The asymmetric current previously identified using voltage probe techniques in maturing Xenopus laevis oocytes has been further investigated using non-invasive ionselective microelectrodes. Three-dimensional fluxes of hydrogen (H+), potassium (K+), and bicarbonate ([special characters omitted]), of varying magnitudes, were found to be hidden within this net 10 pmol current. These fluxes were seen to vary with respect to developmental stage and presence of surrounding follicular tissue. The mechanisms of H + and K+ transport were also examined with P and V-type ATPase pump inhibitors, ortho-vanadate and N-ethyl maleimide (NEM), respectively. A representative flux pattern of H+, K+, and [special characters omitted] ions around fully developed Xenopus oocytes was obtained from this research. H+ effluxes, as well as K+ influxes and effluxes, were detected at the animal, vegetal, and equatorial regions of the membrane. The transport of these two ions was seen to be highly correlated in defolliculated oocytes, with large H+ effluxes accompanied by large K+ influxes. Small effluxes of [special characters omitted] were measured at the animal and vegetal hemispheres. The use of ATPase pump inhibitors also provided information regarding possible transport mechanisms. Vanadate (0.01 mM) and NEM (1 mM) significantly reduced the H+ flux in defolliculated and follicle-enclosed oocytes, respectively. However, the observed K+ fluxes were unaffected by these inhibitors. The combination of these results suggests that ATPase pumps are important only in the transport of H+ ions and more specifically, the presence of a P-type ATPase on the surface of the oocyte membrane and a V-type ATPase in the follicular tissue. The technique of measuring extracellular fluxes from developing cells with ion-selective electrodes was also examined. Construction of more efficient sampling rules, determination of electrode efficiencies using these sampling rules, and calibration of the carbonate electrode were some of the advances resulting from this research. In addition, the implementation of a multiple head ion probe provided simultaneous data collection of more than one ion type from the same oocyte, allowing for the study of ion-ion interactions and the determination of bicarbonate flux Patterns with H+ and carbonate electrodes.

Eccentric muscle contractions induce ultrastructural disruption, prolonged loss of maximal strength (MVC) and range of motion (ROM), increase in muscle soreness (SOR) and muscle proteins, and secondary damage by inflammation. The overall purpose of this dissertation research was to further examine the relationship between blood glutathione (GSH) levels and the blood creatine kinase (CK) response to eccentric exercise, and the inflammatory response, specifically neutrophil function. Study I examined the effect of resting GSH levels on CK activity after eccentric muscle contractions, and Study II examined the effect of α-lipoic acid (LA) supplementation on resting GSH levels in plasma. Study III examined the effect of eccentric muscle contractions on neutrophil function by examining gene expression of neutrophils after eccentric muscle contractions to determine which genes are up-regulated or down-regulated during initiation of inflammation. It was concluded from Study I that subjects in the low GSH group experienced less muscle damage than subjects in the high GSH group, and these changes for the low GSH group were consistent with less secondary damage due to inflammation. Study II concluded that LA supplementation for 4 weeks may not be enough to increase GSH levels in low GSH group. Study III concluded that neutrophils initiate several processes including inflammation, cytoskeletal regulation, transcription, and metabolism in response to eccentric muscle contractions with the evidence of up- or down-regulation of genes.

Adoptive transfer has been an important tool for the study of immune responses in mice. In vivo demonstrations and in some cases the discovery of the nature and role of cell populations and subpopulations in the immune responses of this animal have often depended on the use of some version of adoptive transfer. Because lines of syngeneic animals are essential for the systematic use of adoptive transfer techniques, mice and rats are the only animal models in which this system has been used. Genetic barriers prevent its use in the study of human immunology and, until recently, in studies of the immunology of animals of agricultural interest. The advent of cloning has made it possible to derive cloned lines of cattle (Cibelli et al., 1998). As members of the same clone demonstrate complete histocompatibility, it should be possible to transfer cells from immunized to unimmunized members of the same clone. During the course of this study we were able to show that by means of adoptive transfer it was possible to confer the ability to mount secondary responses upon primary immunization from an immunized donor to another member of the clone via the blood transfusion. Furthermore, we were able to adoptively transfer increased immune responsiveness from immunologically mature primed adult members of the clone to young immunologically immature recipients. All these data point to the fact that after primary immunization all recipients generated an antigen-specific antibody response with the characteristics of a secondary immune response, indicating the active functioning of memory cell pools transferred from the donors. We showed that antigen-specific polyclonal responses could be transferred by means of a single blood transfusion, which indicated that using blood transfusion it is possible to transfer multiple clones of antigen-specific B cells. Finally, by use of a medically relevant antigen for donor immunization, we were able to demonstrate the suitability of this technique for the adoptive transfer of antibody responses of biomedical and biodefense interest. We were able to show the kinetics of antibody generation and class switching as well as polyclonality of the humoral response formed in recipients. These results demonstrated that the advent of cloning has brought to experimental bovine immunology access to the investigational power of adoptive transfer and other approaches that require the transfer of cell populations from manipulated donors to recipients. I believe that the use of adoptive transfer for the study of B-cell sponsored responses will invite the extension of this approach to in vivo study of areas such as bovine regulatory T cells and other cell dependent phenomena that are particularly well-studied by adoptive transfer.

Fertilization initiates a series of intracellular calcium [Ca 2+]i oscillations in the oocyte that promote its exit from and completion of meiosis. In mouse oocytes, the [Ca2+] i oscillations arrest at the pronuclear stage and this is accompanied by a down-regulation in the inositol 1,4,5-trisphosphate receptor type 1 protein (IP3R-1). The goal of this dissertation was first, to investigate the signaling pathway responsible for IP3R-1 degradation during fertilization. Eggs were either incubated in or injected with several compounds that induce egg activation, and the IP3R-1 protein content evaluated using Western blot analysis. Exposure to ethanol or ionomycin, which induce a single [Ca 2+]i rise, failed to induce down-regulation of the IP3R-1. However, injection of porcine sperm factor (pSF), which presumably stimulates IP3 production, or adenophostin A, an IP3R-1 agonist, both induced degradation of the receptor. Exposure to thimerosal, that modulates the IP3R without stimulating IP3 production, also initiated down-regulation. [Ca 2+]i oscillations induced by SrCl2, which does not trigger IP3 production, failed to evoke down-regulation. Degradation of the IP3R-1 appears to be mediated by the proteasome pathway because it was inhibited by lactacystin, a specific proteasome inhibitor. It was therefore propose that persistent stimulation of the phosphoinositide pathway by sperm during fertilization leads to down-regulation of the IP3R-1. The second goal of this research was to investigate the factor(s) involved in the arrest of [Ca2+]i oscillations seen at PN formation. This was achieved by generating parthenotes with similar IP3R-1 numbers and similar kinase activity at three different stages of the cell cycle, metaphase II (MII), pronuclear (PN), and the post pronuclear (PPN) stages. These models allowed us to study the effect of different parameters on PN [Ca2+]i arrest: (1) Cell cycle, (2) IP3R-1 number and sensitivity, and (3) content of intracellular Ca2+ stores. Results from these experiments demonstrate that the cell cycle determines the ability of the egg to exhibit [Ca2+] i oscillations by controlling the decrease in sensitivity of the IP3R-1 at the PN stage. This desensitization appears to be due to the inability of the IP3R-1 to undergo a conformational change and this phenomenon seems to be regulated by presently unknown factors.

The objective of this dissertation was to identify and characterize diversity within the expressed primary immunoglobulin heavy chain (IgH) repertoire in cattle. Determinations relied heavily upon different comparative analysis strategies focussing on both germline and expressed IgH gene segment sequences. We determined that the early fetal expressed IgH repertoire is constituted by as few as 2–3 VH genes and a single JH gene (multiple D). VH gene use increases with age, though IgH expression is restricted to members of a single VH family and primarily one JH gene (>300 sequences analyzed). All isolated germline VH genes also belong to a single VH family, corresponding to that in the expressed repertoire. Therefore, germline-encoded VH (and JH) sequence polymorphism is low, making limited contributions to overall IgH sequence diversity. In sharp contrast, bovine CDR3 regions exhibit extremely high levels of heterogeneity both in terms o f sequence and hypervariable lengths. A major fraction of IgH diversification occurs after rearrangement, most likely via untemplated somatic hypermutation. Nucleotide substitutions within the JH-Cμ intron, which does not support gene conversion due to a lack of known donor sequences, were consistent with USH on multiple levels, including hotspot targeting, the ratio of transitions to transversions, preferential nucleotide substitutions and potential strand bias. Sequence diversity levels varied with time among immunologically relevant tissues. Early fetal spleen and late fetal ileum appear to be two important sites of B cell diversification during their respective developmental stages. Patterns of IgH expression suggest that spleen is the early site of substantial gene rearrangement, and source of B cell emigrants, which subsequently populate other peripheral tissues including liver, ileum, and bone marrow.

