Human tissue kallikrein-related peptidases (KLK) are fifteen genes located on chromosome 19q13.4, encoding hormonally regulated, secreted serine proteases with trypsin/chymotrypsin-like activity. I identified expression of many KLKs in tissues throughout the female reproductive system and in cervico-vaginal fluid (CVF). The female reproductive system is hormonally regulated during the menstrual cycle, suggesting KLKs may also be regulated by these hormones. Measurement of KLKs levels in CVF and saliva samples throughout the menstrual cycle revealed a peak in expression following ovulation in both fluids. Progesterone levels rise during this period suggesting KLK regulation by progesterone during the menstrual cycle. Using proteomic techniques, I resolved the CVF proteome to identify potential KLK substrates. Among 685 proteins identified, several cell-cell adhesion molecules, cervical mucins and defense-related proteins were found. KLKs play a role in the desquamation of skin corneocytes through cleavage of cell-cell adhesion proteins. The vaginal epithelium undergoes cyclical changes during the menstrual cycle involving desquamation of cells upon rising progesterone levels. The post-ovulatory peak in KLK expression suggests that KLKs may contribute to cell desquamation during the menstrual cycle. Cervical mucus acts to block the uterus from vaginal microorganisms. Around ovulation, cervical mucus loses viscosity to facilitate sperm passage through the cervix. Proteolytic enzymes are thought to aid in this mucus remodelling. Our immunohistochemical studies localized KLK expression to the mucus secreting cervical epithelium and I investigated KLK processing of cervical mucin proteins in vitro. KLKs 5 and 12 were found to cleave mucins, suggesting their potential involvement in cervical mucus remodelling. CVF plays a role in host defense. KLKs are known to process the antimicrobial cathelicidin protein in skin and I investigated whether KLKs may also process antimicrobial proteins found in CVF. KLK5 was found to cleave defensin-1 alpha, in vitro, suggesting KLKs may aid in defense of the female reproductive system. Here I provide evidence of potential physiological roles for KLKs in the female reproductive system: in desquamation of vaginal epithelial cells, remodelling of cervical mucus and processing of antimicrobial proteins. These findings suggest KLKs may function in female fertility, in pathological conditions such as vaginitis and in host defense.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Includes bibliographical references (leaves 102-135) Also available in print.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 102-118).

No description available.

Specific Drosphila tRNA Ser4,7 plasmids were identified and isolated by hybridization with purified [¹²⁵I] tRNA Ser4,7 molecules. Seven clones were isolated carrying the Drosphila Ser tRNA Ser4,7 gene and were further characterized by restriction endonuclease digestion; agarose gel electrophoresis and hybridization with individual purified [¹²⁵I] tRNA Ser4,7 molecules. The results show that five different DNA fragments have been isolated, four which code for a single, specific isoacceptor, and one which appears to code for two different isoacceptors. Two plasmids which initially contained multiple Hind III inserts upon primary isolation were recloned to contain single Hind III inserts containing the tRNA Ser4,7 gene. One of these recloned plasmids contained a smaller tRNA Ser4,7 gene carrying insert than did its original multiple insert isolate. Small tRNA Ser4,7 gene carrying restriction fragments were labelled with T4 polynucleotide kinase and [³²P] ATP, strand separated, and electroeluted, in preparation for nucleotide sequencing. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function - proteolysis as well as nonproteolytically mediated chemotaxis.

Malaria is an infectious disease caused by Plasmodium parasites and transferred by Anopheles mosquitos. Due to Artemisinin resistance, new druggable targets identification and new drug development are urgently needed. Serine hydrolases (SHs) are one of the largest classes of enzymes having important roles in life processes. The deadliest malaria parasite, P. falciparum, encodes more than 50 SHs including proteases, lipases, esterase and others, while only several of them have been characterized. The study of uncharacterized SHs will shed light on future drug development to treat malaria. In this study, we applied chemical biology and genetic approaches to identify SHs important for the pathogenic asexual stage growth of P. falciparum parasites. We mainly focused on a depalmitoylase essential for merozoite invasion and lysophospholipases (LPLs) essential for acquiring fatty acids (FAs) from the host. Identifying essential metabolic enzymes will benefit the treatment to malaria. We focused on metabolic SHs and identified two SHs were refractory to knock out. We studied a likely essential SH named PfABHD17A, which is a human depalmitoylase homolog. PfABHD17A is localized on the rhoptry, an organelle essential for invasion. We expressed the recombinant PfABHD17A, conducted inhibitor screen and discovered that human depalmitoylase inhibitor ML211 inhibits PfABHD17A in vitro. ML211 inhibits merozoite invasion but not egress, which together with the localization of PfABHD17A on the rhoptries, suggested that PfABHD17A is essential in merozoite invasion. We also purified PfABHD17A and verified that PfABHD17A may exhibit depalmitoylase activity in vitro. LPLs are important for asexual stage parasites acquiring FAs from the host. The P. falciparum genome includes 17 putative LPLs while LPLs responsible for hydrolyzing FA from lysophosphatidylcholine (LPC) in the asexual stage are currently unknown. Using a chemical biology approach, we identified serine hydrolase inhibitor AKU-010 inhibits LPC hydrolysis effectively. Using activity-based protein profiling (ABPP) and genetic approaches, we identified that AKU-010 inhibits a series of SHs including Exported Lipases (XLs), Exported Lipases Homolog (XLH) and Plasmodium falciparum prodrug activation and resistance esterase (PfPARE). We generated a series of knockout parasite lines on the AKU-010 targets and identified that red blood cell (RBC)-localized XL2 and cytosolic XLH4 contribute to most LPC hydrolysis activity in the asexual stage. XLs and XLHs are important for parasites using LPC for growth and contribute to detoxification from accumulated LPC. XL2 and XL4 together are essential for parasite growth under high LPC concentration medium, such as human serum. XL/XLH-deficient parasites could still acquire FA from LPC, which is mainly contributed by parasite membrane- localized PfPARE. PfPARE has little impact on parasite growth and LPC metabolism with the existence of XLs and XLHs but is important after the loss of XLs and XLHs. Parasites deficient in PfPARE, XLs and XLHs have little ability to release FA from LPC and cannot use LPC as FAs source for growth. In summary, we identified metabolic SHs mediating protein depalmitoylation and lipid metabolism and in asexual stage Plasmodium falciparum, which may benefit future drug development to treat malaria. / Doctor of Philosophy / Malaria is an infectious disease caused by Plasmodium parasites and transferred by mosquitos. New druggable target identification and drug development are urgently needed to deal with the malaria issue. We focused on an understudied enzyme superfamily termed serine hydrolase (SHs), which includes more than 50 members in the deadliest malaria parasite, P. falciparum. We identified that several druggable enzymes, which can mediate protein depalmitoylation and lipid metabolism, are important for parasite growth in the pathogenic stage. Identifying essential metabolic enzymes will benefit the treatment to malaria. We screened eleven SHs and discovered that two of them are likely essential in the pathogenic stage. We focused on one human depalmitoylase homolog termed PfABHD17A. We screened the inhibitors on PfABHD17A and used the inhibitor to suggest that PfABHD17A is essential for the growth of pathogenic stage parasites. We also identified lipases important for acquiring fatty acids (FAs) from the host. Using chemical biology and genetic approaches, we discovered that three lipases are important for acquiring FAs form the host in the pathogenic stage. Inhibiting these enzymes may kill the parasite in the host.

D-serine metabolism is implicated in schizophrenia pathophysiology. This is based on reduced D-serine levels in the disorder, its ameliorative effects therapeutically and the potential genetic contributions of its metabolic enzymes, D-amino acid oxidase (DAO) and serine racemase (SRR). D-serine is a gliotransmitter and the N-methyl D-aspartate receptor (NMDAR) co-agonist. Thus, altered D-serine metabolism may contribute to NMDAR hypofunction in schizophrenia. The research in this thesis was designed to investigate D-serine metabolic enzymes further through studying their distribution, their expression in schizophrenia and their effect on NMDARs. The regional and cellular distribution of DAO and SRR in rodent and human brain were investigated using immunohistochemistry. Both enzymes were found within frontal cortex, hippocampus and cerebellum. In rodent frontal cortex, SRR expression was neuronal suggesting D-serine is not always glia-derived. In the human this was not the case, highlighting possible species differences. DAO in the rodent and human cortex was robustly detected, challenging previous views. In rodent cerebellum, both enzymes were neuronal and glial and in human, predominantly glial. In schizophrenia, DAO and SRR expression were investigated using western blotting and real-time PCR. DAO expression was elevated in the cerebellum in the disorder, without an accompanying change in SRR. In the dorso-lateral prefrontal cortex (DPFC), DAO and SRR mRNAs were unchanged in schizophrenia but SRR protein was significantly increased. The elevation in DPFC SRR protein was not replicated however in a second study. To investigate the effects of D-serine metabolic enzymes on NMDARs, an in vitro model of altered SRR expression was developed, but its use hindered through technical complications. The data detailed demonstrate new findings of DAO and SRR’s distributions in the brain and highlight novel potential roles for these enzymes. In addition, the data provide some paradoxical findings including DAO’s cortical expression. The investigations in schizophrenia lend to robust demonstrations of DAO’s elevated cerebellar expression in the disorder. However, its roles therein and that of DAO and SRR on NMDAR function remain unclear.

