Three major topics will be discussed in this dissertation. The first is an optical biosensor for specific diagnosis of bacterial skin and wound infection, followed by a paper microfluidic assay and accompanying monitoring device for monitoring blood coagulation and determining patient-specific heparin and protamine dosing. The final work to be discussed is ongoing work involving the detection of circulating tumor cells (CTCs) using a paper microfluidic detection platform. All of these works involve the development of biosensors for the simultaneous advancement and simplification of diagnosis and analysis of blood and bacterial infection. The aims of each of these projects included significantly decreasing the time to diagnosis and decreasing the reagents, laboratory space, personnel, and other resources needed for detection and diagnosis. The first works are focused on the design, development, and testing of an optical biosensor for the immediate detection of bacterial skin and wound infection, including diagnosing the specific species of bacteria responsible for the infection. The optical biosensor developed allows for diagnosis of a bacterial infection on skin or in a wound in as little as three seconds, in a contact-free, reagent-free manner. The second work focused on the design, development, and testing of a paper microfluidic assay and accompanying Raspberry Pi-based monitoring device for use before, during, and after surgeries requiring the use of cardiopulmonary bypass. The assay monitors the extent of blood coagulation of a whole blood sample and determines patient-specific dose response curves of an anticoagulant and its reversal agent. The final work discussed focuses on developing a paper microfluidic assay for the detection of CTCs from whole blood samples. The goal of this work is to detect multiple morphologies of CTCs from whole blood samples to provide insight on patient prognosis in a rapid, low resource manner.

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Aspartame is a dipeptide sweetener that can be found in most of the sugar-free products available on the market today. The FDA approved the use of aspartame, but ever since the safety of the consumption of aspartame has been questioned. Thus the aim of this thesis was to determine the effects of aspartame ingestion on the blood coagulation system and the blood filtering organs (liver and kidneys) of the rabbit. The protocol for obtaining blood from a rabbit as well as successful administration of aspartame was perfected. The rabbit was proven as best experimental model, when compared to a mouse, for studying the effects of aspartame on coagulation and haemostasis. The effects of aspartame were determined by: 1.) measuring the factors from the different coagulation pathways, namely the common pathway (factors II, V, X and fibrinogen); factors in the intrinsic pathway (factors VIII, IX), as well as factor VII, found in the extrinsic pathway. The prothrombin time (PT; measures how long blood takes to form a clot) and activated partial thromboplastin time (aPTT; measures recalcification time of plasma) was also measured; 2.) The ultrastructure of the fibrin networks, platelet morphology and endothelial lining were studied; 3.) The histological morphology of the leukocytes, liver and kidney were examined. Results obtained indicated that F VII, X and VIII were decreased with a prolonged prothrombin time. The concentration of circulating fibrinogen increased significantly, which corroborated with results obtained for the ultrastructure of the fibrin networks. The degree of fibrin fibre formation increased the higher the concentration of aspartame and the degree of platelet aggregation occurring, decreased with the increase of aspartame concentration. It is hypothesized that the amount of circulating serotonin decreased. The endothelial lining of the rabbits were damaged with the nuclei appearing apoptotic. The endothelial lining and their tight junctions play an integral part in the functioning of the BBB, in synchronization with cAMP (complexity of tight junctions, decreased due to decreased amount of serotonin), thus it appeared as though the BBB was compromised. The morphology of the leukocytes were altered, specifically that of the eosinophils and heterophils. The granules inside the eosinophils of the aspartame treated rabbit appeared to have increased and were more clearly visible, while the granules in the heterophils appeared to have decreased. The total number of leukocytes also decreased. Thenormal histological morphology of both the liver and kidney were affected by aspartame. Damage to the hepatocytes and their subsequent arrangement were noted. The visceral layer of the capsule of Bowman appeared thickened and the cuboidal epithelium lining the proximal convoluted tubule was also damaged The final judgment and conclusion of the results obtained in this thesis regarding the consumption of abuse doses of aspartame, was that aspartame could lead to bleeding disorders (especially in genetically predisposed individuals), suppressed immunity and a compromised BBB. Trouble can occur with formation of the glomerular filtrate and absorption of fluid from the proximal convoluted tubule, which could result in high blood pressure and an increased probability of dehydration respectively. / Thesis (PhD (Anatomy))--University of Pretoria, 2008. / Anatomy / PhD / unrestricted

