbidade em pacientes imunocomprometidos, especialmente em transplantados de medula óssea, e pode manifestar diversas complicações que incluem hepatite, doença gastrointestinal e pneumonia intersticial ou a denominada "Síndrome Viral por HCMV"caracterizada por febre, leucopenia e trombocitopenia. O HCMV pode também ter um efeito imuno-modulador, fazendo da infecção por esse vírus um fator de risco importante para o desenvolvimento de rejeição ao enxerto aguda e crônica e para co-infecção com outras herpesviroses. A detecção do genoma do HCMV pela PCR (Reação em Cadeia da Polimerase) é específica e sensível, e pode ser usada como uma poderosa ferramenta para o diagnóstico precoce da infecção causada por este vírus. Variações em regiões funcionalmente relevantes do genoma do HCMV têm sido utilizadas como marcadores genéticos em diversos estudos clínicos para diferenciar as linhagens do vírus e associá-las com a patogênese viral e com as manifestações clínicas no paciente. A glicoproteína B (gB) é a maior glicoproteína do envelope do HCMV e tem sido relacionada à entrada na célula hospedeira, transmissão célula-a-célula, e conseqüentemente à fusão das células infectadas. A amplificação do gene gB pela PCR combinada com análise de restrição por RFLP em regiões polimórficas deste gene são eficientes para a identificação dos genótipos do HCMV, tornando possível a distinção de pelo menos 4 padrões eletroforéticos. Por outro lado, a determinação da carga viral em pacientes imunologicamente afetados tem sido associada como marcador ou preditor do desenvolvimento de doença por HCMV órgão-específica. Sendo assim, a determinação da carga viral, especificamente nestes pacientes, é fundamental para a supervisão da terapia antiviral. Além disso, os valores da carga viral estão relacionados aos níveis de imunossupressão, à patogênese do HCMV e ao grupo de pacientes e/ou ao tipo de transplante e podem indicar o início da administração da terapia antiviral.O método de real-time PCR (RT-PCR) foi aplicado para a quantificação do genoma do HCMV em amostras clínicas e a detecção e posterior quantificação do DNA do HCMV em amostras de soro por esta técnica é capaz de distinguir entre pacientes com infecções sintomáticas daqueles com infecções inativas ou latente.Avanços têm sido feitos na prevenção da doença por HCMV após o transplante de medula óssea, inclusive a administração profilática, por períodos prolongados, de antivirais como o Acyclovir e o Ganciclovir e como conseqüência, pode originar linhagens resistentes relacionadas principalmente a dois genes virais: a fosfotransferase viral (UL97) e a DNA polimerase viral (UL54). Sabendo-se da importância da identificação das linhagens do HCMV em pacientes transplantados de medula óssea e da possível relação com a infecção e apresentação clínica; da relevância em determinar a carga viral como preditor de doença; e finalmente, da detecção de linhagens resistentes aos agentes antivirais disponíveis, este estudo avaliou, prospectivamente, pacientes transplantados de medula óssea em seguimento no Hemocentro/UNICAMP. Além disso, teve como principais objetivos: determinar a prevalência dos genótipos do HCMV e avaliar uma possível associação com a apresentação clínica nesses pacientes; determinar a carga viral para o monitoramento da terapia antiviral; e identificar e correlacionar mutações que conferem resistência ao Ganciclovir com carga viral e apresentação clínica. Foram incluídas na casuística, 169 amostras de DNA de sangue periférico e 187 amostras de DNA de soro de 22 pacientes transplantados de medula óssea. Dentre as 47 amostras de DNA de sangue periférico HCMV positivas, 42 foram genotipadas e observamos a prevalência do genótipo gB1 (47%) como descrito em literatura, e embora sem comprovação estatística, notamos a tendência deste genótipo com melhor prognóstico. Aplicamos a RT-PCR em 96 amostras de DNA de soro de 12 pacientes transplantados de medula óssea seguidos no Ambulatório de Hematologia, e observamos que o método é adequado para a avaliação da carga viral neste grupo de pacientes. No entanto, é necessário estabelecer um valor de corte a fim de se utilizar esta metodologia para obtenção de um valor que seja preditivo de doença e para o monitoramento do tratamento dos pacientes. Este método mostrou-se mais preciso que a ?nested?-PCR no mesmo tipo de amostra. Além disso, identificamos 8 novas mutações no gene UL97, uma delas pode estar relacionada à resistência viral ao Ganciclovir. Dentre os polimorfismos identificados, 3 parecem estar relacionados ao genótipo gB1 e possivelmente podem ser utilizadas como marcadores genéticos para a genotipagem do HCMV. Para o gene UL54 foram identificadas 5 novas mutações na região IV do gene e que geralmente é relacionada à resistência ao Ganciclovir. Nós concluímos que a determinação da carga viral é importante, mas não é o único modo de avaliar a eficiência do tratamento antiviral. Dessa forma, a avaliação de outros parâmetros moleculares, como a genotipagem e mutações relacionadas à resistência aos antivirais, são informações complementares e devem ser consideradas para o monitoramento da evolução clínica em pacientes transplantados de medula óssea
