Experiments were conducted to determine whether endogenous progesterone regulates synthesis and/or secretion of luteal oxytocin (OT). In experiment 1, mature ewes (n=5 per group) were assigned randomly to control or mifepristone (RU 486) treatment groups. Ewes were injected twice daily s.c. with vehicle or 10 mg RU 486 from days 5-7 of the estrous cycle (estrus=day 0). On day 8, following an i.v. prostaglandin F₂α (250 μg cloprostenol) challenge, venous samples were collected at frequent intervals to determine plasma OT concentrations. Plasma OT in RU 486-treated animals did not differ significantly from those of the control animals (P>0.05). In Experiment 2, ewes were injected s.c. daily with vehicle or 175 mg RU 486 from days 2-5 of the estrous cycle followed by a prostaglandin F₂α (250 μg cloprostenol) challenge on day 6. Four of five RU 486-treated ewes exhibited "split-estrus" (estrous behavior through 36 hours and again 84 to 108 hours after the onset of initial estrus). There was no significant difference in mean plasma OT or progesterone levels between treatment groups (P>0.05). Mean mature corpus luteum (CL) weights of control and RU 486-treated ewes on day 6 did not differ (394.8 ± 28.8 vs. 319.5 ± 48.3 mg; P>0.05). Mifepristone-treated ewes contained mature CL, new CL (2 of 4 ewes), and/or preovulatory follicles (≥ 10 mm, 2 of 4 ewes). Average interestrous interval for RU 486-treated ewes was 9 days longer than that of control animals (26.2 ± 2.9 vs. 17 ± 0.5 days; P<0.025). A subsequent study was conducted to determine the effects of gonadotropin-releasing hormone (GnRH)-stimulated release of luteinizing hormone (LH) on luteal OT and progesterone production in beef heifers. Ten heifers with normal estrous cycles were assigned randomly in equal numbers to a control and treatment group. On day 2 of the estrous cycle (estrus=day 0) heifers were injected with either physiological saline or 100 pg GnRH every 4 hours for 56 hours. Samples were collected 0 min pre- and 180 min post-GnRH challenge for progesterone analysis. Sixty hours after the initial injection of GnRH or saline, heifers were challenged with an i.v. injection of 500 pg prostagland in F₂α (cloprostenol) and blood was collected at frequent intervals for OT analysis. Luteal OT synthesis was suppressed (P<0.01) in heifers receiving repeated injections of GnRH compared to saline-treated control animals. Progesterone secretion was significantly greater in saline-treated animals compared to GnRH-treated animals pre- and post-challenge (1.0 ± 0.06 vs. 0.93 ± 0.11 ng/ml and 1.16 ± 0.05 vs. 0.96 ± 0.13 ng/ml, respectively; P<0.05). / Graduation date: 2003

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The type VII secretion system is a protein export pathway linked to diverse phenotypes in both Actinobacteria and Firmicutes. The Actinobacterial subtype of the T7SS, referred to as T7SSa, has been shown to play a critical role in various aspects of Mycobacterial life including virulence, conjugation, and metal homeostasis. The T7SSb of Firmicutes bacteria on the other hand has similarly been shown to influence virulence but by the direct growth inhibition of competitor bacteria. Structure-function analyses of the T7SSa apparatus as well as various effectors and chaperones have begun to build a more mechanistic understanding of how T7SSa functions. In contrast, we know little of how the T7SSb functions despite its noted importance to both pathogens and environmental bacteria such as Bacillus, Staphylococcus, Enterococcus, and Streptococcus. During my thesis work, I have addressed several gaps in our understanding of T7SSb function. The three major questions that I have studied are: (1) how do T7SSb immunity proteins inhibit the toxicity of their cognate toxins, (2) how does the T7SSb export effectors through the thick Gram-positive cell wall, and (3) what is the role of chaperone proteins in facilitating T7SSb effector export? / Thesis / Doctor of Philosophy (PhD) / Bacteria require space and various nutrients to survive and grow and must therefore compete against other bacteria for access to these resources. To gain advantage over their competitors, many bacteria have developed molecular weapons that target and kill other closely related bacteria. Some of these weapons take the form of protein secretion machines that export antibacterial toxins. Gram-positive bacteria use the type VIIb secretion system (T7SSb) to inhibit the growth of other Gram-positive bacteria. In this work, I explore several aspects of T7SSb including: (1) how toxins are inhibited by immunity proteins, (2) how toxins are secreted through the cell envelope, and (3) how toxins are recognized by the secretion apparatus. The goal of this work is to better understand how T7SSb functions at the molecular level.

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Colon cancer occurs in over 150,000 men and women in the United States each year and is fatal about one third of the time. There are many well characterized genetic transformations which commonly occur during colon carcinogenesis, including mutations in the Adenometous Polyposis Coli (APC), Kirsten RAS (K-RAS) and p53 genes among many others. There are also many alterations in gene and protein expression which are not yet completely elucidated. We have identified kallikrein 6 (KLK6) as a gene whose expression is dependent on cancer associated proteins such as K-RAS, SRC and caveolin-1 (CAV-1). KLK6 is a member of the kallikrein protein family which consists of 15 secreted serine proteases. Like other types of proteases, kallikreins have been demonstrated to play a role in cancer progression and they hold promise as potential cancer biomarkers. The up-regulation of KLK6 was first identified by performing a microarray analysis on a mutant K-RAS transfected Caco2 cells. The activated K-RAS transfected cells expressed significantly more KLK6 than the mock transfected controls. Pathways downstream of K-RAS were found to induce KLK6 gene expression, including the p42/44 MAPK and the PI3-K/AKT pathways. Caveolae, plasma membrane associated structures, and their principle protein component, CAV-1, positively influence both KLK6 gene expression and KLK6 protein secretion in HCT116 colon cancer derived cells. Finally, it is shown that KLK6 increases the invasive potential of cells through laminin and Matrigel. Because KLK6 plays a role in cancer progression it may serves as a novel therapeutic target. Additionally, KLK6 holds potential for use as a serum biomarker for multiple types of cancer, including colon cancer for colon and other types of cancer.

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The type II secretion system (T2SS) is widespread in Gram-negative bacteria that cause disease in animals and plants. In human and animal pathogens toxins are secreted (e.g. cholera toxin) and in plant pathogens lytic enzymes that breakdown the plant cell wall are exported in to the extracellular milieu (e.g. pectate lyase). Structurally the T2SS comprises at least 11 core proteins that form three major subassemblies spanning the inner-membrane, periplasmic space and outer-membrane: (i) the inner-membrane platform and associated cytoplasmic ATPase (E); (ii) the pseudopilus, which consists of five pseudopilins, G to K; and (iii) a large, pore-forming outer-membrane complex secretin D. The inner-membrane platform comprises three single transmembrane helix proteins, and one three transmembrane helix protein, OutF. The evidence from cryo-electron microscopy on the related type IVa pilus machine (T4PS) places the protein corresponding to OutF at the centre of this platform. This platform is responsible for assembling the pilus and for communicating between the periplasm and the cytoplasmic ATPase. To date, no high-resolution structure of a full-length OutF/PilC family protein is available. A low-resolution electron microscopy reconstruction of isolated PilG (PilC ortholog from Neisseria meningitides T4PS) showed a tetrameric two lobed structure. Here I report the results of studying the structure of the inner-membrane protein OutF from Dickeya dadantii and the complete inner-membrane platform comprising 9 proteins: OutEFGHIJKLM. This work involved cloning the corresponding operon, purifying the proteins, and using crystallography and electron microscopy. Key results reported here are the crystal structure of the first cytoplasmic domain of Dickeya dadantii, OutF65-172 and a preliminary three-dimensional model of the Dickeya dadantii inner-membrane platform. This model, and higher-resolution models to come, will provide valuable information about the oligomeric state, and arrangement of the inner-membrane proteins. These studies will help us to understand how the type II secretion system works.