Master of Science / Department of Diagnostic Medicine and Pathobiology / Melinda J. Wilkerson / Failure of passive transfer (FPT) is one of the main reasons for increased mortality rate in newborn calves and diagnosis is dependent on determination of serum IgG concentrations (diagnosis is based on < 1 g/dL of total IgG). Several qualitative assays are available, but the reference method, single radial immunodiffusion assay (SRID), albeit quantitative measures only one subclass at a time. We set out to develop a competitive multiplex microsphere flow cytometry assay to measure bovine IgG1 and IgG2 concentrations in 30 serum samples acquired from newborn Holstein calves prior to and 24 hours after ingestion of colostrum and to compare the values with SRID. A triplex bead assay was created by mixing three distinct sets of Quantum plex carboxylated fluorescent microspheres that were coated with purified bovine IgG1, IgG2 or albumin using a two step chemical reaction. The triplex protein coated beads were reacted with a cocktail of sheep anti-bovine IgG1 and IgG2. Evaluation of analytical specificity demonstrated cross reactivity between anti-bovine IgG2 and IgG1 coated beads that precluded determination of IgG2 > 0.5 g/dL. Cross reactivity between anti-IgG1 and IgG2 coated beads was minimal and did not affect IgG1 concentrations between 0.15 to 1.2 g/dL. A competitive linear decrease in the fluorescence intensity was observed in the triplex assay when 2-fold dilutions spanning a concentration range of 12 mg/dL – 100 mg/dL of either purified bovine IgG1 or IgG2 were included as a competitive inhibitor of the reaction. Precolostral serum samples from 29 calves were determined to be < 0.4 g/dL by SRID. Standard calibrants for the flow assay were prepared from two fold serial dilutions of purified bovine IgG (stock concentration 10 g/dL) using a precolostral calf serum pool as the diluent. The standard calibrants (IgG1 was 1.0- 0.16 g/dL and IgG2 was 3.4 – 0.22 g/dL) were used as the inhibitors in a triplex assay to develop a standard curve for unknown samples. Dilutions of bovine reference serum containing known amounts of IgG1 (1.2 – 0.15 g/dL) and IgG2 (1.6 – 0.2 g/dL) was used as positive control. The intra Intra-assay and inter-assay precision of the mutiplex assay was good (coefficient of variation < 10%). Since the IgG2 concentrations of post colostral samples were below detection limit, only IgG1 values were compared to the SRID. The agreement between triplex microsphere assay and SRID for IgG1 was poor with a mean bias of 0.743 g/dL towards triplex microsphere assay (95% confidence interval of 0.382 to 1.105 g/dL). Method comparison studies between total IgG determined by SRID and the gamma-globulin fraction determined by serum electrophoresis indicated that the SRID calculated higher values than the protein method (mean bias of -1.4 g/dL, 95% confidence interval was -1.8 to -1.05 g/dL). We hypothesized that the positive bias for the microsphere assay was explained in part by the use of dilution factors, use of standards that had a low analytical range, and erroneously high standards used in the SRID method.

