Single Canadian mothers of Jamaican heritage share experiences about their children's education

This thesis examines the experiences of four single Canadian mothers of Jamaican heritage with respect to their children’s education. Four themes suggested in the literature—beliefs, practices, barriers, and supports—guided the research. The interviews with the mothers largely confirmed previous research in the field. As such, all the mothers believed that it was a shared responsibility between parents and teachers in supporting children’s education. The mothers’ practices included primarily at-home support and to a lesser extent at-school support but did not include strict discipline. The barriers most salient for these mothers were lack of time and resources. To help overcome these barriers, the mothers relied on domestic kin networks. From these findings, the thesis provides implications for both research and practice.

The Influence of Copper Binding on the Stability of the SCO Protein from Bacillus subtilis

Every aerobic organism expresses cytochrome c oxidase to catalyze reduction of molecular oxygen to water, and takes advantage of this energy releasing reaction to produce an electrochemical gradient used in cellular energy production. The protein SCO (Synthesis of cytochrome c oxidase) is a required assembly factor for the oxidase, conserved across many species. SCO is implicated in the assembly of one of two copper centres (ie., CuA) of cytochrome oxidase. The exact mechanism of SCO’s participation in CuA assembly is not known. SCO has been proposed to bind and deliver copper, or alternatively to act in reductive preparation of the CuA site within the oxidase. In this body of work, the strength and stability of Cu(II) binding to Bacillus subtilis SCO is explored via electronic absorption and fluorescence spectroscopies and by calorimetric methods. An equilibrium dissociation constant (Kd) of 3.5x10-12 M was determined as an upper limit for the BsSCO-Cu(II) interaction, via differential scanning calorimetry. In the first reported case for a SCO homolog, dissociation kinetics of Cu(II) from BsSCO were characterized, and found to be dependent on both ionic strength and the presence of free Cu(II) in solution. Further differential scanning calorimetry experiments performed at high ionic strength support a two-step model of BsSCO and Cu(II) binding. The implications of this model for the BsSCO-Cu(II) interaction are presented in relation to the mechanism of interaction between SCO and the CuA site of cytochrome c oxidase.

TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

CONTRIBUTION OF A SPERM PROTEIN, PAWP, TO THE SIGNAL TRANSDUCT PATHWAY DURING VERTEBRATE FERTILIZATION

PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early and later egg activation events including meiotic resumption, pronuclear formation and egg cleavage. Based on the fact that calcium increase is universally accepted as the sole requirement for egg activation, we hypothesized that PAWP is an upstream regulator of the calcium signaling pathway during fertilization. Intracellular calcium increase was detected by two-photon laser scanning fluorescence microscopy following microinjection of recombinant PAWP into Xenopus oocytes, bolstering our hypothesis and suggesting the involvement of a novel PAWP-mediated signaling pathway during fertilization. The N-terminal of PAWP shares a high homology to WW domain binding protein while the C-terminal half contains a functional PPXY motif, which allows it to interact with group I WW domain proteins. These structural considerations together with published data indicating that PPXY synthetic peptide derived from PAWP inhibits ICSI-induced fertilization led to the hypothesis that PAWP triggers egg activation by binding to a group I WW domain protein in the oocyte. By far-Western analysis of oocyte cytoplasmic fraction, PAWP was found to bind to a 52 kDa protein. The competitive inhibition studies with PPXY synthetic peptide, WW domain constructs, and their point mutants demonstrated that the interaction between PAWP and its binding partner is specifically via the PPXY-WW domain module. The 52 kDa protein band crossreacted with antibodies against group I WW domain protein YAP in Western blot assay, indicating that this 52 kDa PAWP binding partner is either YAP or a YAP-related protein. In addition, the far-Western competitive inhibition studies with recombinant GST fusion protein YAP and another WW domain-containing protein, TAZ, demonstrated that the binding of PAWP to its binding partner was significantly reduced by TAZ, providing evidence that TAZ could be the 52 kDa protein candidate. Mass spectrometry was employed to identify this PAWP binding partner candidate. However, due to the low abundance of the candidate protein and the complexity of the sample, several strategies are still needed to enrich this protein. This study correlates PAWP induced meiotic resumption and calcium efflux at fertilization and uncovers a 52 kDa candidate WW domain protein in the oocyte cytoplasm that most likely interacts with PAWP to trigger egg activation.

A Quantitative Investigation of Selected Reactions in the Fibrinolytic Cascade

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.

Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

Giovanni Battista Montano as Architectural Draughtsman: Recording the Past and Designing the Future

Giovanni Battista Montano (1534-1621), who was born in Milan and trained as a woodcarver, relocated permanently to Rome in the early 1570s where his interest in sculpting was replaced by intense study of the city’s antique monuments and ruins. Although Montano carried out several sculptural and architectural projects during his time in Rome, it is his surviving corpus of drawings that testifies to his passion of exploring ancient architecture through the medium of drawing. While Montano was not famous during his lifetime, a large body of his intriguing designs became celebrated and widely circulated after his death thanks to the 1624 publication of Montano’s designs by his loyal pupil, Giovanni Battista Soria. Montano’s lifelong work differs from virtually all of his predecessors and contemporaries in its “fantastical” and ornamental nature. This thesis explores Montano’s artistic training as it relates to his later interest in imaginatively reconstructing antique buildings, along with his disregard for archaeological or historical accuracy. The subject matter upon which Montano focused is discussed, along with his objective in creating a large corpus of half-historical, half-invented drawings. His drawing techniques are explored with specific reference to the largest group of extant Montano drawings, today housed in Sir John Soane’s Museum, London, England, and also in reference to three original Montano drawings in the Centre Canadien d’Architecture/Canadian Centre for Architecture, Montréal. Also explored is the legacy and impact of Montano’s drawings and the later publications of his designs on the works of Roman Baroque architects, specifically Borromini and Bernini. This thesis ultimately attempts to understand the impact of the intellectual and artistic environment surrounding Montano in late sixteenth and early seventeenth century Rome, his drawing techniques, his choice of subject matter, and the reception that his unique works received from contemporary artists and intellectuals, along with those of the following generation.

Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.

Delamination of oil paints from acrylic grounds

Many modern artists paint in oil or oil-modified alkyd paints over acrylic grounds. In some cases the oil based paints do not remain adhered to the ground. In a set of composite samples of oil or alkyd paints, over acrylic grounds, naturally aged for nine years, some of the samples delaminated. Samples were analyzed with X-ray fluorescence (XRF), inductively coupled plasma (ICP), Fourier transform infrared - attenuated total reflectance (FTIR-ATR), scanning electron microscopy (SEM), pyrolysis gas-chromatography mass-spectrometry (PY-GC/MS), laser desorption/ionization mass-spectrometry (LDI-MS), atomic force microscopy (AFM) and other methods, in order to find what the delaminating ones have in common. In addition, two examples of severely delaminating paintings were examined, to confirm the results from the laboratory-prepared samples. Results indicate the main cause of delamination is metal soaps in the oil paint and particularly zinc soaps. There is some evidence that metal soaps were more concentrated at the interface between the layers and this disrupted the adhesion. The ground is a minor consideration as well, rougher grounds providing better adhesion than smooth ones.

COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.

THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

HAMSTER OVIDUCTIN ENHANCES TYROSINE PHOSPHORYLATION OF SPERM PROTEINS DURING CAPACITATION

Capacitation is essential for fertilization of ovulated oocytes. Capacitation is correlated with activation of a signal transduction pathway leading to protein tyrosine phosphorylation, an essential prerequisite for fertilization. Oviductin has been shown to bind to the acrosomal cap and the equatorial segment region of the sperm head. In light of findings reported in previous studies, we hypothesized that estrus stage-specific oviductin (EOV) enhances tyrosine phosphorylation. Immunofluorescent detection by light and confocal microscopy and immunogold labeling by electron microscopy and surface replica techniques were used to localize tyrosine phosphorylated proteins to the equatorial segment region and midpiece after incubation in medium in the presence or absence of EOV. In the presence of EOV, an increase in tyrosine phosphorylation in the equatorial segment region was observed as early as 5 minutes after incubation. On prolonging incubation in medium containing EOV immunostaining further increased, indicative of increased levels of tyrosine phosphorylation of sperm proteins as capacitation proceeds. Regardless of the presence or absence of EOV, phosphotyrosine expression was observed along the tail, specifically at the midpiece. However, this reactivity was enhanced in the presence of EOV. Western blot analysis of NP-40 extractable and non-extractable sperm proteins confirmed these observations. NP-40 extractable sperm proteins (25, 37, 44kDa) and non-extractable sperm proteins (70, 83, 90kDa) showed increased intensity when sperm were capacitated in the presence of EOV after 5-, 60-, 120- and 180-minutes of capacitation. Mass spectrophotometric analysis identified enolase, ATP-specific succinyl CoA, succinate CoA ligase, zona pellucida binding protein, heat shock protein 90, aconitase and hexokinase as proteins that undergo enhancement in tyrosine phosphorylation in the presence of EOV. The proteins identified are known to be involved in specific functions including cellular metabolism, molecular chaperoning and normal sperm development. In summary, the present investigation has provided new evidence showing that sperm capacitated in vitro in the presence of EOV display an enhanced expression of tyrosine phosphorylation compared to sperm incubated in capacitating medium alone. These results indicate that inclusion of oviductin in media used for in vitro fertilization (IVF) may improve success rates of IVF by enhancing the signaling pathways involved in sperm capacitation.

Functional Characterization of TAFI mutants Resistant to Activation by Thrombin, Thrombin-Thrombomodulin or Plasmin

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo.