Glycogen storage diseases (GSD) are inherited metabolic disorders that affect glycogen use and storage. People with GSD Ia lack the enzyme glucose-6-phosphatase (G6Pase). As a result, these people are unable to convert liver glycogen to free glucose and develop severe hypoglycemia. Patients with GSD also develop growth retardation, hepatomegaly, renomegaly, hypertriglyceridemia, hypercholesterolemia, and hyperlactacidemia. No cure for GSD Ia currently exists. Patients are treated symptomatically with repeated naso-gastric feedings and glucose infusions to maintain normal blood glucose concentrations. Despite treatment, the underlying enzymatic defect remains. Gene therapy holds the promise of correcting this metabolic defect, thus providing a true cure for GSD Ia. Gene therapy uses modified virus particles to deliver a replacement functional G6Pase gene to the patientâs liver. Our group is using two viral vectors, adeno-associated virus (AAV) and helper-dependent adenovirus (HDAd), for gene therapy in dogs with an inheritable form of GSD Ia. We have treated three GSD Ia dogs with the AAV vector and two GSD Ia dogs with the HDAd vector. Vector-treated dogs were able to maintain normal blood glucose concentrations and unlike their untreated counterparts, survived for several years. These promising results provide hope that gene therapy may emerge as an effective treatment for people with GSD Ia.

A series of experiments were conducted to determine physiologic mechanisms of mucosal repair in the ischemia-injured intestine. The first experiment (Chapter III) investigated the contributory role of individual Cl- channels in the recovery of barrier function in ischemia-injured porcine ileum. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (TER) were measured in response to PGE2 and pharmacologic inhibitors of epithelial Cl- channels. Overall, results from these studies demonstrate that ClC-2-mediated intestinal Cl- secretion restores TER in ischemia-injured intestine. Chapter IV entails a study aimed at more directly investigating the role of ClC-2 in mucosal repair by evaluating mucosal repair in ischemia-injured intestinal mucosa mounted on Ussing chambers treated with the selective ClC-2 agonist, lubiprostone. Results from this suggest that activation of ClC-2 with the selective agonist, lubiprostone, stimulated elevations in TER and reduction in mannitol flux in the Ischemia-injured intestine. In Chapter V, experiments focused on the role of individual NHE isoforms in the recovery of barrier function in ischemia-injured porcine ileum. Results from this study demonstrate that inhibition of NHE2 activity, possibly via EBP50, induces recovery of barrier function in ischemic-injured intestine

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is persistent in the environment and the food chain resulting in a chronic exposure to human populations. The effects of this known human carcinogen are evaluated by measuring various TCDD-inducible endpoints. Cytochrome P450 1B1, a recently identified TCDD responsive gene, shows inducibility in both the rat and humans that is similar to Cytochrome P450 1A1, a widely accepted marker for TCDD responsiveness. In order to evaluate a Sprague Dawley rat model to predict human tissue effects with easily accessible human tissue, such as peripheral blood lymphocytes (PBLs), rat PBL and granulocyte CYP1B1 and CYP1A1 expression were compared to expression among several tissues. Two treatment groups were used: an acute group, dosed with a 540 ng/kg single daily dose over a 3-day period followed by a day 4 sacrifice, and a chronic group, dosed biweekly with 7.6 ng/kg/day for 15 weeks. Competitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to show the different levels of expression of CYP1B1 and CYP1A1 in resting PBLs, granulocytes and various other tissues. CYP1B1 and CYP1A1 were shown to be inducible in liver, adrenal gland, lung, kidney, granulocytes and in PBLs. However, a lower induction for PBLs and granulocytes was observed compared to the highly inducible tissues such as liver and lung. Furthermore, male rats showed a 200-fold induction in CYP1B1 expression in PBLs and 16-fold induction in granulocytes in the chronic group following TCDD exposure compared to a 2 fold induction in PBLs and 2-fold induction in granulocytes for the female rats of the same dosing group. The differences in inducibility between PBLs, granulocytes and tissue may be due to the different levels of TCDD that reach each tissue due to the pharmacokinetics dependent upon that tissue. With the different expression patterns and low copy number of CYP1B1 and CYP1A1 in PBLs and granulocytes in Sprague Dawley rats, their use as surrogates for expression in tissues of interest does not correlate with recent published data for human studies on PBLs exposed to TCDD. Therefore, the comparison needs to be further investigated and the use of PBLs better established as a surrogate for expression.

The regulation of intestinal barrier function is of importance in various clinical situations. For example, in cases of ischemia/reperfusion injury, there is a disruption in the gastrointestinal barrier. This may lead to release of pro-inflammatory cytokines and neutrophil (PMN) infiltration. It has been demonstrated that COX-2, which is upregulated during inflammation, and its downstream product PGE2 are important for recovery of barrier function, yet their regulation has not been fully characterized. Study 1: To study the effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation to ischemic-injured porcine ileal mucosa. Since COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5x10⁶ PMNs to ischemia-injured ileal mucosa significantly enhanced transepithelial resistance (TER), an effect inhibited by the selective COX-2 inhibitor NS-398 and an IL-1beta receptor antagonist. Western blots revealed up-regulation of COX-2 in response to PMNs, which was inhibited by an IL-1beta receptor antagonist. Real time PCR revealed increased mRNA COX-2 expression, which preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2. Study 2: Mitogen activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli, including IL-1beta. The three primary MAPK signaling pathways are the extracellular regulated kinases (ERK 1&2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Because COX-2 expression is regulated in part by MAPK?s, we postulated that MAPK pathways would play an integral role in recovery of porcine ischemia-injured ileal mucosa. Treatment of tissues with the p38 MAPK inhibitor SB-203580 or the ERK 1&2 inhibitor, PD-98059, abolished recovery. Western blots revealed that SB-203580 inhibited upregulation of COX-2 whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER by SB-203580 or PD-98059 was overcome by exogenous PGE2. The JNK inhibitor, SP-600125, significantly increased TER and resulted in COX-2 upregulation. Thus, COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways respectively. Alternatively, ERK 1&2 appears to be involved in COX-2-independent reparative events. Study 3: In previous studies, we had shown that PGE2 restored barrier function via a signal transduction pathway involving Cl^- secretion and recovery of interepithelial tight junctions (TJs). To study these mechanisms, we utilized human colonic T84 cells. We postulated that PGE2 induced chloride secretion would precede increases in TER associated with re-distribution of critical proteins to TJs. T84 cells were grown to confluence, but utilized at lower TER values (200-500 ohms.cm²) to simulate our ileal mucosal model of ?leaky? restituted epithelium. Basolateral application of PGE2 induced transient increases in Isc, indicative of chloride secretion, followed by sustained increases in TER. Basolateral application of the Na/K/2Cl cotransporter inhibitor bumetanide, abolished the PGE2-induced rise in Isc and subsequent elevations in TER. PGE2 induced a shift in claudin-1 from the Triton-X soluble to insoluble fraction, beginning 4-hour after PGE2 administration, which was prevented by bumetanide. Alternatively, there were no changes in occludin or claudins-3 and -5. Immunoflourescence demonstrated that PGE2 increased accumulation of claudin-1 at the apical lateral membrane. Additionally, we showed that PGE2 increased tyrosine phosphorylation of claudin-1 within 30 min, an effect prevented by bumetanide. Therefore, PGE2-induced chloride secretion in T84 cells is directly linked with increases in TER, and these elevations in TER are associated with phosphorylation of claudin-1 and a shift in claudin-1 to the tight junction.

The purpose of this research has been to determine whether an extended estrogen response element half-site (ERRE) contributes to the differential estrogen responses of the human and mouse lactoferrin estrogen response element (ERE) in the context of their natural promoters. This research utilized molecular biology techniques to evaluate gene activation. Transfections of MCF-7 cells showed that liganded ER-alpha activates transcription of the human lactoferrin ERE 4-fold higher than the mouse lactoferrin ERE in the context of their natural promoters. Since the ERRE of the human lactoferrin gene naturally occurs 18 bp upstream from the ERE and is absent in the mouse lactoferrin gene promoter, we created a chimeric mouse lactoferrin CAT reporter, which now encodes the ERRE in the identical location as in the human lactoferrin gene. The addition of the ERRE in the mouse lactoferrin gene rendered this reporter extremely responsive to estrogen stimulation. We also demonstrated that the conformation of the estrogen receptor bound to the ERE alone or in the presence of ERRE differed and that the ERRE influenced the selectivity of coactivators in liganded ER-alpha-mediated transcriptional activity. Like the lactoferrin gene ERE, most known natural estrogen response elements are imperfect palindromes that differ from the consensus by at least a 1 base pair change and confer different levels of ER transcriptional activation compared to the consensus ERE. In contrast to our transient transfection data showing a lower estrogen response of the mouse lactoferrin ERE compared to the human lactoferrin ERE in the context of their natural promoters, in vivo data showed that the gene is robustly transcribed in response of estrogen in both species. Therefore, this research model can be applied to studies of genes that have different hormone responses in vivo versus in vitro in an attempt to identify non-typical estrogen response elements that influence ER-alpha-mediated transactivation.

