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As an excellent organism for genetic study and with the many useful genetic manipulation tools developed in the past two decades, Drosophila melanogaster is an important model organism that has been utilized to understand the genetic regulation of various developmental processes. Here, we focus on the development of the Drosophila CNS midline cells to pursue a long term goal to understand (1) the regulation of programmed cell death during nervous system development and (2) the interaction between nervous system and the mesoderm structures during early embryonic development. Programmed cell death is an essential part of the development of the nervous system for vertebrate and invertebrate animals (reviewed by Oppenheim 1991). In the Drosophila CNS midline, about 70% of the glia cells die during a critical period. We found that the elimination of unwanted midline cells during embryonic nervous system development is a very well coordinated process, which ensures that specific number of midline glia survive. The others are relocated and executed by proteolytic cascade, followed by engulfment and further degradation by macrophages. We observed that three cell death regulatory genes (rpr, hid and grim) in the 75C1,2 region of the 3rd chromosome are all expressed in midline cells that are chosen to be executed. Using several mutants and genetic deficiencies, we found that in order for the appropriate number of midline cells to be eliminated, the functions of multiple genes in the 75C1,2 region are required. To further understand this cell death process, we targeted the expression of rpr and/or hid to midline cells. We found that the two genes show synergism in inducing ectopic cell death, further suggesting that they may functionally interact to regulate the proper cell death pattern. Inside the embryo, the development of the nervous system is not a isolated process. The developing nerve cells interact with the mesoderm during early embryonic development. We found that the CNS midline cells are required for the differentiation of a specific group of mesoderm cells, the dorsal median cell. The well conserved epidermal growth factor (EGF) signaling pathway is at least partially responsible for this CNS midline regulation of mesoderm cell development. In addition, we found that the proper anterior/posterior location of the dorsal median cells depends on the function of wingless or patched segmental polarity gene, and that these two genes might act through regulating the spatial differentiation of the CNS midline cells.

The purpose of this study was to determine if administering two painful stimuli used to assess motor responses in patients with brain injury, nail bed compression (NBC) and supraorbital pressure (SOP), had an effect on intracranial pressure (ICP), mean arterial pressure (MAP), cerebral perfusion pressure (CPP), heart rate (HR), and motor responses, in unconscious patients. Thirteen unconscious adult male and female subjects with brain injury were enrolled in the study. All subjects had normal ICP and CPP, and were hemodynamically stable. After collection of baseline values of the dependent variables, NBC and SOP were delivered on both sides of the body while ICP, MAP, CPP and HR were recorded from the bedside monitor. Subjects were recorded on video tape for motor responses to NBC and SOP and later given a motor score on the Glasgow Coma Scale. Pressures used to administer NBC and SOP were measured in psi by devices constructed for purposes of this study. Data were analyzed using MANOVA statistical procedures to detect differences within subjects on all physiological values from baseline. Findings from this study were that NBC and SOP administered on both sides of the body resulted in statistically significant increases in ICP, MAP, CPP, and HR from baseline for a brief, yet unsustained time period (p = $<$0.05). The ICP had returned to baseline in 30 seconds, the MAP and CPP within four minutes, and HR by the second minute after administration of NBC and SOP. ANOVA statistical procedure was used to detect differences in motor scores when NBC and SOP were given. There were no statistical differences between motor scores on the GCS with NBC or SOP. The mean pressures that were measured on NBC and SOP were 77.11 $\pm$ 30.98 and 86.1 $\pm$ 8.54 psi, respectively. These data suggest that NBC and SOP do have an effect on physiological indices of cerebral perfusion by an increasing ICP, MAP, CPP, and HR for a brief period of time but return to baseline quickly. Therefore, administering painful stimuli to evaluate motor responses in unconscious patients who have normal ICP and CPP is probably safe. Also, the data suggest that NBC and SOP produce similar motor responses and could both be used to assess unconscious patients.

In this study nuclear and ooplasmic maturation of prepuberal calf oocytes was evaluated to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the metaphase II stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following in vitro fertilization, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. Delayed formation of sperm aster and asynchronous pronuclear formation characterized these anomalies. Microfluorometry was used to characterize the Ca$\sp{2+}$ responses of calf oocytes to the addition of agonists. The addition of thimerosal demonstrated the presence of Ca$\sp{2+}$ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trishphosphate (InsP$\sb3),$ used to test the sensitivity of the InsP$\sb3$R, released significantly less Ca$\sp{2+}$ in calf than cow oocytes. Furthermore, injection of a porcine sperm factor elicited Ca$\sp{2+}$ release in calf oocytes, however, these responses did not exhibit the frequency or amplitude known to be characteristic of cow oocytes. These results suggested that the Ca$\sp{2+}$ content of the intracellular stores was similar, but the sensitivity of the Insp$\sb3$R may be different. The activity of histone H1 and MAP kinases, which are required for the initiation and completion of meiosis was evaluated, and the presence and maturation of the InsP$\sb3$R system. Results show that following in vitro maturation, the activity of both histone H1 and MAP kinases, as well as the relative amount of the InsP$\sb3$R protein, were substantially lower in prepuberal calf oocytes when compared to oocytes of adult cattle. Together, these findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation.

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.