The tumor-suppressor gene, TP53, plays a major role in surveillance of cellular and genomic integrity, functioning in cell cycle regulation, repair of damaged DNA, and deletion of malfunctioning or defective cells. The latter is achieved through apoptosis, a mechanism of programmed cell death, which is important in both normal morphogenesis and suppression of tumorigenesis. The loss of p53 function in breast cancer has been the subject of intensive study, however, not much is known about how p53 functions in the normal mammary gland to mediate its protective apoptotic responses. The aim of this dissertation was to examine p53-mediated cell death in the murine mammary epithelial cells in response to two distinct apoptotic stimuli: exposure to ionizing radiation and detachment from an appropriate substratum. The first component of this work examined the role p53 plays in mediating apoptosis in response to gamma-radiation during distinct stages of post-natal mammary gland. The second part of this dissertation focused on whether detachment of epithelial cells from their extracellular matrix results in cell death that is p53-dependent or -independent. Results from these experiments demonstrate a requirement for functionally active p53 in radiation-induced cell death, and show that the proliferative capacity of the mammary gland at time of radiation exposure predicts the robustness of this apoptotic response. In contrast, detachment from substratum can trigger both p53-dependent and -independent cell death in mammary epithelial cells. However, apoptosis induced by the specific abrogation of β1 integrin-ligand interaction appears to proceed through a p53-dependent mechanism.

The purpose of this study was to investigate the effects of selective isotonic muscle fatigue upon the neuromuscular coordination control mechanisms underlying ballistic limb movement in males and females. Eight males and eight females served as subjects and were tested across eight days which involved the monitoring of three inertial load conditions (L0 = no load, L1 = 1.5, L2 = 3.0 $\times$ moment of inertia) of a Class B, ballistic forearm flexion movement. Following the first three practice days, selective isotonic muscle fatigue was imposed at one of two intensities, high (6RM) or low (20RM) resistance, in either the agonist (biceps brachii) or antagonist (triceps brachii) muscle group. Exercise days involved pre and post testing of the movement task interspersed by fatiguing isotonic resistance exercise. Kinematic, temporal and quantitative integrated electromyographic pattern and strength measures were analyzed. All measures were reliable (R = 0.14-0.98). Eight days of practice generated significant decreases (p $<$ 0.05) in movement time (MVT). Males exhibited a larger decrease across days (L0 = 14.9%) as compared to females (L0 = 4.7%). Although, males generated a 25% faster MVT than females, both sexes demonstrated a similar reduction in MVT improvements as inertial loading increased. Practice effects manifested in agonist and antagonist temporal pattern measures were pronounced. Several substantial sex differences persisted following the eight days of practice. Antagonist delay (T2D) was significantly (p $<$ 0.05) shorter (32.9%) in males. Across days, males reduced this delay (13.5%) while females exhibited a large (43.2%) increase. The increase in T2D exhibited by females represents an uncoupled temporal association between the decelerating action of the antagonist and the point of maximum acceleration. This modification contributed to the comparatively small improvements in MVT displayed by females across days; explaining the MVT differential observed between the sexes. Low resistance (20RM) selective isotonic muscle fatigue generated the largest decrease in maximum isometric strength (13.6%-21.1%) with low resistance (20RM) flexion fatigue increasing MVT to the greatest extent (15.6%). Males responded more dramatically to isotonic muscle fatigue. However, comparisons of fatigue modifications in neuromuscular coordination control mechanisms between the sexes warrants caution due to the persistent sex differences observed in stabilized performance.

