A calcificação ocorre pela formação de depósitos de cálcio em diferentes matrizes envolvendo fatores mecânicos, químicos e biológicos. Alguns compósitos, polímeros e proteínas são utilizados na formação de matrizes por promover maior eficiência no processo de mineralização. Estima-se que a fibroína da seda apresente também esta finalidade. A fibroína é uma proteína fibrosa extraída do casulo do bicho-da-seda (Bombyx mori), que pode ser processada como filme, membrana, esponja, pó, gel e aplicada em ossos e cartilagens, enxertos vasculares, reparação de nervos e córnea, como sistema de liberação de drogas, suturas, ligamentos, peles, tendões e substrato para cultura de células. Nesse trabalho houve a preparação de micropartículas de fibroína da seda através de dois procedimentos distintos, um por borrifamento em N&sup2 e outro por borrifamento em Na2HPO4 e o processo de calcificação realizado foi por imersão alternada de soluções tamponadas de cálcio e fosfato. As caracterizações realizadas foram Espectroscopia de Absorção no Infravermelho (FT-IR), Análise Termogravimétrica (TGA), Microscopia Eletrônica de Varredura (MEV), Espectroscopia de Energia Dispersiva (EDS) e Calorimetria Exploratória Diferencial (DSC). Os resultados obtidos mostraram que a calcificação das micropartículas de fibroína ocorre pelas duas metodologias empregadas. O teor de calcificação foi de aproximadamente 29% para micropartículas borrifadas em N&sup2 e de aproximadamente 80% para as micropartículas borrifadas em Na2HPO4. As micropartículas de fibroína calcificadas, não apresentaram transição térmica até a temperatura de 120°C, possibilitando a esterilização em autoclave a seco. / Calcification occurs by the formation of calcium deposits in different matrices involving mechanical factors, chemical and biological. Some composites, polymers, and proteins are used in forming matrices to promote higher efficiency in the process of mineralization. It is estimated that the silk fibroin also present for this purpose. The fibroin is a fibrous protein extracted from silkworm cocoon silkworm (Bombyx mori), which can be processed as film, membrane, sponge, powder, gel and applied in bone and cartilage, vascular grafts, nerve repair and corneal as a delivery system for drugs, sutures, ligaments, skins, tendons and substrate for cell culture. In this work was the preparation of microparticles of silk fibroin by two different procedures, sputter under N&sup2 and in other sputter Na2HPO4 and calcification process was performed by immersion of alternating buffered solutions of calcium and phosphate. The characterizations were performed Absorption Spectroscopy Infrared (FT-IR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and Differential Scanning Calorimetry (DSC). The results showed that the calcification of fibroin microparticles occurs by the two methodologies. The calcified fibroin microparticles showed no thermal transition temperature to 120°C, enabling autoclaving of the microparticles dry

Made available in DSpace on 2017-07-21T14:13:08Z (GMT). No. of bitstreams: 1 Jessica Bitencourt Emilio Mendes.pdf: 1572029 bytes, checksum: 2b2c9dc7481232e1bca34cb744c1ab6d (MD5) Previous issue date: 2011-09-17 / Resveratrol is a potent antioxidant, anti-inflammatory, anticancer, and chemoprotective agent. However, it is sensitive to some external agents such as air, light and oxidative enzymes that can reduce its viability and bioavailability for clinical use. In order to provide a controlled release, the aim of this study was to obtain resveratrol-loaded polyester microparticles and to evaluate their physicochemical properties, antioxidant potential and effect on hemolysis of human erythrocytes. Microparticles of poly(3-hydroxybutyrate-co -3-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) containing resveratrol were successfully prepared by simple emulsion/solvent evaporation. All formulations showed suitable encapsulation efficiency values higher than 80%. PHBV microparticles revealed spherical shape with rough surface and presence of pores. PCL microparticles were spherically shaped with smooth surface. Fourier-transformed infrared spectra demonstrated no chemical bond between resveratrol and polymers. X-ray powder diffraction patterns and differential scanning calorimetry analyses indicated that microencapsulation led to a drug amorphization. These PHBV/PCL microparticles delayed the dissolution profile of resveratrol. Release profiles were better fitted to biexponential equation. The hypochlorous acid scavenging activity and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay confirmed that the antioxidant activity of resveratrolloaded into PHBV/PCL microparticles was kept, but was dependent on the microparticle morphology and dissolution profile. Resveratrol-loaded PHBV/PCL microparticles showed no cytotoxic effect on red blood cells. These results support an experimental basis for the use of resveratrol-loaded PHBV/PCL microparticles as a feasible oral drug delivery carrier for controlled release of resveratrol, being an attractive alternative in chronic diseases prevention. / O resveratrol é um potente agente antioxidante, anti-inflamatório, anticancerígeno e quimiopreventivo. No entanto, é sensível a algumas condições externas e ambientais, tais como: ar, luz e enzimas oxidativas, que podem reduzir a sua viabilidade e a sua biodisponibilidade para o uso clínico. Com o propósito de se elaborar sistemas de liberação modificada, o objetivo deste estudo foi a obtenção de micropartículas baseadas em poliésteres, contendo resveratrol, e a avaliação das características físico-químicas, do potencial antioxidante e do efeito sobre a hemólise de eritrócitos humanos desses materiais. As micropartículas de poli(3-hidroxibutirato-co-3 hidroxivalerato) (PHBV) e poli(-caprolactona) (PCL) contendo resveratrol foram preparadas com sucesso pelo método de emulsão simples/evaporação do solvente. Todas as formulações mostraram valores de eficiência de encapsulação adequados, superiores a 80%. As micropartículas de PHBV revelaram formato esférico com superfície rugosa e presença de poros. As micropartículas de PCL apresentaram formato esférico com superfície lisa. Os espectros de infravermelho com transformada de Fourier não demonstraram nenhuma ligação química entre o resveratrol e polímeros após a microencapsulação. As análises de difração de raio-X e de calorimetria exploratória diferencial indicaram que a microencapsulação conduziu a uma amorfização do fármaco. As micropartículas de PHBV e de PCL foram efetivas no controle da liberação do resveratrol. Os perfis de dissolução apresentaram o melhor ajuste para a equação biexponencial. Os ensaios de inibição do ácido hipocloroso e de descolaração do radical catiônico 2,2-azinobis(3-etilbenzotiazolina-6-ácido sulfônico) confirmaram a manutenção da atividade antioxidante do resveratrol presente nas micropartículas de PHBV e de PCL, mas dependente da morfologia das micropartículas e do perfil de dissolução. As micropartículas de PHBV e de PCL contendo resveratrol não apresentaram efeitos citotóxicos sobre os eritrócitos humanos. Esses resultados sugerem que as micropartículas poliméricas elaboradas a partir do PHBV e da PCL, contendo o fármaco, têm viabilidade como sistemas de liberação controlada por via oral do resveratrol, sendo uma alternativa interessante na prevenção de doenças crônicas.

Progression and regression of renal fibrosis is observed in patients with diabetic nephropathy (DN). The underlying pathways, especially those that promote regression of fibrosis, remain poorly understood in part due to the fact that most rodent DN models only mirror the early features of human DN. Another obstacle for optimizing treatment strategies is that albuminuria, the current gold standard biomarker of renal damage in DN, often lacks sensitivity and specificity for identification of those patients with diabetes who are at risk of a rapid decline in renal function. A novel DN model, in which diabetes was induced with streptozotocin in Cyp1a1mRen2 rats and hypertension was generated by inducing renin transgene expression with dietary indole-3-carbinol (I-3-C), mimicked many of the key biochemical, pathological and transcriptomic changes observed in the kidney of patients with DN. Recently, the model was extended to include a ‘reversal phase’ in which glycaemia was tightly controlled and blood pressure normalized for eight weeks after an ‘injury phase’ of 28 weeks. The present study aims to employ this novel rodent model to examine pathways activated in the kidney during and following reversal of hyperglycaemia and hypertension and to identify new biomarkers that might complement albuminuria in assessing risk of renal deterioration in patients with diabetes. Methods Tissue and urinary specimen from the Cyp1a1mRen 2 model of DN were analysed by realtime-PCR, Western-Blot, ELISA and staining techniques including immunohistochemistry, immunofluorescence and zymography. To establish in-situ zymography a model of ureteric obstruction was used. Urinary peptidomic analysis as well as measurement of urinary exosomes and microparticles was performed in the model and in patients with DN utilizing liquid chromatography/tandem mass-spectrometry, nanoparticle tracking analysis (NTA) or flow cytometry. Results Tight control of blood glucose and blood pressure during an 8 week ‘reversal phase’ did not significantly reverse the degree of renal fibrosis accrued during a 28wk ‘injury phase’. However, it did result in a reduction in expression of genes encoding myofibroblast markers and