Master of Science / Department of Kinesiology / Craig A. Harms / Diaphragmatic fatigue is known to limit endurance performance during heavy exercise in humans. Previous reports have shown that diaphragmatic fatigue is reduced in rats with N-acetylcysteine (NAC; a nonspecific antioxidant) infusion, suggesting that oxidative stress contributes to this fatigue. However, it is not known if oral supplementation of NAC will reduce respiratory muscle fatigue during heavy exercise in humans. Therefore, the purpose of this study was to determine the effect of an acute oral dose of NAC on respiratory muscle fatigue during whole body heavy exercise. Eight healthy, non-smoking men (22+/-2 yrs), with no history of cardiovascular or lung disease, completed baseline pulmonary function tests followed by an incremental cycle VO[subscript 2peak] test. A randomized, double blind crossover design was then used where subjects were given either placebo (PLA) or NAC (1800 mg) 45 min prior to a 30 minute constant load (85% VO[subscript 2peak]) discontinuous (six-five minute stages) or continuous (cycle until volitional exhaustion) exercise test. Tests were separated by approximately one week. Maximum pressures (inspiratory, PImax; expiratory, PEmax) and venous blood samples (plasma lactate and total plasma glutathione) were made prior to- and following each 5-min of exercise in discontinuous tests and pre- and post-exercise in continuous tests. Subject's VO[subscript 2peak] was 43+/-5 ml/kg/min. There was no difference (p>0.05) in PImax between NAC (127.9+/-34.1 cmH[subscript2]O) or PLA (134.1+/-28.1 cmH2O) at rest. During exercise, PImax was significantly lower ([similar to]14%) in 6 of 8 subjects with PLA compared to NAC at minutes 25 and 30 of the discontinuous test indicating respiratory muscle fatigue. With NAC, PImax did not change (p>0.05) from rest throughout exercise indicating no respiratory muscle fatigue. There was no difference (p>0.05) in PEmax, plasma glutathione, lactate, oxygen uptake (VO[subscript 2]), ventilation (VE), heart rate (HR), or rating of perceived exertion between PLA and NAC at rest or during exercise. Time to exhaustion was not different (p>0.05) during the continuous tests (PLA: 1263 + 334 sec; NAC: 1047 + 136 sec). These results suggest that an acute dose of NAC reduces respiratory muscle fatigue during high intensity exercise but does not alter other ventilatory or metabolic indices. The significance of this reduced respiratory muscle fatigue with NAC on whole body exercise performance remains to be determined.

The genetic stability of the rabies glycoprotein (G) expression cassette of AdRG1.3 was examined using both in vitro and in vivo models. For the in vitro study, the AdRG1.3 vaccine was serially passaged 20 times in 293 cell culture and from the twentieth passage a total of 67 AdRG1.3 virus clones were obtained. The G gene expression cassette (including an SV40 polyadenylation signal sequence) along with flanking human adenovirus type 5 sequences were amplified from these clones using PCR to generate an amplified product approximately 2.25 kb long. These products were then purified and subjected to DNA sequence analysis. No changes were observed in the 1870 nucleotide sequence window (containing both the G gene (1572 nt) and the SV40 polyA sequence (132 nt)) of any of the 67 clones. Findings show that the G gene insert is stably expressed in a conformationally appropriate form by the recombinant HAd5 vector. The genetic stability of the G gene cassette of AdRG1.3 was also evaluated upon in vivo passage using five independent series of cotton rats (Sigmodon hispidus). From the fifth in vivo passage of AdRG1.3, a total of 105 virus clones were obtained from these five independent series of animals. The complete G gene expression cassette was amplified from these clones by PCR and sequenced as for the in vitro study; no base changes were observed in the targeted 1870 nucleotide sequence window of any of these clones. Therefore, these results show that the G gene expression cassette of AdRG1.3 remains stable within the adenovirus vector upon passage of the vaccine both in cell culture and in animals. (Abstract shortened by UMI.)

acase@tulane.edu

This dissertation is a report on a study of the elastic properties of the human mandible. Using a pulse transmission technique, it was determined that the elastic stiffness 76 specimens, from various locations in the ramus and corpus of four mandibles material exhibited symmetry properties which were characteristics of anisotropic material In order to facilitate an understanding of the coordinate system used in determining the elastic stiffnesses of the specimens, a simplistic model can be employed. Assume that a section of the human mandible is projected point-by-point on to a straight line with the left wisdom tooth at one extreme, and the right wisdom tooth at the other Now consider this projection of the mandible to approximate a right circular cylinder. The x$\sb3$-direction is defined to be along the length of the cylinder, axial direction. The circumferential direction is designated by x$\sb2$ and the radial by x$\sb1$ The similarity between mandibular and femoral bone is quite remarkable. Femoral bone is slightly more dense and stiff, but the similarities far outweigh the differences. The average density of mandibular bone is 1799 kg/m$\sp3$ 95% of that of femoral bone. The average stiffness coefficient for mandibular bone is 96% of that of femoral bone. It would appear from this comparison that the mandible is in its elastic properties very much like a long bone bent in the shape of a parabola. The average technical constants for mandibular and femoral bone show a distinct parallelism It was determined from a study of the human mandible by region that the lingual region, surface in the proximity of the tongue, is most dense and stiff while the buccal, surface nearest the cheek, is the least stiff. Similarly, it was found that average specimens from mandibles with teeth were denser and stiffer than those from mandibles without teeth. (Abstract shortened with permission of author.) / acase@tulane.edu