A recent decline in Atlantic salmon (Salmo salar) populations have led to their listing as an endangered species and is related to the contamination of rivers due to the high application rates of pesticides in agriculture. Four experiments were designed to study the effect of pesticides on several life history stages of Atlantic salmon: (i) yolk-sac larvae (YSL) were exposed for 21 d to sub-lethal levels (<100 μg l-1) of hexazinone (HEX) and atrazine (ATZ) in fresh water (FW). After a year smolts were exposed to 24 h seawater challenge (SW); (ii) smolts were exposed for 21 d to sub-lethal levels (<100 μg l-1) of HEX and ATZ in FW. After 21 d smolts were exposed to 24 h SW; (iii) YSL were exposed for 12 d to 10 μg l-1 of ATZ, HEX, chlorothalonil (CTL), and phosmet (PHO) at pH 6.5 and 5.0; and (iv) YSL were exposed to either 1 or 10 μg l-1 of ATZ, CTL, PHO and HEX or binary combinations of each. We measured the hormones involved in smolt development (cortisol, GH, IGF-I, T4 and T3) and cholinesterase activity in larvae, plasma ions (Cl-, Mg2+, Na+, Ca2+) in FW and after SW challenge, and Na+, K+-ATPase activity at both life stages. In conclusion ATZ causes ionoregulatory, growth and endocrine disturbance and reduces salinity tolerance of Atlantic salmon smolts. Plasma cortisol was affected in smolt exposed to ATZ and HEX as YSL and after a second ATZ exposure. Exposure of YSL to ATZ, HEX and low pH caused faster opercular movement, which suggests a higher energetic demand and/or a respiratory impact. Low pH causes ionoregualtory disturbance in YSL. Low pH and combinations of pesticides causes few synergistic effect and in most cases the effect was additive. Body size was smaller in YSL exposed to low pH and to a combination of pesticides. Disruption of nerve transmission was found in YSL exposed to the insecticide PHO alone or in combination with low pH, ATZ, CTL or HEX. In the wild these impacts may compromise their growth, competitive ability and predator avoidance affecting their survival and population recruitment.

Episodic acidification and its associated aluminum (Al) toxicity has been identified as a possible cause of Atlantic salmon decline in the northeastern United States including Maine where several salmon rivers are listed as endangered. During precipitation events such as snowmelts and storms, rivers and streams in this region experience episodic pulses of low pH and elevated inorganic Al which can damage the gill epithelium of fish leading to ion regulatory disturbances. To date, the impacts of episodic acid/Al on the physiology of Atlantic salmon undergoing critical life-stage transitions such as the parr-smolt transformation remain largely unknown. In this dissertation, I have used both laboratory and field studies to demonstrate that Atlantic salmon smolts are particulary vulnerable to ion regulatory disturbances during episodic acid/Al exposure. In particular, short-duration (days) exposures to acid and low levels of inorganic Al can impair the seawater tolerance of smolts in the absence of detectable impacts on freshwater ion regulation demonstrating the extreme sensitivity of the smolt hypoosmoregulatory system. I have also presented evidence that loss of seawater tolerance occurs through alterations in gill ion transporter expression, chloride cell dynamics, and several endocrine systems including the growth hormone-insulin-like growth factor I, interrenal and thyroid systems. Many of these alterations are likely involved in the upregulation of ion uptake mechanisms as part of acclimation to acid/Al in freshwater which may come as a direct cost to the ability to maintain ion homeostasis in seawater. The results presented here have important implications for salmon populations in regions affected by episodic acidification. Smolts with compromised seawater tolerance may experience delayed migration, decreased seawater preference and increased susceptibility to predation. This is likely to increase mortality during downstream migration, seawater entry, and marine residence ultimately leading to population level effects. Furthermore, these results support the idea that the freshwater experience of smolts may have a direct impact on survival in the marine environment.

In rodents, there exists a strong interaction between the reproductive and circadian systems. For this thesis the female hamster was used as a model for the study of this interaction. Studies described in chapter II investigated whether the circadian regulation of reproductive processes may be through direct input of the suprachiasmatic nucleus (SCN) to neurons containing estrogen receptor (ER) and/or to neurons containing luteinizing hormone releasing hormone (LHRH). The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was applied to the SCN and double label immunocytochemistry for PHA-L and either ER or LHRH was carried out. Both ER- and LHRH-immunoreactive cells show appositions with SCN efferents or with efferents of the subparaventricular nucleus and the retrochiasmatic area. Results suggest that the circadian system can regulate reproductive processes via input to LHRH- and/or ER-containing neurons. Studies described in chapter 111 investigated whether effects of estrogen on circadian rhythms may be exerted through estrogen-binding systems afferent to the SCN. Immunocytochemistry for ER and the retrograde tracer cholera toxin B subunit, after its application to the SCN, demonstrated that some areas contain relatively high percentages of SCN afferent neurons which show ER immunoreactivity. Retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Furthermore, using a combined retrograde and anterograde tracing technique, SCN input to some SCN afferent neurons was demonstrated. However, no evidence of reciprocity between single ER-immunoreactive cells and the SCN was found. Results indicate the existence of estrogen binding systems afferent to the SCN which might mediate the effects of gonadal steroid hormones on circadian rhythms. Studies in chapter IV analyze the effects of blockade of SCN axonal output by local unilateral application of tetrodotoxin (TTX) on the LH surge. Injections of TTX on either the morning or the afternoon of proestrus were unable to block the LH surge. Results favor the interpretation that the SCN output signal responsible of the circadian gating of the LH surge occurs before the onset of the light period on the day of proestrus.