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of β-factor Xlla as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. A second human liver cDNA library was screened by colony hybridization with ³²P-labeled cDNA clones obtained from the first screen and two identical clones were isolated. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for the signal peptide sequence, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly A⁺ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, were identified three peptide bonds that are cleaved by kallikrein during the formation of β-factor Xlla. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A human genomic phage library was screened by using a human factor XII cDNA as ahybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kbp in size and is comprised of 13 introns and 14 exons. Exons 3 through 14 are contained in a genomic region of only 4.2 kbp with introns ranging in size from 80 to 554 bp. The multiple regions found in the coding sequence of FXII that are homologous to putative domains in fibronectin and tissue-type plasminogen activator are contained on separate exons in the factor XII gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' flanking region of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple start sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thirty thousand colonies of a bovine liver cDNA library were screened with a mixture of synthetic oligodeoxyribonucleotides coding for bovine factor X. Five positive colonies were identified, and plasmid DNA was isolated. Cleavage with restriction endonucleases showed that these plasmids (designated pBXl-5) contained inserts of 1530 bp, 770 bp, 700 bp, 1100 bp and 930 bp. DNA sequence analysis of the plasmid with the largest insert (pBXl) confirmed that bovine factor X cDNAs had been cloned. The cDNA sequence predicts that factor X is synthesized as a single chain precursor in which the light and heavy chains of plasma factor X are linked by the dipeptide Arg-Arg. The cDNA sequence also predicts that factor X is synthesized with a preproleader peptide. It is proposed that at least five specific proteolytic events occur during the conversion of preprofactor X to plasma factor Xa. A human liver cDNA library was screened by colony hybridization with a bovine factor X cDNA probe. Three of the positive plasmids contained overlapping DNA that coded for most of human factor X mRNA. A second human liver cDNA library was screened by in situ hybridization with 32P-labeled human factor X cDNA clones obtained from the first screen. Several clones were isolated that contained longer inserts. DNA sequence analysis of these clones allowed the prediction of the amino acid sequence of the precursor form of human plasma factor X. From these studies, it is predicted that human factor X is synthesized as a single polypeptide chain precursor in which the light and heavy chains of plasma factor X are linked by the tripeptide Arg-Lys-Arg. The cDNA sequence also predicts that human factor X is synthesized as a preproprotein having an aminoterminal leader peptide of 40 amino acid residues. A comparison of the amino acid sequences of human and bovine factor X shows high sequence identity around the calcium- binding regions and catalytic regions but low sequence identity around the nonfunctional regions. A human genomic phage library was screened with a human factor X cDNA as a hybridization probe. Thirty-two overlapping phage clones were isolated. Characterization of six of these clones indicates that over 32 Kbp of contiguous sequence is represented. DNA sequence and restriction map analysis shows that the factor X gene is comprised of at least 8 exons and 7 introns. No clones representing the 5' untranslated region and the prepeptide of the leader sequence were identified. Two further genomic phage libraries and two libraries specific for the 5' region of the factor X gene were screened, but no 5' end clones were obtained. Restriction enzyme mapping and Southern blot analysis indicate that thus far, the human factor X gene maps to 24 Kbp of the human genome. Comparison of the factor X gene with other vitamin K-dependent blood coagulation factor genes reveals homologous exon organization. Within the blood coagulation serine proteases factor X, factor IX, factor VII, and protein C form a closely related gene family. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Orientador: Joyce Maria Annichino-Bizzacchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T01:02:06Z (GMT). No. of bitstreams: 1 DePaula_LaisIvinaSilva_M.pdf: 1198632 bytes, checksum: 0ba443fa4dbfb9c64ad09eacb865b1bb (MD5) Previous issue date: 2015 / Resumo: Problemas associados à aterosclerose são a causa mais comum de morte na população ocidental. A aterosclerose é uma doença inflamatória, e existe uma associação da coagulação com processos inflamatórios e complicações da doença arterial. Estudos populacionais inicialmente demonstraram uma diminuição do risco de infarto agudo do miocárdio em homens hemofílicos, quando comparados com controles pareados por idade e sexo. Contudo estudos subsequentes não confirmaram esses achados, havendo inclusive um estudo que mostrou aumento do risco de doença aterosclerótica e infarto em pacientes hemofílicos. Um dos fatores que deve ter tido importante papel nessa mudança foi o aumento da sobrevida dos pacientes a partir de 1999, com o advento de terapêuticas mais eficazes. Este estudo teve como objetivo investigar o papel da deficiência grave de fator IX (FIX), no desenvolvimento da doença aterosclerótica, em um modelo de dislipidemia, utilizando animais da linhagem C57Bl/6 deficientes de apolipoproteína E (APOE -/-). Comparamos grupos de animais com deficiência de apoE com ou sem deficiência de FIX (hemofílicos B - HB), através da avaliação quantitativa das lesões, na raiz e em toda sua extensão aórtica; e caracterização histológica das lesões na raiz aórtica por imunofluorecência. Nossos resultados mostraram que os animais com hemofilia B e deficiência de apoE apresentaram o mesmo grau de formação de placa aterosclerótica quando comparados com o grupo controle (APOE -/-), com 8 e 22 semanas de dieta. Portanto, a deficiência do fator IX não protegeu contra a formação de lesões ateroscleróticas na raiz e em toda extensão aórtica. O mesmo ocorreu na avaliação qualitativa, quando comparamos a porcentagem total de macrófagos e de células musculares lisas. Independente dos seus elevados níveis de colesterol e triglicerídeos. Contudo, foi evidente a diminuição na quantidade de macrófagos M1, através da produção de IL - 1?, em comparação aos camundongos controles, deficientes apenas de apoE. Um dado intrigante é que apesar de termos observado uma diminuição de macrófagos M1 nos animais hemofílicos, isso não foi acompanhado por uma diminuição nas lesões ateroscleróticas. Portanto, apesar da hipocoagulabilidade e do perfil de resposta dos macrófagos, nossos resultados sugerem que outros fatores foram mais importantes no desenvolvimento da aterosclerose. Nossos resultados corroboram os achados mais recentes de que pacientes hemofílicos também são susceptíveis ao desenvolvimento de doença aterosclerótica, incluindo doença coronariana / Abstract: Problems associated with atherosclerosis are the most common cause of death in the western population. Atherosclerosis is an inflammatory disease, and there is a coagulation associated with inflammatory processes and complications of arterial disease. Population studies initially demonstrated a decreased risk of acute myocardial infarction in men hemophiliacs compared with controls matched for age and sex. But subsequent studies have not confirmed these findings, including having a study that showed increased risk of atherosclerotic disease and stroke in hemophilia patients. One of the factors that must have played an important role in this change was the increased survival of patients since 1999, with the advent of more effective therapies. This study aimed to investigate the role of severe deficiency of factor IX (FIX), in the development of atherosclerosis, dyslipidemia in a model using animals C57BL strain / 6 deficient apolipoprotein E (APOE - / -). Groups of animals compared with apoE-deficient or without FIX deficiency (hemophilia B - HB) by quantitative evaluation of lesions in the root and aortic its entire extension; and histological characterization of the lesions in the aortic root by immunofluorescence. Our results showed that animals with hemophilia B and apoE deficiency showed the same degree of atherosclerotic plaque formation when compared with the control group (APOE - / -), with 8 and 22 weeks of diet. Therefore, factor IX deficiency does not protect against the formation of atherosclerotic lesions in the aortic root and throughout extension. The same occurred in the qualitative evaluation, when comparing the total percentage of macrophages and smooth muscle cells. Whatever your high cholesterol and triglycerides. However, it was apparent decrease in the amount of M1 macrophages by IL - 1? compared to control mice, apoE deficient only. A given intriguing is that although we observed a decrease of M1 macrophages in hemophiliacs animals, this was not accompanied by a decrease in atherosclerotic lesions. Therefore, despite the hypocoagulability and macrophage response profile, our results suggest that other factors are more important in the development of atherosclerosis. Our results support the latest findings that hemophilia patients are also susceptible to developing atherosclerosis, including coronary heart disease / Mestrado / Fisiopatologia Médica / Mestra em Ciências