extracellular matrix (ECM) proteins. Genes that were up-regulated during both injury and reversal phases were implicated in adaptive immunity, phagocytosis, lysosomal processing and degradative metalloproteinases (MMPs). Paradoxically MMP activity was massively reduced during both injury and reversal phases. This may be due to an elevated level of tissue inhibitor of metalloproteinase-1 (TIMP-1) protein in both phases. After separating TIMP1 from MMP in renal tissue homogenates from animals of both the injury and reversal phases using gel electrophoresis, MMP activity was restored above that of controls. For biomarker discovery peptidomic analysis was performed on urine from rats at baseline and during the injury and reversal phases of the Cyp1a1mRen2 model of DN and from patients with moderately advanced DN and from normal controls. The use of two different search and analyse tools (Maxquant, Progenesis QI) resulted in the discovery of significantly altered peptides in the urine in rodent and human DN. Further studies focused on peptides derived from those proteins for which the corresponding gene was similarly regulated in the DN model and in human DN. Urinary epidermal growth factor (uEGF) matched these criteria as the reduction of excretion during the injury phase in the DN model was paralleled by reduced EGF protein expression in renal tissue. Key biomarker candidates identified in the first two chapters were measured in urinary specimens of patients from the Edinburgh Type 2 Diabetes study (ET2DS) to test translational utility. MMP7 and other candidates, such as osteopontin or vascular endothelial growth factor (VEGF) were not of value in predicting renal outcomes. Reduced uEGF was significantly associated with increased mortality rate. In a subgroup of 642 study participants who were normoalbuminuric and had a preserved renal function at baseline, a lower uEGF to creatinine ratio was a risk factor for either developing an estimated glomerular filtration rate less than 60 ml/min per 1.73m2, rapid (over 5% per annum) decline in renal function or the combination of both. The latter remained significant after correction for other covariates. Addition of uEGF resulted in a marginal improvement in a model derived from traditional risk factors for predicting rapid decline and the composite end-point. Urinary microparticle (20nm-1000nm) analysis was established in the rodent DN model and translated to patients with DN. Total urinary exosomes (20nm-100nm) or exosomes derived from specific renal cell types including podocytes and tubular cells, increased during the injury phase in the Cyp1a1mRen2 model followed by a decrease after reversal phase. In a pilot study comprising participants with advanced chronic kidney disease, the urinary exosome concentration correlated with renal function. In the ET2DS an increased exosome concentration at baseline indicated a higher risk for renal deterioration during four years follow-up even after correction for baseline eGFR. Urinary microvesicles (100nm-1000nm) concentration increased during the injury phase in the DN model though correlation with renal function in humans was only significant if kidney-specific marker (podocalyxin) positive microvesicles were measured. Conclusion Normalisation of hyperglycaemia and hypertension in the DN model allows the study of genetic and protein regulation during the injury and reversal phases. ECM-production but not ECM-degradation genes are down-regulated during the reversal phase. The lack of reduction in ECM during the reversal phase might be caused by persistently reduced MMP activity due to the presence of TIMP-1. Targeting TIMP might be a treatment strategy to promote reduction of renal fibrosis. For the first time, the analysis of urinary peptidomics was integrated with previous transcriptomic findings in the Cyp1a1mRen2 model and patients with DN for biomarker discovery. The approach was validated using different analysis tools and successfully identified candidate markers which were increased or reduced in DN. Candidates included uEGF, which identified patients with DN who were at risk of a rapid decline of renal function. Though the marker requires further confirmation in other cohorts, it might be especially useful for patients with type 2 diabetes, in whom renal decline is often uncoupled from the development of albuminuria. Finally, the DN model helped to develop the methodology of microparticle analysis. For the first time a potential prognostic value of urinary exosome analysis in patients with diabetes has been demonstrated. Future work will include further optimisation of the methodologies, including labelling of microparticles with multiple antibodies and increasing study participant numbers.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.