The skeletal elements of cartilaginous fishes are composed of a thin layer of mineralized tissue, ‘prismatic cartilage’, overlaying a hyaline cartilage core. Cartilage, even with a surface layer of mineralization, is far less stiff and strong than bone. Nevertheless, several species of stingray, including Rhinoptera and Aetobatus, subsist by crushing hard-shelled mollusks and crustaceans in their cartilaginous jaws. The jaws of these stingrays are composed of a previously undescribed form of cartilage. This tissue, ‘trabecular cartilage’, has mineralized struts which run through the central hyaline core. The struts, or trabeculae, are hollow tubes, made of calcified blocks, arranged as in a brick chimney. They serve to prevent buckling and bending of the jaws while prey is being crushed. The struts are present in late term embryos, indicating that feeding on hard prey does not cause them to form. As the animal grows the struts lengthen and thicken though they do not appear to become more numerous. Trabecular cartilage appears to have evolved at the base of the clade containing the hard prey specialists. This clade also includes Manta, a planktivorous species, which retains trabecular cartilage.

One of the key aspects of the immune system is the ability to prime cells by vaccination. The γδ T lymphocytes represent a significant population of cells in the peripheral blood of cattle that have largely been ignored in this regard. Here we have explored the potential for WC1+ γδ T cells to be primed by vaccination. We hypothesize that the γδ T cells that exhibit a recall response in vitro following in vivo priming represent a unique population. Utilizing a Leptospira borgpetersenii vaccine, we examined the recall responses of WC1+ and CD4+ T cells to leptospiral antigen. The WC1+ γδ cells were the major responding population to antigen for the first few weeks following in vivo priming, with the CD4 T cell response only began to overtake them after a booster dose was administered. The primed WC1+ γδ T cells displayed a unique pattern of surface marker expression when stimulated with antigen compared to mitogen-stimulated cells, and which paralleled that observed on the CD4 T cells that responded to antigen. Additionally, chemokine receptor expression was assessed in both ex vivo and antigen-stimulated WC1 and CD4T cells. Ex vivo CD4+ and WC1+ T cells differed with regard to chemokine receptor transcript expression while the antigen-activated cells had very similar patterns of expression. Both subsets expressed genes typical of TH1-polarized cells, but differed with regard to transcripts for co-stimulatory molecules expressed. TCR usage by the antigen-responsive WC1+ γδ T cells from vaccinated animals was evaluated. The antigen-responsive cells had transcripts for several different Vγ and Vδ gene segments with very limited usage of J genes and highly variable CDR3 sequences. These results did not differ greatly from those obtained with nondividing cells or with ex vivo peripheral blood mononuclear cells suggesting little if any enrichment for a specific TCR type. These results support the hypothesis that WC1 + γδ T cells and CD4 αβ T cells that respond to leptospira antigen are likely to differ mainly with regard to how they are activated and the pathogen molecules that activate them, rather than with regard to their effector functions.

Assessments of stress typically involve either point samples (blood or saliva) reflecting a moment in time or state samples (urine of feces) reflecting several hours or a day. Currently, there is no way to assess chronic levels of stress without using repeated sampling procedures which are both time consuming, expensive, and possibly stressful. The purpose of this dissertation was three fold: (1) to develop a more chronic estimate of stress by measuring cortisol concentrations in hair, (2) to determine using this measure, how rhesus macaques responded to the prolonged stress of relocation, and (3) to determine the role of a proposed modulatory protein corticosteroid binding globulin (CBG) in measuring stress. Results demonstrate that cortisol can be quantified in hair and that stress reactivity can also be measured in hair. Rhesus monkeys responded to a major life stress (relocation) with a significant increase in cortisol in both hair and serum and with substantial behavioral changes. CBG concentrations failed to show alteration initially following relocation, however a significant increase was observed one year later. The findings demonstrate the importance of the cortisol/CBG relationship during a prolonged stress paradigm. Our data also provide further evidence that the free cortisol index (cortisol:CBG ratio) may be a better indicator of stress reactivity when compared to the commonly used serum cortisol concentrations.