INTRODUCTION: The menstrual cycle and pregnancy are processes unique to women. Both these processes involve various hormones as well as the coagulation system. Throughout normal pregnancy, platelet activation and increase in blood coagulation factors contributes to the hypercoagulable state observed on a physiological level. METHODS: Fibrin networks and platelets were analysed by electron microscopy and flow cytometry to determine any differences found in different phases of pregnancy compared to healthy control individuals. The fibrin networks from different phases of the menstrual cycle as well as different phases of pregnancy were investigated. RESULTS: It was found that ultrastructural changes in fibrin fiber morphology result from estrogen changes during the menstrual cycle. During pregnancy the minor thin fibers were prominent and thick matted layers of coagulant formation were evident. A large quantity of protein globular clusters similar to those seen in the menstrual cycle was present. Changes observed in platelet ultrastructure during pregnancy showed pregnancy-specific modifications. Platelets were activated and internal organelles showed variation from control participants. Flow cytometric analysis of platelets verified pregnancy-specific modifications. Close interactions between platelets and erythrocytes were evident. CONCLUSION: The female body is equipped to handle alterations in the coagulation system as can be extrapolated from the pregnancy-specific modifications. This study is the first to show alterations in fibrin network and platelet ultrastructure during and after pregnancy when compared to non-pregnant controls. The physiological changes during normal pregnancy can be used as a standard for comparison to abnormal or ailing pregnancy. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Physiology / Unrestricted

Fluid resuscitation with crystalloid and colloid solutions is a common treatment in perioperative medicine. However, a variety of unbalanced or balanced solutions is used in clinical practice and there is still a vivid debate going on regarding the selection of optimal fluid with minimal negative effect on coagulation. The goal of the dissertation was to investigate the adverse effect of balanced crystalloids and colloids on whole blood coagulation measured by method of rotational thromboelastometry. In the first phase of the work we had assessed the adverse effect of balanced crystalloid, hydroxyethyl starch and gelatin after dilution of blood with the solution in vitro. Parametrs of EXTEM and FIBTEM tests were evaluated by using rotational thromboelastometry. In the second phase of the work we evaluated the negative effect of infusion solution after dilution in vivo. We had obtained blood samples from 30 patients during knee arthroscopy before and after administration of 500 ml of crystalloid or hydroxyethyl starch or gelatin. Parametrs of EXTEM and FIBTEM tests were evaluated by using rotational thromboelastometry.In compliance with the results of the dissertation, hydroxyethyl starch has the most obvious negative effect on clot formation followed by gelatin and finally by crystalloids. Based on...

On vessel wall injury the complex process of blood coagulation is set off. It is composed of vasoconstriction, primary hemostasis, secondary hemostasis and fibrinolysis. This work enriches current model of primary hemostasis of Storti. The previous model used ALE formalism for tracing of development of platelet plug. The phase field method is used for tracing of the development of interface blood-thrombus. Storti's primary hemostasis was extended to capture the fact, that the platelets can be activated in the blood flow in the area of reactive surface not only by influence of chemical agents like thromboxane, ADP and thrombin but also by their exposure to elevated values of shear stress. In our first approach we deal the emerging thrombus as a fluid with very high viscosity. In the second approach it was assumed, that platelet plug develops as a viscoelastic material according to constitutive equations of clot introduced by Kempen. In this manner platelet clot matures into blood clot. In both approaches the blood is represented as a non-Newtonian fluid. The framework of the phase field method was applied also to the model of high shear rate thrombosis of Weller. Weller's original model of Weller took advantage of the cylindrical symmetry of computational domains for its computations, hence the...

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