We investigate the temperature and size distribution of Ag, Ge, CdSe and ZnS nanoparticles undergoing UV excimer laser pulses. A two laser pulse experiment is designed to monitor nanoparticle size before and after laser interaction. We study HRTEM images and measure the ablation and fluorescence spectra of particles before and after evaporation. Results show that the nanoparticle mean radius decreases from 3.4 ± 0.2 nm to 2.6 ± 0.2 nm, from 4.3 ± 0.1 nm to 3.5 ± 0.1 nm, and from 3.1 ± 0.2 nm to 2.6 ± 0.2 nm for Ag, Ge and CdSe, respectively. No ZnS nanoparticle size reduction was observed. Theoretical models for nanoparticles undergoing laser heating show that temperatures above the bulk and nanoparticle material melting point reduce the nanoparticles size by a factor of 0.3 and suggest recondensation before collection. For CdSe nanoparticles collected on dry substrates and solvents, blue shifted fluorescence (PL) peaks support the size reduction. / text

Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text

Σκοπός της παρούσας διπλωματικής εργασίας είναι η εύρεση γεωμετρικών χαρακτηριστικών των αιμοσφαιρίων με ψηφιακή επεξεργασία της σκεδασμένης μονοχρωματικής ακτινοβολίας. Αποτελείται από 5 κεφάλαια. Σε αυτά περιλαμβάνεται η ανάπτυξη, η υλοποίηση και τα αποτελέσματα τεχνικών για την ψηφιακή ανάλυση κι επεξεργασία ιατρικών εικόνων κάτι που παρουσιάζει ιδιαίτερο επιστημονικό ενδιαφέρον. Oι αλγόριθμοι που αναπτύχθηκαν υλοποιήθηκαν με τη βοήθεια του MATLAB 7.1. O κώδικας προγραμματίστηκε από τον Κύριο Aποστολόπουλο και τα αποτελέσματα της εξομοίωσής τους αξιολογήθηκαν σε συνεργασία με τον καθηγητή Κύριο Δερματά. Στο κεφάλαιο 1 επιχειρείται μια εμπεριστατωμένη ανασκόπηση της διεθνούς βιβλιογραφίας πάνω στα ερευνητικά θέματα που άπτονται του “ευθέως προβλήματος” ως θεωρητικό μοντέλο. Αποτελείται από έξι υποκεφάλαια με το πρώτο να αφιερώνεται στη θεωρητική ανάλυση της σκέδασης της ηλεκτρομαγνητικής ακτινοβολίας (ΗΜ). Στο δεύτερο υποκεφάλαιο γίνεται βιβλιογραφική ανασκόπηση εργασιών που ασχολούνται με τη φυσιολογία και τα γεωμετρικά χαρακτηριστικά των ερυθροκυττάρων, ενώ στο τρίτο υποκεφάλαιο αναλύεται το “ευθύ πρόβλημα σκέδασης”. Στη συνέχεια αναλύεται η σκέδαση ανθρώπινου αιμοσφαιρίου από Ηe-Νe laser και από ηλεκτρομαγνητική ακτινοβολία. Τέλος παρατίθενται εικόνες από laser, οι οποίες προέκυψαν από πειράματα του Κυρίου Τσινόπουλου. Στο κεφάλαιο 2, αναφέρονται βασικές έννοιες ηλεκτρομαγνητισμού και σκέδασης ηλεκτρομαγνητικών κυμάτων. Αρχικά ορίζεται το ηλεκτρομαγνητικό φάσμα και η οπτική Η/Μ ακτινοβολία, η δομή της ύλης και των ατόμων και η απορρόφηση του φωτός από ερυθρά αιμοσφαίρια. Στο τέταρτο υποκεφάλαιο γίνεται μια εκτενής αναφορά στη θεωρία του ηλεκτρομαγνητισμού, τις εξισώσεις Maxwell και τις μακροσκοπικές ιδιότητες του μέσου, για κενό χώρο, ισότροπο και ανισότροπο μέσο καθώς επίσης και οι συνοριακές συνθήκες. Τέλος διατυπώνεται η κυματική μορφή των εξισώσεων Maxwell. Στο κεφάλαιο 3 παρουσιάζονται εφαρμογές παρόμοιων πειραμάτων με το “ευθύ πρόβλημα” σκέδασης ηλεκτρομαγνητικής ακτινοβολίας σχετικά με τη σκέδαση μικροσωματιδίων. Αρχικά αναλύονται εκτενώς τεχνικές της βιοτεχνολογίας και ιατρικής, όπως η ακτινοδιαγνωστική, η αθηροσκλήρωση και η βρογχοσκόπηση. Στη συνέχεια παρατίθενται εφαρμογές πέραν της ιατρικής οι οποίες παρουσιάζουν ιδιαίτερο ενδιαφέρον στη βελτίωση του βιοτικού επιπέδου. Ανάμεσα σ’ αυτές είναι ο προσδιορισμός του μεγέθους βακτηριδίων και η χρήση του Radar. Στο κεφάλαιο 4 περιγράφεται η γενική μορφή του “αντίστροφου προβλήματος” ηλεκτρομαγνητικής σκέδασης. Αρχικά παρατίθενται ο ορισμός του προβλήματος, τα πλεονεκτήματα και τα μειονεκτήματα. Στη συνέχεια παρουσιάζεται εκτενώς η επαναληπτική μέθοδος του “αντίστροφου προβλήματος” ηλεκτρομαγνητικής