Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-112).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-86).

The spray freezing into liquid (SFL) and thin film freezing (TFF) processes were utilized to produce 300 nm protein particles with surface areas on the order of 31 - 73 m²/g and 100% protein activities. Despite a cooling rate of ~10²-10³ K/s in SFL and TFF, the particle sizes and surface areas were similar to those observed in the widely reported process, spray freeze-drying (SFD), where cooling rates reach 10⁶ K/s. In SFL and TFF, the thin liquid channels between the ice domains were sufficiently thin and freezing rates of the thin channels sufficiently fast to achieve the similar particle morphologies. Therefore, the extremely rapid cooling rate in the SFD process was not necessary to form the desired submicron protein particles. In SFL and TFF the surface area/volume ratio of the gas-liquid formed on the liquid protein formulations (46-600 cm⁻¹) was 1-2 orders of magnitude lower than in SFD (6000 cm⁻¹), leading to far less protein adsorption and aggregation. This larger exposure to the gas-liquid interface resulted in lower protein activities in SFD. Although protein stabilities are high in conventional lyophilization, cooling rates are on the order of 1 K/min resulting in large 30 to 100 [mu]m sized particles. Thus, the intermediate cooling rate regime for SFL and TFF, relative to SFD and lyophilization, offers a promising route to form stable submicron protein particles of interest in pulmonary and parenteral delivery applications. The rod-shaped protein particles produced by SFL and TFF are beneficial for forming suspensions stable against settling in hydrofluoroalkanes (HFA) for pressurized metered dose inhaler (pMDI) delivery. The flocculated rods are templated by atomized HFA droplets that evaporate and shrink to form particles with optimal aerodynamic diameters for deep lung delivery. Fine particle fractions of 38-48% were achieved. This novel concept for forming stable suspensions of flocs of rod shaped particles, and templating and shrinking the flocs to produce particles for efficient pMDI deep lung delivery is applicable to a wide variety of drugs. / text

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Physiology / Master / Master of Philosophy

Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions. In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506. In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection. MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding. The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers. / published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Influenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development. The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells. A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated. In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Spermatozoa experience a series of physiological alternation upon traveling from the testis to the epididymis in order to acquire motility and capacity necessary to recognize and to fuse with an oocyte. Further maturation and modification of the spermatozoa occur along the journey in the female reproductive tract in a process known as capacitation. p53 protein is a well-known transcription factor involved in guarding of genome integrity by triggering cell cycle arrest and cell death upon DNA damage. p53-binding protein 1 (53BP-1) binds to p53 and enhances p53-mediated transcriptional activity. It has been reported that human spermatozoa produce 53BP-1 for DNA repair in response to oxidative stress. 53BP-1 is expressed in pre-implantation embryos. The current study aimed to identify the expression and subcellular location of 53BP-1 proteins in mouse spermatozoa by immunofluorescence staining. Increased expression of 53BP-1 proteins was found in the swim-up and capacitated mouse spermatozoa. The immunoreactivity was localized to the sperm head and midpiece, suggesting possible roles of 53BP-1 in sperm function. It was also found that mouse oocytes and 1-cell embryo also expressed 53BP-1. The potential function of 53BP-1 in early embryo development was also investigated using 53BP-1 antibody neutralized sperm in in-vitro fertilization. There was no significant difference between the development rate of embryos fertilized with spermatozoa transduced with anti-53BP-1 antibody and those that transduced with the control goat IgG. Further studies are needed to investigate the role of sperm 53BP-1 at later stage of embryo development and implantation. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model. The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx). Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins. Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation. The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle. In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy