<p> Inherited bleeding disorders can be caused by mutations affecting platelet, coagulation, or fibrinolytic proteins. Quebec platelet disorder (QPD) is a rare, autosomal dominant disorder associated with increased expression of the fibrinolytic enzyme urokinase plasminogen activator (uPA) in platelets. Individuals with QPD experience delayed-onset bleeding after hemostatic challenges that is attenuated with fibrinolytic inhibitor therapy. The aims of this thesis were to: 1) determine if increased platelet uPA contributes to QPD clot lysis in vitro; 2) investigate whether QPD individuals have increased urinary uPA, as some individuals experience hematuria; and 3) map the genetic locus of QPD, and look for the putative mutation. Studies of clot lysis indicated that QPD platelets induce a gain-of-function defect in fibrinolysis when platelets are incorporated into clots. This suggests that accelerated fibrinolysis may contribute to QPD bleeding. Studies of urinary uPA in QPD showed that uPA is not increased, indicating that hematuria in QPD is likely a consequence of increased platelet uPA. This finding also suggests that uPA overexpression in QPD may be megakaryocyte-specific. Linkage studies showed that QPD is strongly linked to a 2 megabase region on chromosome 10 that harbors the uPA gene, PLA U. No mutations in PLA U or its regulatory regions were identified; however, a common haplotype for a 32.5 kilobase region around PLA U, including inheritance of a rare, linked polymorphism, suggests this is the most likely locus for QPD. mRNA studies in QPD platelets showed that QPD selectively increases expression of the linked PLAU allele, without similar increases in megakaryocyte progenitors or in saliva. These findings implicate a cis-mutation near PLA U as the cause of QPD. This thesis provides novel insights on the fibrinolytic abnormality in QPD blood, and on the QPD genetic locus. which will be important for identifying the precise mutation that converts normally prohemostatic platelets to profibrinolytic cells. </p> / Thesis / Doctor of Philosophy (PhD)

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This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A pré-eclâmpsia (PE) é classificada em de início (EOPE) e tardio (LOPE) quando presente antes ou após 34 semanas de gestação, respectivamente. Este estudo transversal investigou as diferenças e possíveis associações existentes entre os perfis antropométricos, hemodinâmicos, hematológicos e bioquímicos na pré-eclâmpsia entre os grupos EOPE e LOPE. O estudo incluiu 65 voluntárias admitidas em um hospital universitário no Brasil sendo 29 normotensos (grupo C) e 36 com pré-eclâmpsia (13 com EOPE e 23 com LOPE). O grupo LOPE apresentou um aumento significante de peso e aumento limítrofe no índice de massa corporal ao final da gestação em relação aos outros grupos, o que é compatível com a origem metabólica, associada à obesidade, atribuída a esta forma da doença. As mulheres grávidas do EOPE apresentaram uma redução limítrofe no número de eritrócitos e uma diminuição signi-ficante no número de plaquetas e um aumento significante de reticulócitos, ferro sérico e fer-ritina quando comparados ao grupo C e LOPE. O grupo EOPE demonstrou um aumento significante na estabilidade osmótica dos eritrócitos em relação aos demais gru-pos. A análise hemodinâmica por ultrasonografia Doppler da artéria oftálmica mostrou que ambos os grupos de mulheres grávidas com PE apresentaram alterações compatíveis com a ocorrência de hiperfluxo no território orbital. Essas alterações hemodinâmicas foram associa-das a alterações nos índices hematimétricos. / Preeclampsia (PE) is classified in early- (EOPE) and late-onset PE (LOPE) when pre-sent before or after 34 gestation weeks, respectively. This transversal study aimed to investi-gate the differences and possible associations existing in the anthropometric, hemodynamic, hematologic and biochemical profiles of late- and early-onset preeclampsia. The study in-cluded 65 volunteers admitted to a tertiary hospital in Brazil, 29 normotensive and 36 with preeclampsia (13 with EOPE and 23 with LOPE). Pregnant women with LOPE presented greater weight gain and borderline increase in body mass index at the end of gestation in rela-tion to the other groups, which is compatible with the metabolic origin, associated with obesi-ty, attributed to this form of the disease. Pregnant women with EOPE presented a borderline reduction in the number of erythrocytes and a significant decrease in the number of platelets, in addition to a significant increase in reticulocytes, serum iron and ferritin when compared to normotensive pregnant women and pregnant women with LOPE. A significant increase in osmotic stability of erythrocytes was observed in the EOPE group in relation to groups. He-modynamic analysis by Doppler ultrasonography of the ophthalmic artery showed that both groups of pregnant women with PE presented alterations compatible with the occurrence of hyperflow in the orbital territory. These hemodynamic changes were associated with changes in hematimetric indices / Tese (Doutorado)

Cell cycle regulation is an important part for the detection and destruction of mutated and dysregulated cells as it is a natural protection against degenerative diseases and cancer. The ability of the body to detect and destroy these cells is a vital part in maintaining homeostasis in the body. Once cells have circumvented this line of defence, dismantling these cells would become very difficult.  Research into new ways to target and destroy mutated cells are constantly evolving in hopes of being able to control and direct lysis of target cells using therapeutic drugs. One of the possibilities for such a method are Cholesterol Dependent Cytolysins (CDC), specific proteins found in bacteria. These proteins are dependent on the ability to bind to cholesterol in cell-walls to form pores that lyse and effectively destroy cells.   This project aims to study the structure and mechanistic details of pore formation by CDC mitilysin using cryogenic electron microscopy (cryo-EM). Mitilysin was purified by affinity chromatography and its pore formation ability was confirmed by calcein release assay and hemolysis assay. The pore structures of mitilysin were observed by transmission electron microscopy (TEM) using liposomes composed of both 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as model membranes. Detergent screening directed separation of pores from liposomes; so that they could be visualized by cryo-EM. While these steps were optimized and proven successful, they were time-consuming. An initial 3D-model of pore-structures was rendered, but no molecular characteristics could be determined at the end of the allotted time. The study does lay the ground steps for obtaining the complete structure of mitilysin pores.