Dans le présent travail, le rôle de la glie dans l’expression de la douleur cancéreuse et de la douleur neuropathique a été étudié de façon comparative. Le modèle animal de douleur cancéreuse a été obtenu par injection osseuse dans le tibia, chez la rate Sprague-Dawley, de cellules de carcinome mammaire de type MRMT-1. Le modèle de douleur neuropathique a été obtenu chez le rat Wistar par ligature des nerfs spinaux L5 et L6. Les données obtenues par l’analyse du comportement douloureux en réponse à la stimulation par des filaments de von Frey ont permis de quantifier l’allodynie et l’hyperalgésie mécaniques statiques. La douleur chronique, hors stimulation nociceptive, a été mesurée à l’aide d’un test d’impotence. Les agents pharmacologiques ont été administrés par voie intrapéritonéale ou par voie intrathécale, à l’aide d’un cathéter implanté de façon chronique. L’analyse des comportements nociceptifs après stimulation par filaments de von Frey montre que l’inhibition fonctionnelle transitoire de la glie spinale par le fluorocitrate est sans effet sur la douleur dans les deux modèles. Dans les deux modèles, l’expression des réponses douloureuses dépend de l’activation des récepteurs NMDA spinaux. L’administration par voie intrathécale d’une seule dose de D-aminoacide oxydase, qui dégrade la D-sérine, co-agoniste endogène du récepteur NMDA, réduit l’allodynie et l’hyperalgésie chez les rats neuropathiques et l’allodynie chez les rats cancéreux. Les effets d’un traitement chronique par le fluoroacétate chez les rats neuropathiques sont réversés par l’administration intrathécale de D-sérine. La D-sérine altère légèrement le seuil nociceptif chez les rats cancéreux. Aucun des agents pharmacologiques utilisés ne réverse la réduction d’appui du membre lésé chez les rats cancéreux ou neuropathiques. Ces résultats montrent que, chez le rat, la douleur neuropathique comme la douleur osseuse cancéreuse dépend de la co-activation des récepteurs NMDA spinaux par un de ses ligands endogènes, la D-sérine, mais que seule la douleur neuropathique requiert une glie spinale fonctionnelle. Ils suggèrent donc un rôle différentiel de la glie dans la physiopathologie de ces deux types de douleur chronique / The present work has investigated the role of glia upon pain symptoms in a well established peripheral neuropathic pain model and a bone cancer pain model. The neuropathic pain model was obtained by right L5-L6 spinal nerve ligation in male Wistar rats. Bone cancer pain was induced by injecting MRMT-1 rat mammary gland carcinoma cells into the right tibia of Sprague-Dawley female rats. Mechanical allodynia and hyperalgesia were quantified using von Frey hairs and ambulatory incapacitance using dynamic weight bearing. Drugs were administered either acutely or chronically using osmotic pumps, through intrathecal catheters chronically implanted in experimental animals. Using von Frey hair stimuli, we found that transient inhibition of glia metabolism by intrathecal injection of fluorocitrate was ineffective in both models. In both models, pain symptoms required spinal NMDA receptor activation. Intrathecal administration of a single dose of D-aminoacid oxidase, which degrades D-serine, a co-agonist of NMDA receptors, reduced mechanical allodynia and hyperalgesia in neuropathic rats and allodynia in cancer rats. The effect of chronic fluoroacetate in neuropathic rats was reversed by acutely administered intrathecal D-serine, which had only a slight effect in cancer rats. None of these compounds altered the functional disability shown by neuropathic or cancer animals and measured by the dynamic weight bearing apparatus. These results show that neuropathic pain and cancer pains depend upon D-serine co-activation of spinal NMDA receptors but only neuropathic pain requires functional spinal cord glia in the rat. Glia may thus play different roles in the development and maintenance of chronic pain in these two situations.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.