Abstract: Human Cytomegalovirus (HCMV) remains a significant cause of morbidity in immunocompromised patients, especially in bone marrow transplant recipients. It may manifest severe complications including hepatitis, gastrointestinal disease, and interstitial pneumonitis or as so-called ?HCMV viral syndrome? with fever, leukopenia, and thrombocytopenia. The HCMV may also has an immunomodulatory effect, potentially making HCMV infection an important risk factor for the development of an acute and chronic allograft rejection and for coinfection with other herpesviruses. The detection of the HCMV genome by PCR (Polymerase Chain Reaction) is specific and sensitive. Besides this, it can be used as a powerful tool for the early diagnoses of the infection caused by this virus. Variations in functionally relevant areas of the HCMV genome have been used as genetic markers in numerous clinical studies to differentiate the HCMV strains and to associate them with the viral pathogenesis further with the patients? clinical manifestations. The glycoprotein B (gB) is the major glycoprotein of HCMV?s envelope and it has been implicated in host cell entry, cell-to-cell virus transmission, consequently in the fusion of infected cells. The gB amplification by PCR combined with the restriction analysis by RFLP in polymorphic areas are effective for the identification of the HCMV genotypes, becoming possible the distinction of at least 4 electrophoretic patterns. On the other hand, the determination of the viral load in the immunologically affected patients has been associated as marker or predictor for the development of the organ specific disease by the HCMV. Hence, the determination of the viral load in these specific patients is fundamental for the management of the antiviral therapy. In addition, the viral load values are related to levels of the immune-suppression, the pathogenesis of the HCMV and the group of patients and/or the type of transplant. Furthermore, the viral load values can indicate the beginning of the antiviral therapy administration. A real-time PCR (RT-PCR) assay was applied for quantifying the HCMV genome load in clinical samples and the detection and quantification of HCMV DNA in blood serum through RT-PCR are able to distinguish patients with symptomatic infections among those with latent or inactive infections. Advances have been made in the prevention of HCMV disease after bone marrow transplantation, including prophylactic administration of antivirals such as Acyclovir and Ganciclovir. The HCMV prophylaxis with antiviral in this patients? group is administered for prolonged periods of therapy, consequently it can originate resistant viruses related mainly to two genes: the viral phosphotransferase (UL97) and the viral DNA polymerase (UL54). Ahead the importance of the identification of HCMV strains in bone marrow transplant patients, the HCMV strains performance in the patients? infection and clinical presentation, the relevance of determinating the viral load as a disease predictor, and finally, the detection of the resistant strains to the available antivirals, this study prospectively evaluated bone marrow transplant recipients followed at Hemocentro/UNICAMP. Moreover, it had as main goals: to determine the prevalence of the HCMV gB genotypes, to evaluate a possible gB genotype association with the patients? clinical presentation; to determinate the viral load for monitoring the antiviral therapy, and to correlate Ganciclovir resistant mutations in UL97 and UL54 gene with the viral load and patients? clinical presentation. From 22 bone marrow transplant recipients, DNA samples of peripheral blood (169) and DNA samples of blood serum (187) were included in this casuistic. Among 47 HCMV positive samples, 42 were genotyped. We observed the prevalence of gB1 genotype (47%), as described in the specific literature, however without statistical analysis, the raw data exhibited that gB1 genotype can be related to patients? better prognostics. From 12 followed bone marrow transplant recipients, we applied the RT-PCR in 96 DNA blood serum samples and we observed that the method was accurate for the viral load evaluation in this patients? group. However, it is necessary to establish a crucial cutoff to consider whether a specific value of viral load is a predictive value to cause HCMV disease and to monitor the patients? treatment. This method was more precise than the nested-PCR for blood serum samples. Additionally, we identified 8 new mutations in UL97 gene, one of them can be related to Ganciclovir HCMV resistance. Among all of identified polymorphisms, 3 of them can be related to gB1 genotype and may be used as genetic marker to HCMV genotyping. In the region IV of the UL54 gene, 5 new mutations were identified, and can possibly be related to Ganciclovir HCMV resistance. We concluded that the determination of the patients? viral load is crucial, even so it is not the only way to evaluate the antiviral treatment efficacy. Then, the evaluation of other molecular parameters as genotyping and mutations related to the HCMV antiviral resistance, are complementary information and must be considered to monitor the clinical evolution of bone marrow transplant recipients