<p> Substantial economic losses occur in the swine industry during periods of high ambient temperatures. Heat stress produces physiological changes such as increased body temperature and respiration rate resulting in production losses from decreased reproductive performance, growth rate and feed intake. Heat stress in growing gilts delays puberty and decreases ovarian follicle numbers. In boars heat stress decreases semen quality. Electronically-controlled floor cooling pads were designed and constructed to assist pigs with thermoregulation by removing excess heat from pigs in a production facility. Based on this study, experiments were conducted to further investigate the effects of electronically-controlled cooling pads on physiological and reproductive performances in gilts and boars. A study was conducted on limit-fed gilts at 32°C and 35°C during short-term heat stress. Gilts exposed to short term heat stress at 32°C and 35°C had increased respiration rate, vaginal temperature and skin temperature. Gilts on electronically-controlled cooling pads exposed to short term heat stress at 35°C were able to minimize negative impacts of HS such as reduced respiration rate and vaginal temperature. A study was conducted with 24 boars which were exposed to cyclical heat stress for a duration of 3 days at 32°C and 35°C. Boars exposed to cyclical heat stress for 3 consecutive days at 32°C or 35°C which increased respiration rate and body temperature followed by a decrease in semen quality over several weeks. Boars cooled with electronically-controlled floor cooling pads had reduced physiological effects of heat stress as well as consistent semen quality post HS. The use of electronically controlled floor cooling pads have implications towards minimizing or removing the negative impacts of heat stress in gilts and boars. </p>

Since the production of the first sheep by somatic cell nuclear transfer a great deal of effort has been made to improve efficiency and to understand nuclear reprogramming mechanisms. Unfortunately efficiency remains low, and nuclear reprogramming mechanisms remain uncharacterized. The objectives of this research were to identify factors associated with somatic cell nuclear transfer efficiency and to analyze the transcriptome of blastocyst-stage clone and control embryos and cotyledonary tissue in an effort to elucidate mechanisms responsible for the low developmental efficiency and high post-implantation losses. The experiments reported here identify factors including oocyte source and timing of activation following nuclear transfer that yield improved efficiencies. It was determined the use of cow oocytes for somatic cell nuclear transfer results in improved in vitro development and increased pregnancy rates. These data further indicate prolonged exposure of the donor nucleus to pre-activated oocyte cytoplasm results in increased nuclear fragmentation and reduced developmental efficiency in vitro. Several aberrantly expressed genes were identified in nuclear transfer blastocysts and cotyledons that could impact cloning efficiency. Major histocompatibility complex I and down-regulator of transcription 1 were overexpressed in nuclear transfer blastocysts, and retinol binding protein 1 was overexpressed in nuclear transfer cotyledons. The functions of these genes in immune response, transcriptional regulation, and retinol binding and transport make them attractive candidates for further nuclear transfer research. Expression levels of six developmentally important genes were analyzed in various stages of preimplantation nuclear transfer embryos by real-time polymerase chain reaction to determine the timing of nuclear reprogramming following nuclear transfer. Five of the six genes were aberrantly expressed multiple developmental stages, however by the blastocyst stage only one gene was aberrantly expressed. These data indicate reprogramming is delayed in nuclear transfer embryos resulting in over- or under-expression of developmentally important genes during early embryogenesis. These experiments report factors associated with improved nuclear transfer efficiency; provide insight into potential mechanisms for low developmental rates, abnormal placentation, and fetal loss of clones; and characterize the timing of nuclear reprogramming following somatic cell nuclear transfer.

No description available.

No description available.

No description available.