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Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets. / Physiology

The nitritation-anammox process is an efficient and cost-effective approach for biological nitrogen removal, but its application in treating mainstream wastewater remains a great challenge. The key objectives of this dissertation are to develop nitritation-anammox process to treat wastewater with low-nitrogen strength, understand the fundamental microbiology, and optimize its operation through experimental studies and mathematic modeling. Chapter 2 showed that the nitritation-anammox process has been successfully developed in an upflow membrane-aerated biofilm reactor, where pure oxygen was delivered via gas-permeable membrane module. Chapter 3 demonstrated that hybrid anaerobic reactor (HAR) could be an effective pretreatment method to provide a relatively low COD/N ratio for nitritation-anammox reactor. In Chapter 4, a novel mathematical model has been proposed to evaluate the minimum DO requirement for the nitritation-anammox reactor to achieve the maximum TN removal under various COD/N scenarios (controlled by HRTHAR). Chapters 5 and 6 designed an OsAMX system by linking nitritation-anammox to forward osmosis to remove the reverse-fluxed ammonium while using ammonium bicarbonate as a draw solute. The microbial community structures and dynamics, spatial distributions in these bioreactors were characterized by high-throughput sequencing and fluorescent in situ hybridization techniques. The studies in this dissertation have demonstrated that nitritation-anammox process is a promising alternative for sustainable mainstream treatment with the appropriate pretreatment approach and operation optimization. / PHD

Aerobic granular sludge has been regarded as a promising alternative to the conventional activated sludge which has been used for a century in that granular sludge offers advantages in high biomass retention, fast sludge-water separation, and small footprint requirement. However, this technology has been rarely applied in continuous flow reactors (CFRs) which are the most common type of bioreactors used in water resource recovery facilities across the world. Hence, the overarching goal of this study is to provide advanced understanding of biogranulation mechanism to enable industrial application of this technology. The lack of long-term stability study in CFRs has restricted its full-scale acceptability. The high settling velocity-based selection pressure has been regarded as the ultimate driving force towards biogranulation in sequential batch reactors (SBRs). In this study, this physical selection pressure was firstly weakened and then eliminated in CFRs to investigate its role in maintaining the long-term structural stability of aerobic granules. Given the fact that implementing settling velocity-based selection pressure only can cultivate biogranules in SBRs but not in completely stirred tank reactors (CSTRs), the essential role of feast/famine conditions was investigated. Seventeen sets of data collected from both literature and this study were analyzed to develop a general understanding of the granulation mechanisms. The outcome indicated that granulation is more sensitive to the feast/famine conditions than to the settling velocity-based selection pressure. The theory was tested in a CFR with 10-CSTR chambers connected in series to provide feast/famine conditions followed by a physical selector separating the slow-settling bioflocs and fast-settling biogranules into feast and famine zones, respectively. Along with successful biogranulation, the startup performance interruption problem inherent in SBRs was also resolved in this innovative design because the sludge loss due to physical washout selection was mitigated by returning bioflocs to the famine zone. Then, a cost-effective engineering strategy was put forward to promote the full-scale application of this advanced technology. With this generalized biogranulation theory, pure culture biogranules with desired functions for high value-added bioproducts were also investigated and achieved for the first time in this study, which paves a new avenue to harnessing granulation technology for intensifying waste valorization bioprocesses. / Doctor of Philosophy / Nowadays, the rapid population growth and unprecedented urbanization are overloading the capacity of many wastewater resource recovery facilities (WRRFs). Therefore, there is a need to develop a cost-effective strategy to upgrade the treatment capacity of existing WRRFs without incurring major capital investment. Because conventional activated sludge comes with loose structure and poor settleability, replacing them with dense aerobic granular sludge offers the opportunity to intensify the capacity of existing WRRF tankage and clarifiers through better retention of high bacterial mass that offers not only a fast pollutant removal rate but also a high water-solids separation rate. The aerobic granulation technology turns traditional activated sludge into granular sludge by inducing microbial cell-to-cell co-aggregation. Although this technology has been developed for more than 20 years, its application in full-scale WRRFs is still limited because majority of WRRFs are constructed with continuous flow reactors in which the aerobic granulation mechanism largely remains unknown. Besides, the long-term stability of aerobic granules in continuous flow reactors also remain unstudied, further constraining the full-scale application of the technology. The sensitivity of aerobic granulation to physical selection and biological selection was analyzed in this study. The results concluded that aerobic granulation is more sensitive to the latter but not to the former. Subsequently, this theory was tested in a novel bioreactor setup that creates feast/famine conditions for biological selection. A physical selector was installed at the end of the bioreactor to separate and return the fast- and slow- settling bioparticles to the feast and famine zones, respectively. This unique reactor design and operational strategy provided an economical approach to retrofitting current WRRFs for achieving treatment capacity upgrading without major infrastructure alternation. It also protected the bioreactor startup performance by enhancing the stability of WRRFs in the future application. Last but not least, this updated understanding of aerobic granulation theory was for the first time extrapolated to and verified with the formation of pure culture biogranules harnessed in this study for value-added bioproduct valorization from waste materials.