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

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The study of bone microarchitecture and its molecular aspects may provide new information for better understanding of "bone quality". Histomorphometry is a recommended reference method for bone-dimensional analysis. At the molecular level, possible changes in the process of resorption and bone formation has not been studied in different patterns of bone "normal." Objective: To analyze the correlation between clinical, radiographic and histomorphometric bone-molecular sites for dental implants in humans. Methods: The sample consisted of 44 sites of 32 volunteers implantable. These sites were classified according to three different methods: a classification based only on periapical and panoramic images (PP), the classification of Lekholm and Zarb, based on methods of diagnostic imaging in conjunction with the tactile perception of the surgeon during surgery and classification of Lindh. The bone specimens were removed with the use of trephine during the first drilling site for placement of dental implants. The samples were subjected to staining with hematoxylin-eosin and histomorphometric analysis to obtain the following histomorphometric parameters: trabecular thickness (Tb.Th), trabecular number, bone volume fraction (BV / TV), trabecular bone surface area by bone volume (BS / BV), bone surface fraction and trabecular separation (Tb.Sp). We also made the technique for analysis of proteins immunoistoquímica RANK, RANKL, OPG and osteocalcin (OC) in samples of bone tissue. Results: Rankings PP & L and Z correlated with BV / TV, BS / BV, Tb.Th and Tb.Sp. The classification of Lindh did not correlate with any histomorphometric parameter. L & Z show differences between types when compared to bone BV / TV, BS / BV, Tb.Th and Tb.Sp. We found a weak correlation between ratings PP / L & Z and the expression of regulators of bone metabolism (RANK, RANKL, OPG and OC). Conclusions: It may be that the subjective ratings conculir types of bone are influenced by aspects of histomorphometry and the regulatory molecules of bone remodeling seems not to exert influence in the morphology of the maxilla and mandible.
O estudo da microarquitetura óssea e dos seus aspectos moleculares podem trazer novas informações para a melhor compreensão da qualidade óssea . A histomorfometria é um método de referência recomendado para análise óssea bidimensional. A nível molecular, possíveis alterações das vias de reabsorção e formação óssea ainda não foram estudadas nos diferentes padrões de osso normal . Objetivo: Analisar a correlação entre aspectos clínico-radiográficos e histomorfométrico-moleculares de sítios ósseos para implantes dentários em humanos. Material e métodos: A amostra foi composta por 44 sítios implantáveis de 32 voluntários. Estes sítios foram classificados de acordo com 3 diferentes métodos: uma classificação baseada somente em imagens periapical e panorâmica (PP); a classificação de Lekholm e Zarb, baseada em métodos de diagnóstico por imagens em conjunto com a percepção tátil do cirurgião durante a cirurgia e a classificação de Lindh. Os espécimes ósseos foram removidos com o uso da trefina durante a primeira perfuração do sítio para colocação dos implantes dentários. As amostras foram submetidas à técnica de coloração com hematoxilina-eosina e análise histomorfométrica para obtenção dos seguintes parâmetros histomorfométricos: espessura trabecular (Tb.Th), número de trabéculas, fração de volume ósseo (BV/TV), área de superfície óssea trabecular pelo volume ósseo (BS/BV), fração de superfície óssea e separação trabecular (Tb.Sp). Também foi realizada a técnica da immunoistoquímica para análise das proteínas RANK, RANKL, OPG e Osteocalcina (OC) nas amostras de tecido ósseo. Resultados: As classificações PP e L&Z apresentaram correlação com BV/TV, BS/BV, Tb.Th e Tb.Sp. A classificação de Lindh não apresentou correlação com nenhum parâmetro histomorfométrico. L&Z demonstrou diferença entre os tipos ósseos quando comparado a BV/TV, BS/BV, Tb.Th e Tb.Sp. Foi encontrada uma fraca correlação entre as classificações PP/L&Z e a expressão dos reguladores do metabolismo ósseo (RANK, RANKL, OPG e OC). Conclusões: Pode-se conculir que as classificações subjetivas dos tipos ósseos são influenciadas pelos aspectos histomorfométricos e que as moléculas reguladoras da remodelação óssea parecem não exercer influência nos aspectos morfológicos da maxila e mandíbula.

Carbon nanotubes have received a lot of attention since their introduction in 1991 because of novel properties that show substantial promises for use in many applications. Their usage depends on the strength of our knowledge of their properties. In this work, molecular mechanics approach is used to study the mechanical properties of multi-wall carbon nanotubes. In particular this paper investigates fundamental frequency of double walled carbon nanotubes. Carbon nanotubes are big and long molecules that can be regarded as mechanical structures. In modeling of multi-walled carbon nanotubes, two distinct atomic bonds are required to be modeled, i.e. covalent bonds between the neighboring carbon atoms in the same layer and Van der Waals bonds between close atoms in neighboring layers. In this approach, for modeling of each wall; covalent bonds are modeled by beam joints such that atoms are considered to be concentrated masses at the ends. Interactions of neighboring walls that are mainly due to Van der Waals forces are treated to be truss rods in modeling. The most challenging aspect of modeling is to define truss rod properties as they are highly nonlinear. We utilized an averaging method for finding truss rod properties. Finite Element Method is employed to obtain Fundamental frequencies. Results are compared to available researches and a close agreement is observed. Results indicate that by increasing aspect ratio, fundamental frequency of double walled nanotubes decrease. In addition, double walled carbon nanotubes have higher fundamental frequencies at clamp-clamp in comparison to clamp-free condition; however, this difference becomes negligible as aspect ratio increases.

The paper has presented the result of tests and researches realized at our university and Oncology Clinical Hospital from Oradea, Radiotherapy Section, about improving of biolubrication between cartilages in relative moving of synovial joints with osteoarthritis, having slow evolution under non-conventional treatment of irradiation with gamma ray. By radioactive molecular treatment of synovial joints with gamma ray, type hinge of knee and spheres of disorder hip (gon-arthrosis and cox-arthrosis), changed molecular structures of porous cartilages and of synovial fluid in contact. All these due to partial recovering of mechanical-elastic and damper system that was spoiling, with a great reducer of pains, altering some orthopedic and non-conventional treatments at overweight patients, which are been impossible by surgery. This paper has presented a theoretical model of human body subjected the applied and conjunction forces, which explained the damping of vibrations and shocks inside of synovial joints by elastic modulus-E of bone cartilages in contact and variation of dynamic viscosity-η of synovial fluid. This work had proposed to promoted osteoarthritis therapy by using irradiation with gamma ray, being ones of the most modern active molecular treatments by using irradiation with particles in radiotherapy from neurological field.

Abstract Countless organisms in nature have adapted high-aspect-ratio micro-/nano-fibrillar arrays on their functional surfaces for achieving special and often optimized functionalities using earthly abundant materials. At the core of nanoscience and nanotechnology, rationally mimicking nature offers a promising route to create multifunctional superstructures that capture organisms and biological materials’ intriguing responsive and self-adjusting properties. Prior work has demonstrated that hierarchical vertically aligned multi-walled carbon nanotube (VA-MCNT) arrays can achieve ten folds of adhesive force comparing to the fibrillar structures of the gecko toe pads. However, little is known with regard to their wettability at the ultimate atomistic level, and how this may influence the adhesive performance and/or self-cleaning capabilities, despite water condensation and bridging are common phenomena at this length scale. In present study, molecular dynamics (MD) simulations were performed using Large-Scale Atomic / Molecular Massively Parallel Simulator (LAMMPS). Results indicate that commonly believed hydrophobic defect free CNTs (i.e., carbon sp2 hybridization without any dangling bonds) become super-hydrophilic at this length/temporal scale. The critical factors that influence the number of H-Bonds in water are: i) tube-tube spacing; and ii) shape/size and position of the water nanodroplet; and iii) how many droplets exists and how many nanotubes are bridged by the droplets. Chirality has little effect on the water interfacial behaviors. Future work will focus on the effect of water condensation and bridging on the adhesive and self-cleaning properties of carbon-based bio-inspired fibrillar dry adhesives considering defects and saline water.