The frog neuromuscular junction is a unique model that allowed us to selectively remove cellular components from the neuromuscular junction and create preparations with varying degrees of nerve terminal stability. We found further evidence that frog terminal Schwann cells communicate with their cellular partners, as terminal Schwann cells responded with changes in number or morphology as a result of changes in synaptic integrity. Terminal Schwann cells divided at synaptic sites in response to a regenerating nerve terminal. Terminal Schwann cells also had morphological changes in response to changes in status of their cellular partners; they extended processes in response to removal of the nerve terminal. Orientation and length of these processes was profoundly affected by the presence or absence of muscle fiber and nerve terminal. Similar to observations at the mammalian neuromuscular junction, terminal Schwann cells appear to play a role in reinnervation, as frequently regenerating nerve terminals were within the confines of terminal Schwann cells and their processes. I also investigated the organization of actin within preparations with varying amounts of nerve terminal stability, including developing nerve terminals and regenerating adult nerve terminals that were forming either stable or unstable connections. Previously, F-actin stained target-deprived nerve terminals in a ladder-like pattern and was concentrated in the nonrelease domains (Dunaevsky and Connor 2000). I found that β-actin was similarly distributed and localized to the nonrelease domains of nerve terminals at intact neuromuscular junctions. Further, association of actin with these particular domains appeared to be important for nerve terminal stability. As nerve terminals acquired increasing stability during development, they acquired this domain specific distribution of F-actin. Additionally, although synaptic sites with stable regenerating nerve terminal acquired this ladder-like pattern of F-actin, it was very rare for unstable regenerating nerve terminals to do so. I also tested the dynamic nature of F-actin with pharmacological perturbation. F-actin at nonrelease domains was found to be very stable. This stability of the F-actin based cytoskeleton further suggests that F-actin at the nonrelease domains of nerve terminals may play a role in the stability of motor nerve terminals.

Nutrition greatly affects the reproductive success in many insects. Also, previous studies have shown this nutrition-reproduction connection is mediated via endocrine control. In Phormia regina, a proteinaceous meal is essential for egg maturation. Recent discovery of a midgut hormone, from liver-fed P. regina, established a link between feeding and endocrine function which leads to oogenesis. This midgut hormone is the first such insect gut hormone governing oogenesis. Whether or not this hormone is released into the hemolymph, however, is not determined. To demonstrate the existence of the midgut hormone in the hemolymph, hemolymph was partially purified and its oogenesis-stimulating effect tested. A gonadotropic effect was observed in the hemolymph extracts prepared from 5 and 6h post-liver-fed females. The midgut hormone appears to circulate in the hemolymph to reach its target, presumably the brain neurosecretory cells. Injection of partially purified midgut hormone caused a significant volume increase and changes in the distribution of stainable materials of the type-A brain median neurosecretory cells (MNCs). It is conceivable that these cellular changes reflect the synthesis/release of neurohormones by the type-A MNCs. Thus, results suggested that midgut hormone is hemolymph-borne and has a cerebrotropic function. The possible role of the brain after being stimulated by midgut hormone was investigated. In vitro studies showed that the brain releases, at 8h after a protein meal, a factor(s) with a strong allatotropic effect (i.e., causing the corpus allatum (CA) to produce 6.9 times more juvenile hormones (JHs) than the control CA). The characteristics of this allatotropic factor(s) were estimated with different experiments. First, Manduca sexta allatotropin (Mas-AT)-immunopositive substances were observed in the lateral neurosecretory cells (LNCs) and the neurosecretory cells located between the pars intercerebralis and oesophagus foramen. Second, synthetic Mas-AT stimulated JH biosynthesis by the fly's CA (2.64-fold activation when stimulated with 20 pmol per test). Third, when brain-released factor(s) was neutralized with anti-Mas-AT antiserum both in vivo and in vitro, the CA still synthesized a considerable quantity of JH. This result suggested that there may be additional non-Mas-AT allatotropic agent in P. regina.