The tristetraprolin family consists of mRNA destabilizing genes that bind to target mRNA and degrade them. In this context, TIS11 gene family is of primary interest in the spontaneous fetal loss seen in pigs. I hypothesized that the tristetraprolin family regulates these genes in this model. Higher levels of the TIS11 family transcripts in endometrium and trophoblast samples from healthy conceptuses at gd20 were found than in tissues from arresting conceptus attachment sites and significantly higher levels of TIS11 family transcripts were expressed in trophoblast samples associated with arresting conceptuses at gd50 compared to healthy endometrium and trophoblast. TIS11 proteins were expressed and localized at both maternal and fetal sides of maternal-fetal interface. These results provide a basis for understanding some aspects of gene regulation by mRNA destabilizing factors such as tristetraprolin at the maternal-fetal interface and how these factors may play a role in porcine pregnancy and fetal loss. / Department of Biomedical Sciences, NSERC, Ontario Pork, Bioniche Life Sciences Inc.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of human senile dementia. Alzheimer’s represents a significant public health concern, having widespread social and economic implications. Consequently, protocols for early detection and therapeutic intervention (both behavioral and pharmacologic) constitute important targets for medical investigation. Furthermore, contemporary research depends upon comprehensive neurobehavioral assessment and advanced statistical and computational analytic methodologies for characterizing AD-associated sensorimotor and cognitive impairment, as well as evaluating therapeutic efficacy. This dissertation introduces data mining-based techniques (decision trees, neural networks, support vector machines) for behavioral analysis in both nontransgenic and Alzheimer’s transgenic mice, to evaluate the cognitive benefits of long-term caffeine treatment. Both treatment and transgenic effects are identified through advanced statistical (discriminant analysis) and data mining approaches. In addition, a novel mouse-based cognitive assessment paradigm, adapted from a human interference learning AD-diagnostic protocol, is implemented to evaluate both genetic (GRK5) and therapeutic (GM-CSF) effects in mice, against an Alzheimer’s transgenic background. Data mining techniques are shown to be comparable to con ventional statistical analyses, often providing complementary diagnostic information. Indeed, comparisons between data mining-based and multivariate statistical analyses, with respect to groupwise discriminability, support the use of both methodologies in neurobehavioral research. Future work involving both data mining-based and multivariate statistical analyses of cognitive-behavioral data is discussed, emphasizing the need for longitudinal studies, repeated-measure designs, and spatiotemporal modeling for evaluating the time-course of both human AD and AD-like pathology in transgenic mouse models.

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24h with LPS (100ng/ml) or LPS (100ng/ml)+IFNγ (1μg/ml). Cells were subsequently stimulated with LPS or HKSa (10μg/ml) for 24h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα (3070±711pg/ml and 217±9pg/ml, respectively) and IL-6 (237±8.9pg/ml and 56.2±2.9pg/ml, p<0.05, n=3, respectively) in control cells compared to basal levels (<25pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p<0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580pg/ml, p<0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076pg/ml, p<0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470pg/ml, p<0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

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