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Serine protease inhibitors (serpins) are a large superfamily of proteins that regulate critical proteolytic pathways by inhibiting serine proteases. Tick-encoded serpins are thought to play a vital role in the feeding process. To determine the relationship of Amblyomma americanum serpin 6 (S6) to tick feeding regulation, this study attempted to define the biological significance of this molecule through transcription and protein expression profiles, biochemical characterization of recombinant s6 (rS6), and the effects of in vivo post-transcriptional gene silencing on blood meal acquisition and fecundity. Transcriptional analysis revealed that S6 mRNA is ubiquitously expressed in unfed and partially fed ticks through the initial 5 days of the feeding period. S6 mRNA abundance in dissected tick organs showed a 3.7, 3.4, and 1.7- fold upregulation from 24 h to 96 h in the salivary gland (SG), midgut (MG) and the carcass (CA) remnant after removal of SG, MG respectively before downregulating at 120 h. Native S6 protein is downregulated in response to tick feeding, with correlation between transcription and protein expression profiles only consistent from the unfed to 48 h. Similarly, S6 protein expression in dissected female tick tissues is reduced as feeding progresses, with S6 being identified in SG, MG, ovary (OV), and CA from 24 h until 72 h. Biochemical characterization of S6 was not achieved, as rS6 did not form an irreversible complex when incubated with chymotrypsin or trypsin. Although complete silencing of S6 and S6/S17 mRNA was achieved, post-transcriptional gene knockdown had no effect on tick feeding efficiency or fecundity. These findings have been discussed in regards to the development of a vaccine against A. americanum and necessary future studies have been suggested for further characterization and assessment of biological significance.

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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.

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.

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.