LD2668 .T4 1962 L44

INTRODUCTION: Traumatic brain injury (TBI) is a major public health problem, causing approximately 52,000 deaths from 1.7 million injuries in the United States annually, with a combined direct and indirect economic cost estimated at $60-75 billion per year. Traumatic subarachnoid hemorrhage (tSAH), a subtype of closed head injury, has a high prevalence within TBI—evident in up to two-thirds of moderately and severely brain injured patients. tSAH is also associated with poor clinical outcomes; some research suggests mortality and unfavorable outcome rates are two-to-three times higher in patients with tSAH, based on brain imaging, compared to those without. To date, no pharmacological treatment has been conclusively shown to improve outcomes in humans for either moderate or severe TBI or for specific tSAH injury. The aim of this study was to assess whether the effect of PROG was substantially different in study TBI patients with evidence of tSAH on initial brain imaging compared to those that did not have evidence of tSAH. METHODS: ProTECT III clinical trial data was used for an exploratory, post hoc subgroup analysis to determine the effect of the hormone progesterone (PROG) on outcome. Study subjects with any abnormality on baseline brain imaging were included in the analysis and two subgroups, tSAH positive (+tSAH) and tSAH negative (–tSAH), were selected. The primary outcome evaluated was a favorable/unfavorable dichotomy derived from the 6-months post-injury Extended Glasgow Outcome Scale (GOSE) assessment, which evaluates both mortality and functional outcomes. Risk ratios (RRs) were calculated for the total sample and each of the two subgroups and used as statistical evidence for interaction between PROG and tSAH. RESULTS: All subjects from the original ProTECT III trial cohort (N=882) with no abnormalities found on baseline computed tomography (CT) image (n=125) or missing image (n=1) were excluded from this analysis. Subjects with one or more abnormalities noted on CT (+CT, n=756) were then divided into subgroups based on presence (n=582) or absence (n=174) of tSAH. Subjects with +tSAH were more severely injured than –tSAH (mean Rotterdam CT score 3.3 vs. 2.2; 3.1 overall) and had a lesser proportion of favorable outcomes (47.4% vs. 74.3%; 53.6% overall). Compared to placebo, patients treated with progesterone had marginally better likelihood of favorable outcomes (risk ratio among +tSAH 1.06, 95% confidence interval [CI], 0.89 to 1.26; and RR among –tSAH 1.02, 95% CI 0.85 to 1.22). A multivariable model, adjusted for baseline differences in treatment group covariates did not yield substantially different results for the effect of progesterone on favorable outcomes (+tSAH 1.07; 95% confidence interval [CI], 0.84 to 1.36, –tSAH 1.08; 95% CI 0.75 to 1.56, +CT 1.06; 95% CI 0.87 to 1.29). CONCLUSION: Our study demonstrated that progesterone did not result in different effects in patients with or without tSAH than those without based on initial brain imaging. This investigation supports previous research findings; tSAH is correlated with more severe injury and worsened outcomes. Concomitant injuries found in +tSAH group are likely worsening the outcomes over –tSAH, but this was not evaluated here. More complex statistical modeling should be used on this data to determine if it provides evidence that tSAH is an independent prognosticator of unfavorable outcome or merely associated with more severely injured patients.

Introduction: The human endometrium has a remarkable capacity for efficient cyclical repair following the inflammatory process of menstruation. Defective postmenstrual repair may contribute to the common complaint of heavy menstrual bleeding (HMB). The mechanisms and factors involved in endometrial repair are still to be fully elucidated. Endometrial function is governed by the ovarian hormones and pre-menstrually progesterone levels decline as the corpus luteum regresses. Consequently, the synthesis of prostaglandins (PG) is increased, namely PGE2 and the potent vasoconstrictor PGF2α. Subsequent vasoconstriction of endometrial spiral arterioles is believed to result in a transient hypoxic episode in the upper endometrial layer. Therefore, the aims of this thesis were to determine (i) the endometrial expression of putative repair factors across the menstrual cycle (ii) the regulation of these factors by hypoxia, PGE2 and PGF2α (ii) the role of hypoxia inducible factor (HIF)-1α in endometrial repair and (iii) differences in endometrium from women with objectively measured HMB (>80ml) and normal controls (<80ml). Methods/Results: Putative repair factors, with known angiogenic, mitogenic and proliferative functions, were identified in human endometrial samples by quantitative reverse transcription PCR and immunohistochemistry. Interleulin-8 (IL-8), vascular endothelial growth factor (VEGF), adrenomedullin (AM), connective tissue growth factor (CTGF) and endothelin-1 (ET-1) were all maximally expressed during the menstrual and/or proliferative phases of the cycle, consistent with the onset of endometrial repair. Endometrial cells and tissue explants treated with 100nM PGE2/F2α and/or hypoxia (0.5% O2) revealed up-regulation of IL-8, VEGF, AM and CTGF. An in vitro progesterone antagonism model revealed that progesterone withdrawal, hypoxia and prostaglandins are all necessary for significant increases in repair factor expression in endometrial tissue. HIF-1α was detected in human endometrium but exclusively in the late-secretory and menstrual phases. Using shorthairpin RNA against HIF-1α, it was determined that hypoxia up-regulated these factors via HIF-1α, whereas PGF2α acted in a HIF-1α independent manner to increase repair factor expression. Finally, whole genome array analysis was performed on menstrual endometrium from women with objectively measured heavy and normal menstrual bleeding to provide an unbiased comparison of gene expression. 259 transcripts displayed significant changes between the two groups. Five candidate genes were validated using Q-RT-PCR. Bioinformatic analysis of the differentially expressed gene set identified bioprocesses that included positive regulation of biological and cellular processes, leukocyte differentiation, regulation of apoptosis and response to stress/hypoxia. The presence of HIF-1α protein was examined in menstrual endometrial tissue nuclear protein extracts by Western blot, revealing significantly decreased levels in women with HMB versus normal controls. Furthermore, the mRNA expression of known target genes of HIF-1α (VEGF, CXCR4) was also significantly decreased in these women. The functional impact of endometrial HIF-1α was assessed using an in vitro angiogenic assay. Silencing of HIF-1α in endometrial cells significantly reduced the angiogenic potential of culture supernatants when compared to untransfected cells or cells transfected with a scrambled sequence. Conclusions: Repair factors are significantly increased in the human endometrium following the onset of menstruation. Progesterone withdrawal, hypoxia via HIF-1α and prostaglandins appear necessary for the regulation of these factors at this time. Menstrual endometrium displays significant differences in gene expression and HIF- 1α protein levels between women with HMB and normal controls. The findings of this thesis contribute to the existing literature on both the physiological process of endometrial repair and the pathogenesis of HMB. Extension of this work may allow the identification of novel therapeutic targets for the treatment of this common, debilitating condition.

Both estradiol and progeterone are proposed autocrine or paracrine regulators of ovarian function in primate species. However, specific receptors for these steroids have not been localized to individual compartments of the primate ovary. Using immunocytochemical techniques, estradiol receptors were detected in the germinal epithelium, but not other structures, of ovaries obtained from rhesus or cynomolgus monkeys during the follicular and luteal phases of the menstrual cycle. In contrast, progesterone receptors were present in stromal and interstitial tissue, the thecal layers of healthy and atretic follicles, as well as the functional corpus luteum. These results are consistent with the concept of a receptor-mediated role for progesterone, but not estrogen, within the predominant gametogenic and endocrine structures, e.g., the follicle and corpus luteum, of the primate ovary. The recent discovery of distinct cell types in the corpus luteum of domestic ungulates has revised concepts on the control of luteal function in these species. Studies were designed to test the hypothesis that the primate corpus luteum consists of cell subpopulations that differ in physical characteristics, function and regulation. Cells enzymatically-dispersed from the monkey corpus luteum at mid-luteal phase of the menstrual cycle differed in size (diameter) and the presence of the steroidogenic enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD). Analysis of dispersed cells for forward and 90° light scatter properties by flow cytometry revealed two distinct continua (Cα and Cβ). These continua were isolated using the sorting capabilities of the flow cytometer. Cα contained single cells of ≤ 15 μm and cell clusters; the cells were typically 3β-HSD-negative nonsteroidogenic. Cβ consisted of single cells that increased in size up to 40 μm and were 3β-HSD-positive. Cβ was divided into two regions (R₁ and R₃) and the cells isolated. R₁ cells were ≤ 15 μm whereas R₃ cells were ≥ 20 μm. Basal progesterone and estrogen production by R₃ cells was greater than that produced by R₁ cells (as determined by radioimmunoassay of the incubation media). Relative stimulation of progesterone production by hCG, cAMP or PGE₂ was not different between R₁ and R₃ luteal cells. These results support the hypothesis that the primate corpus luteum consists of distinct cell subpopulations which differ in size and steroidogenic capacity. However, the cell types which secrete progesterone are typically responsive to gonadotropin and PGE₂, possibly via a cAMP-mediated pathway.

An estrus synchronization trial was conducted to determine the efficacy of adding an injection of gonadotropin releasing hormone (GnRH) at initiation of the controlled intravaginal drug releasing device (CIDR®) progesterone synchronization protocol in heifers. Nulliparous (n=121) beef heifers were randomly assigned to one of two treatment groups. All heifers received a CIDR® implant at the initiation of the breeding season. Half of the heifers (Select Synch) received an injection of GnRH. Heifers in the Select Synch treatment group had a lower numerical response (76.7% versus 88.3%) to treatment (detected in heat) and an overall lower artificial conception rate (46.0% versus 53.3%), but no statistical difference was detected. Days to conception and artificial insemination conception rates for both groups were similar for all heifers inseminated. Three hundred multiparous Hereford, crossbred and composite beef cows were assigned to one of two breeding groups (Early and Late) based on calving date and randomly assigned to receive an injection of GnRH at the time of CIDR® insertion (Select Synch). The addition of GnRH did not impact the percentage of cows detected in estrus or days to conception. Conception rates were not affected by the addition of GnRH (Select Synch), however cows in the early breeding group were more likely to become pregnant (58% versus 45%) by artificial insemination (P<0.02). An experiment evaluated the efficacy of the CIDR® protocol to synchronize estrus in Arizona Holstein dairy cows (n=696). Cows assigned to the CIDR® protocol (n=337) received a CIDR® insert at the end of the voluntary waiting period (55 days). CIDR®s were removed and an injection of prostaglandin was administered seven days after insertion. There was no difference due to CIDR® treatment in number of services per conception or first service conception rate. CIDR® treatment reduced days to first service, days open at first service, and days open (P<0.02). Warm season had a deleterious effect on number of services, days to first service, first service conception rate and days open (P<0.0001). In summary, estrus synchronization improved postpartum reproductive performance; however, thermal stress continues to be a major barrier to reproductive efficiency.

A thesis submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. / In the past 25 years, a large volume of data was collected on steroid hormone receptors and their role in the action mechanisms of steroid hormones. Efforts were made to use the principles, derived from fundamental investigations into the actual function of steroid hormone receptors in target tissues, (e.g. breast and uterine tissues) to provide a comprehensive understanding of the nature of hormone responsive tumours (e.g. breast tumours) and to formulate successful endocrine treatment regimes for e.g. breast cancer patients. Although some measure of success was achieved with antihormonal drugs like tamoxifen, clomiphene, medroxyprogesterone acetate, etc. a statistical analysis of patient response data has revealed disappointingly low median response times for breast cancer patients. A number of reasons can be offered in explanation of the discrepancies between theory and practice, however, it was felt that the two most obvious ones are the lack of a proper animal model, closer to man on the species scale, and insufficient understanding of the actual in vivo mechanisms of steroid hormone action. For this very reason it was decided to launch an in depth investigation into the properties and functions of steroid hormone receptors in the biological action of steroid hormones, especially the sex steroid hormone receptors, like the oestrogen and progesterone receptors. Furthermore, it was decided to employ a nonhuman primate, Cercopithecus aethiops pygerythrus, commonly called the Vervet monkey, as the animal model, instead of the more common Sprague-Dawley rat. Since the field of investigation defined is both broad and deep, it is obvious that this thesis could not contain all the answers. So, in view of the obvious limitation in time and man-power, the prime object for this thesis was defined as the laying of sound foundations for future research on the topic specified above. Thus, it was decided to concentrate on the molecular properties of the uterine oestrogen and progesterone receptors of the Vervet monkey. The basic arsenal of biochemical technology used in protein chemistry (competitive protein binding assays, sucrose density gradient analysis, size exclusion chromatography, ion exchange chromatography, chromatofocussing, isoelectric focussing) and available in the Institute of Life Sciences, Faculty of Medicine, University of Pretoria at the time of this investigation, was employed. In view of the information generated in the course of the study, attention was focussed upon a small, but important aspect of the mechanism of steroid hormone action, namely the process of activation or tranformation of receptor- ligand complexes and their interaction with the nuclear compartment. Since disturbance(s) in these processes in abnormal uterine tissues may exist, a limited study of the oestrogen and progesterone receptors in human uterine tissues was undertaken, because no abnormal Vervet monkey uterine tissues were available. Unfortunately it was also extremely difficult to obtain an abundance of pathological human uterine tissues necessary for an adequate study. The investigator consequently had to be content to study only a small number of pathological uterine specimens. Thus the latter part of this thesis only skims the surface of what could have been a major fait accompli with possible clinical significance. / WHSLYP2017

Thesis advisor: Charles Hoffman / The study of mammalian cAMP signaling has often been confounded by the fact that ten different genes encode adenylyl cyclases (ACs) that produce cAMP from ATP and 16 different genes encode phosphodiesterases (PDEs) that hydrolyze cAMP to AMP. In this study, mammalian AC cDNAs were cloned and integrated into strains of the fission yeast Schizosaccharomyces pombe that lack their endogenous AC to determine the basal activity of all ten AC isoforms. In addition, response to the stimulatory mammalian Gsα was determined by co-expression of a mutationally-activated form of the human GNAS1 gene. AC activity was assessed using an fbp1-GFP reporter that is repressed by cAMP production and PKA activity. Results confirm that all ten isoforms have detectable basal activity, and AC1-9 definitively respond to Gsα stimulation. When matched with a sufficiently potent mammalian phosphodiesterase (PDE), strains expressing mammalian ACs make good candidates for small molecule high throughput screening (HTS) to detect AC inhibitors. A 100,000 compound screen was recently performed to detect AC and Gsα inhibitors as well as PDE activators. A promising “hit” was progesterone, which has been previously suggested to inhibit ACs in Xenopus. Initial results suggest that progesterone inhibits AC1 and the closely-related AC3. These data demonstrate the utility of using S. pombe as an effective platform for identifying inhibitors of both basal and GNAS1-stimulated AC activity. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

The hormone withdrawal hypothesis of postpartum depression (PPD) attributes the onset of depressive symptoms to the rapid postpartum withdrawal of the ovarian hormones estradiol and progesterone that occurs during the first five days following childbirth. Although a number of human and non-human animal studies have supported the hormone withdrawal hypothesis, several studies have failed to support this hypothesis. The current research was designed to test the hormone withdrawal hypothesis of PPD using a novel translational research design that includes a series of experimental animal studies and a longitudinal human study. It was hypothesized that estradiol and progesterone withdrawal would cause increased behavioral despair, anhedonia, and anxiety in the rodent studies. In the human study, it was hypothesized that 1) decreases in estradiol would be associated with increases in negative affect and decreases in positive affect; 2) decreases in progesterone would be associated with increases in anxiety; and 3) these associations would be stronger in women with a past episode of PPD compared to those without a history of PPD. In the animal studies, rats were ovariectomized and administered ovarian hormones or placebo (i.e., hormone administration), followed by placebo only (i.e., withdrawal). Animals in these experiments were given the forced-swim test to measure behavioral despair; lateral hypothalamic self- stimulation to measure anhedonia; or the elevated plus maze to measure anxiety. In the human study, women made mood ratings and collected saliva samples daily starting in the third trimester and continuing until 10 days postpartum. In the animal studies, withdrawal from estradiol alone was associated with behavioral despair (t=2.26, p=.02) and anhedonia (t=-3.2, p=.007). In the human study, there a significant prospective association between estradiol and negative affect in women who developed PPD (r=-0.34, p<.001). This association, when combined with group status (i.e., history of PPD versus no history of depression), was used to correctly identify 100% of women who developed PPD. The results of this project contribute to evidence of a neurobiological basis for PPD. Estradiol withdrawal represents a promising candidate for further study, particularly with regard to individual differences in sensitivity to hormone withdrawal.

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Due to the thesis embargo no abstract is provided.