Hybrid microfluidic and nanofluidic devices have a variety of applications including water desalination, molecular gates and DNA sieving among several other lab-on-chip uses. Most microfluidic and nanofluidic devices currently are fabricated in glass, silicon, polydimethylsiloxane (PDMS), or with a combination of these materials. In order to impart functionality, metals, polymers or auxiliary components are often integrated with these devices. Ultra-low aspect ratio channels have several advantages including critical dimensions on the nanoscale but increased throughput compared to higher aspect ratio channels with the same critical dimension, which is important for applications where a higher volumetric flow rate is desired. Additionally, theoretical analysis is significantly easier as ultra-low aspect ratio channels can be modeled as 1-D systems. The fabrication methods for achieving low aspect ratios (< 0.005) usually require extensive facilities with several innovative fabrication and bonding schemes being previously reported. In this paper, we report on fabrication and bonding of ultra-low aspect ratio microfluidic and nanofluidic devices with aspect ratios at 0.0005 in glass/PDMS devices in contrast to the previous best reported result of 0.005 achieved in a silica device using stamp and stick PDMS bonding. The simplicity of our approach presents a new pathway to achieving the lowest aspect ratio nanochannels ever reported for channels fabricated using an interfacial layer for bonding. Centimeter long nanochannels on a borosilicate substrate were fabricated by standard UV photolithography followed by wet etching. Surface roughness of the fabricated channels is on the same order as the roughness of the initial substrate (2–3 nm) and therefore can enable fabrication of channels with critical dimensions approaching 15 nm or less. Devices were then bonded using a second borosilicate substrate with a thin PDMS adhesion layer (∼ 2 μm). The PDMS adhesion layer allows rapid, facile, and alignment-free bonding compared to traditional fusion or anodic bonds. Successful verification of device operation and functionality was determined by verifying flow in operational devices and with scanning electron microscopy to confirm bonding for the formation of nanochannels.

Global efforts to reduce CO2 emissions and to tackle the problem of depleting petroleum resources have stimulated the exploitation of alternative fuels in the aviation industry. One crucial aspect amongst others is to investigate the compatibility of alternative fuels with elastomeric materials currently used in gas turbine engines. However, little knowledge about this has been understood so far for commercial aircrafts under real engine conditions. This study combines Stress-Relaxation test and FTIR spectroscopy techniques to look at the effect of alternative fuels on O-rings made from different materials; such as nitrile, fluorocarbon and fluorosilicone. Results indicated that after immerged in fuels for a period of time while simultaneously being compressed at certain temperatures, the fluorocarbon O-rings showed minimum change in the eight types of fuels tested. This meant they are compatible with these fuels, with the nitrile O-rings changing the most. Furthermore, FT-SPK+20% hexanol caused the biggest relaxation in fluorosilicone O-rings but had the smallest effect on nitrile ones while all fuels presented similar behavior in fluorocarbon. FTIR spectrum analysis showed molecular composition changes are dependent on the reactions between different materials and fuels. For fluorosilicone O-rings, the absorbance reduction from 1150 to 1050 cm−1 was caused by the breakdown of (Si-O-Si) while the formation of new O-H bonds enhanced the intensity from 3000 to 2800 cm−1. For fluorocarbon O-rings, obvious increase in absorption could be found in the region from 1400 to 650 cm−1, while from 3000 to 2800 cm−1, the absorption decreased. Multiple linear regression analysis indicated a highly correlated relationship between the chemical structure changes and the force relaxation.

Layered silicate nanolayers can be used as alternative inorganic components for the construction of nanostructured hybrid composites. The clay silicate nanolayers possess stable Si-O bonds and high particle aspect ratios comparable to conventional fibers. Their interlayer surface is easily modified by ion-exchange reaction, and the gallery can be intercalated by organic polymer precursors for the formation of organic-inorganic nanocomposites. Exfoliated clay composites contain single, 1 nm thick layers of clay dispersed in the polymer matrix. Owing to the platy morphology of the silicate layers, exfoliated clay nanocomposites can exhibit dramatically improved properties such as barrier and mechanical properties that are not available for conventional composite materials. Since the clay particles scavenge water, the nanocomposite samples initially absorb slightly higher amounts of water in comparison to the no-clay samples, with the water molecules congregating around the clay particles. On the other hand, the presence of these clay particles still hinders diffusion of water through the sample, thus protecting the structural interfaces. In this work, low viscosity liquid aromatic diglycidyl ether of bisphenol A (DGEBA) epoxy resin Epon 815C was mixed with nanoclay at 60°C for 6 hours. The epoxy-clay mixture was then mixed with curing agent DETA (Diethylenetriamine) at 80°C for 4 minutes and cured at 120°C for 3 hours to produce exfoliated clay — epoxy resin system. These samples were used to first optimize the percent clay level for lowest water uptake, and subsequently immersed in water in stressed condition (flexural stress) to assess the effect of stress on nanocomposite epoxy system for its water uptake behavior. The results revealed up to 33% reduction in water uptake for the stressed samples.