Energy restriction has a high failure rate, which may be related to energy regulating hormones (acylated ghrelin, insulin, etc.) that stimulates energy intake (EI) and suppresses energy expenditure (EE). An alternative is to maintain EI and raise EE through exercise. The impact of short-term exercise training on energy regulating hormones has not been systematically studied. In addition, women do not lose body fat with exercise training, whereas men do lose body fat. The sex difference may be related to energy regulating hormones. The purposes of this proposal were, (1) to determine the independent effects of exercise with and without energy replaced on energy regulating hormones (acylated ghrelin, insulin, etc.) and appetite responses, and (2) to determine whether sex differences exists. Concentrations and area under the curve (AUC) of these hormones were measured in overweight/obese individuals (9M, 9W) during a meal tolerance test after a 1-day baseline. Using a counter-balanced, crossover design, measurements were repeated after 4 consecutive days of exercise (EE = ∼750 kcal/d-1 for men, ∼600 kcal/d-1 for women) with exercise energy added back to maintain energy balance (BAL) and, 4 consecutive days of the exercise without energy added back to maintain energy deficit (DEF). In men, acylated ghrelin AUC was not different between baseline, DEF, and BAL. In men, compared to baseline insulin AUC was lower after DEF (p<0.05), but not BAL. In women, compared to baseline acylated ghrelin AUC was higher after DEF and BAL (p<0.05). Women vs. men had a greater change from in acylated ghrelin after DEF and BAL. In women, compared to baseline insulin AUC was lower after DEF and lower after BAL (p<0.05). Appetite ratings were lower after BAL compared to baseline or DEF in men. In women, there was no change in appetite ratings. In women short-term exercise training regardless of energy state affects energy regulating hormones and appetite in the directions expected to stimulate energy intake and suppress energy expenditure. In men however, exercise alone has no impact on energy regulating hormones or appetite when energy balance was maintained and the effect of energy deficit was less pronounced than in women.

The ability to traverse barriers of high water velocity limits the distributions of many diadromous and other migratory fish species, and is central to effective fishway design. This dissertation provides a detailed analysis of volitional sprinting behavior of six migratory fish species (American shad Alosa sapidissima, alewife A. pseudoharengus, blueback herring A. aestivalis, striped bass Morone saxatilis , walleye Stizostedion vitreum, and white sucker Catostomus commersoni), against controlled water velocities of 1.5–4.5 m · s−1 in a large, open-channel flume. In Chapter 1, I develop models of maximum distance traversed ( Dmax) by fish ascending these flows, accounting for water velocity and other covariate effects. I then demonstrate the application of these models, using them to predict proportions of active migrants capable of traversing a range of distances and flow velocities. Chapter 2 focuses on behavior and swimming performance of American shad, analyzing covariate effects on attempt rate as well as Dmax, and formalizing how rate and distance jointly affect overall rates of passage. Models describe a complex pattern of varying responses of attempt rate and Dmax to hydraulics, temperature, effort expended on and recovery time since the previous attempt. In Chapter 3, I use the effect of swimming speed on fatigue time to calculate an optimal swimming speed that maximizes the over-ground distance fish can traverse, and hence defines their maximum ability to traverse velocity barriers. This speed reduces to a constant groundspeed within a given gait, regardless of the speed of flow. Data from all six species support this view, although only American shad exhibit a clear shift from the optimum prolonged speed to the optimum sprint speed at the predicted critical flow velocity. Throughout this dissertation I make extensive and novel use of statistical techniques developed for survival analysis to analyze and model behavioral data, both with respect to attempt rate and to D max. Chapter 4 provides an overview of these methods and demonstrates their application to a fish passage study of downstream-migrating Atlantic salmon (Salmo salar) smolts. An understanding of the principles described here will help the reader to better understand the findings of the previous three chapters.

This study was designed to assess the energetic cost of upstream migration in American shad (Alosa sapidissima) and to examine physiological changes during migration that relate to swimming performance or energetic efficiency. Overall total stored energy expenditure ranged from 35-60% during upstream migration. Migrating American shad preferentially use energy stores (lipid and protein) in some tissues, such as the skin and its sub-dermal fat layer (depleted by 63%), while sparing other tissue stores such as red muscle protein. American shad generally increased the activity of aerobic and energy mobilization enzymes as much as 60%, while decreasing the activity of anaerobic enzymes as much as 80% during upstream migration. There was a generalized reversal of these enzyme changes seen during migration at the most upriver site sampled. It is suggested that American shad may be able to metabolically prepare for migration prior to its onset and cessation. The data demonstrate that fish migrating in the middle of the migratory period possessed higher (5-42%) total stored energy content than fish migrating early or late in the season, primarily due to elevated lipid in the white muscle and the sub-dermal fat layer. American shad demonstrate a spleen-controlled increase in available blood hemoglobin (22%) and hematocrit likely resulting in increased oxygen carrying capacity during upstream migration. Active and standard metabolic rates of American shad, determined by respirometry, were intermediate between salmonids and fast-swimming perciforms. Active metabolic rate was logarithmically related to swimming speed (r$\sp2$ = 0.26; slope = 0.2) and tailbeat frequency (r$\sp2$ = 0.36; slope = 0.002). Directly determined standard metabolic rate was 71-198 $\rm mgO\sb2kg\sp{-1}h\sp{-1}.$ The energetically optimal swimming speed was 1.45 $\pm$ 0.51 body lengths per second. Using the data from the swimming respirometer, an empirical model of the Cabot Station fish ladder in Turners Falls, MA was constructed which suggests that the impact of the fish ladder on migration is highly dependent on passage time. This study demonstrates that American shad are equipped with a variety of mechanisms for increasing energetic efficiency during upstream migration and highlights the importance of short-term physiological adaption to migration and the ultimate success of an iteroparous, anadromous fish.