σκέδασης. Τέλος μοντελοποιούμε το παραπάνω πρόβλημα και αναλύονται τα προβλήματα που προκύπτουν ως αποτέλεσμα ενός παραδείγματος αναδημιουργίας αεροσκάφους με “αντίστροφη σκέδαση”. Στο κεφάλαιο 5 παρουσιάζονται εφαρμογές “αντίστροφων προβλημάτων” ηλεκτρομαγνητικής σκέδασης, καθώς και τα πλεονεκτήματα και μειονεκτήματά τους. Μερικές από αυτές τις εφαρμογές αναλύονται εκτενώς, όπως η εκτίμηση μεγέθους κοπαδιού ψαριών με αεροφωτογραφία, η επισκόπηση αρχαιολογικής γεωφυσικής και η σκέδαση φωτός δισδιάστατης γωνίας για το χαρακτηρισμό των μικροσωματιδίων αεροσκάφους. Στο κεφάλαιο 6 αναλύεται πειραματικά το “αντίστροφο πρόβλημα” σκέδασης με αναφορές σε βασικές έννοιες που χρησιμοποιούνται στο παρόν πείραμα, όπως η κανονικοποίηση εικόνας, ο DCT (Discrete Cosine Transform) και το νευρωνικό Δίκτυο. Εν συνεχεία εκτιμάται το μέγεθος και η μορφή του RBC με το “αντίστροφο πρόβλημα” σκέδασης. Τα προκύπτοντα μεγέθη μετασχηματίζουμε με Discrete Cosine Transform (DCT) κι εξάγουμε χαρακτηριστικά γνωρίσματα. Στη συνέχεια κανονικοποιούμε τα χαρακτηριστικά γνωρίσματα και προβλέπουμε τα νευρωνικά δίκτυα για το μέγεθος και τη μορφή των RBC. Τέλος εξάγονται πειραματικά αποτελέσματα και συμπεράσματα. Στο τελευταίο κεφάλαιο δίνονται οι μετρήσεις των ερυθροκυττάρων. Γίνεται μια γενική αναφορά στις εικόνες και την ψηφιακή επεξεργασία τους με τη βοήθεια του MATLAB 7.1. Αναφέρονται οι τύποι των εικόνων και η δομή τους καθώς και τα χρήσιμα Φίλτρα Εξάλειψης Θορύβου Εικόνας. Τέλος παρατίθενται οι γραφικές παραστάσεις που προκύπτουν από το Μatlab 7.1 του μέσου απόλυτου σφάλματος (Regression Error) και της επιτυχίας αναγνώρισης σχετικά με τον αριθμό των νευρώνων και σχολιάζονται αναλυτικά. / -

Optical biosensors are desired for the monitoring of various biochemical markers, which are relevant indicators in the treatment and diagnosis of diseases. Specifically, luminescence sensors are favorable for optical interrogation since they are highly sensitive to analyte changes and may be implemented in lifetime or intensity-based systems. In order to develop particle-based fluorescent sensors, poly(2-hydroxyethylmethacrylate) (HEMA) microspheres have been fabricated via membrane emulsification and characterized to evaluate the emulsion method and the overall process of tailoring properties to synthesize spheres of specific mean sizes. A pH-sensitive indicator seminaphthorhodafluors-4F 5-(and-6)-carboxylic acid (SNARF) was immobilized within the microspheres, and resulting sensor particles were exposed to various pH buffers to obtain a pH calibration curve based on intensity measurements. PolyHEMA microparticles were fabricated in a systematic study with measured mean sizes ranging from 8-21 um. Optical and scanning electron microscopy images revealed the formation of spherical, porous particles, which were additionally stabilized with polymer coatings. The lowest coefficient of variation value achieved was 50%, indicating the inability to produce monodisperse particles due to the dispersity of pore sizes in the membrane. SNARF was immobilized within the polyHEMA spheres, and fluorescence was observed when exposing the sensors to different pH buffers on a fluorescence microscope. Ratiometric intensity measurements for the sensor particles were obtained on a spectrofluorometer while flowing pH buffers over the immobilized spheres in a reaction chamber. The peak intensity ratio of the microparticle sensors exhibited a change in 0.9 units when decreasing the pH from 8.4 to 5.5. In the future, these pH sensing particles may be implanted alongside glucose sensing materials in order to provide valuable pH information in understanding the immune response to specific biomaterials and sensing components.