Brachymeria intermedia is a primary parasitoid of pupae of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae). Previous studies established how females determine the acceptability of potential hosts through contact with their cuticular kairomones. This work investigates how olfactory cues initially may lead females to their hosts. Inexperienced females were less likely to walk toward the host than females with oviposition experience; however, a single antennal contact with the host was sufficient to increase the probability of walking to the host suggesting that females learned the odor of their host on the first encounter (Chapter 1). Olfactory conditioning was then demonstrated using a novel odor (Chapter 2). Through a single oviposition experience on their natural host in presence of vanilla odor, wasps were induced to drum and drill in a vanilla-scented paper roll. Although some wasps were conditioned when exposure to odor coincided with pre-oviposition drumming on the host, conditioning was most successful when odor exposure coincided with oviposition. Evidently, conditioning occurred through the formation of an association between the odor and the 'aroused' state underlying host acceptance. Results support the hypothesis that conditioning occurs through a stimulus-arousal association rather than, as is generally assumed, through a stimulus-stimulus association. Wasps were also conditioned to fly toward a source of vanilla odor in a wind tunnel (Chapter 3). Then, upon approaching vanilla-scented paper rolls hung on a vertical cylinder, conditioned wasps landed on them readily, whereas few wasps landed on real pupae. However, more wasps reached pupae or pupal cases than white scented paper rolls. Thus, visual and olfactory cues appeared to mediate the foraging behavior of wasps in conflicting ways. Conditioned wasps flying upwind, along a plume of vanilla odor flew shallow zigzag tracks (Chapter 4). Contrary to male moths flying to sex pheromone, wasps flew similar zigzag tracks along ribbon and turbulent plumes of vanilla odor. When the plume was removed while wasps were flying upwind, wasps either maintained an upwind course, or drifted sideways, flying alternately upwind and downwind before turning around and flying downwind. No wasp casted upon plume loss, as is typical of male moths.

The osmoregulatory physiology of American shad, Alosa sapidissima, was investigated. Tolerance to full strength seawater developed at the larval-juvenile transition (45 d post-hatch) three months prior to seaward migration. Increased seawater tolerance was associated with gill development, proliferation of chloride cells and increased gill Na$\sp+$,K$\sp+$-ATPase activity. Shad lose the ability to osmoregulate in fresh water during autumnal migration (a possible cue) evidenced by declines in plasma chloride (20%) observed in wild juveniles. Gill Na$\sp+$,K$\sp+$-ATPase activity increases during this period. These changes were observed in the laboratory under natural conditions. Plasma chloride dropped 68% and gill Na$\sp+$,K$\sp+$-ATPase activity increased three-fold. Decreased plasma chloride was associated with increased mortality. Chloride cells (on both the primary filament and secondary lamellae in fresh water) increased during autumn as temperature declined. Changes in physiology and chloride cells are delayed and of a lower magnitude when shad in fresh water were held at constant (24$\sp\circ$C) temperature. In seawater, chloride cells on the secondary lamellae (likely ion uptake cells) declined to less than 2% of fresh water levels. Chloride cells (in both fresh and seawater acclimated shad) were shown to be rich in mitochondria and Na$\sp+$,K$\sp+$-ATPase by developing a technique for using specific fluorescent dyes in fixed tissue. In order to differentiate between the mechanisms of branchial ion uptake and excretion, radioligand binding methods were used to quantify Na$\sp+$,K$\sp+$,2Cl$\sp-$ cotransporters in the gill tissue of Atlantic salmon (Salmo salar). However, no high affinity binding was measured. The Na$\sp+$,K$\sp+$,2Cl$\sp-$ cotransporter was immuno-histochemically colocalized with Na$\sp+$,K$\sp+$-ATPase to chloride cells in fresh water and seawater acclimated shad. Western blot analysis was used to characterize a 170-190 kDa protein (Na$\sp+$,K$\sp+$,2Cl$\sp-$ cotransporter) present in greater quantities in seawater acclimated shad and Atlantic salmon than in fresh water acclimated fish. The presence of the cotransporter in the chloride cells of seawater acclimated shad supports the accepted model of ion excretion but the immunolocalization of the cotransporter in chloride cells of fresh water acclimated American shad is unexpected and remains poorly understood.