<p> When examining hatchability of eggs, the two main factors that contribute are fertility and embryonic mortality (EM). A common commercial observation across different turkey lines was that early embryonic mortality (EEM) appeared to be greater in young hens than in older hens. One point that complicates this issue is the ability to discriminate between EEM and infertile eggs. Therefore, there has always been a question about the accuracy of the reported number of infertile eggs. A study was designed to examine the hatch residue from turkey flocks of the same strain at two different ages. One flock was in its first two weeks of egg production and the second flock had been in production for at least 12 weeks. Six hatches with approximately 56,000 eggs total for all hatches combined were examined by macroscopic breakout. There were no differences in fertility or hatchability between the two different hen ages. EEM was greater in younger hens than in older hens. EM occurring during the last week of incubation was greater in older hens than in younger hens. A significant negative correlation was found between fertility and EEM in young hens suggesting that practices that could improve fertility may also minimize EEM. The difference between the two hen ages provided a model to further examine embryonic mortality in turkeys. Preliminary results from commercial resources indicated lower EM after hens were inseminated with higher numbers of sperm cells. It was hypothesized that sperm binding might differ between hen ages and that increasing the number of sperm in the insemination might compensate for lower binding. A study was designed to examine the sperm penetration (SP) of hens at two stages of egg production: the beginning of egg production and wk 12 of egg production. Using both in vivo and in vitro SP assays, the results indicated a larger number of SP holes in the perivitelline layer of the young hens when compared to older hens. The similarity between the in vivo and in vitro assays indicated a sperm binding effect independent of oviduct influences such as sperm storage and release rate. Another study was conducted to examine the female influence on SP and EEM in turkeys. Eggs from females of two different lines were examined at the two different ages examined in the previous studies. Females were mated to a single sire for the duration of the study to examine IPVL SP in the absence of sperm competition among different males. Eggs were used for either in vivo SP analysis or incubated for fertility, hatchability and EM data. Hens inseminated by a singe sire performed differently between the two age periods as demonstrated by a hen by period interaction. The response was inconsistent whereas some hens had increased SP holes, some decreased and some did not change between the two different age periods. Hens in the upper (HI) and lower (LO) third of the flock population based on SP holes were pooled in analyzing sperm hole effects on embryo viability. No differences in fertility or wk 1 mortality were detected between HI and LO hens. However, HI hens had higher hatchability and lower wk 4 EM compared to LO hens. In conclusion, the results of the current study demonstrated a significant female effect on SP. Furthermore, this effect appears to be due to unknown differences in IPVL properties as well as differences due to sperm storage and release rate from the sperm storage tubules. More research is needed to understand how the number of sperm cells relates to both early and late EM. These studies indicate that neither a high nor a low SP number, but that an intermediate number might result in optimal embryo survival.<P>

<p><p>POOLPERM, PARIWAT.  Factors Influencing Semen Quality and Fertilityin Boars. (Under the direction of Drs.Glen W. Almond and William L. Flowers)<p>        The objectives of this researchwere 1) determine the influence of antibiotics on semen quality, 2) determinethe association among insulin-like growth factor I (IGF-I) in seminal plasma,semen quality, and subsequent fertility, and 3) retrospectively study theassociation among semen parameters and sow fertility.  In the firststudy, the effects of gentamicin (GM), amikacin (AM), neomycin sulfate(NM), and penicillin-streptomycin (PS) in semen extender on the percentagesof motile (MOT), morphologically normal sperm (MOR), and sperm with normalacrosome (NAR) were examined.  An in vitro penetration assay was conductedusing sperm cells on day 0 and day 5 of storage.  GM and NM groupsshowed higher (p<0.05) MOT than other groups after 5 days of storage. No differences in penetration rate were found among treatments; however,the penetration rate decreased (p<0.05) on day 5 of storage.<br>        In the second study, ejaculateswere collected and diluted in an extender (Vital?).  Gilts (n=113)and sows (n=375) were inseminated with the extended semen in homogenetic-homospermicregimens.  Farrowing rate (FR), total pigs born (TB) and born alive(TBA) were recorded.  IGF-I was determined in seminal plasma by radioimmunoassay. Concentration of IGF-I from 204 ejaculates was 95.38 ± 3.56 ng/ml (mean± SEM) and total amount of IGF-I/ejaculate was 23.50 ± 1.20 µg.  SeminalIGF-I differed (p<0.05) among genetic lines and had no effect (p>0.05)on MOT, MOR and NAR.  However, IGF-I was associated (p<0.05) withsemen volume, sperm concentration and total number of sperm/ejaculate. No association between IGF-I level in seminal plasma and fertility indiceswas found.<br>        The third study determinedthe associations among insemination parameters with subsequent fertilityof boar semen using data from the second study.  MOR was associated(p<0.05) with fertility parameters.  TB and TBA were associatedwith age of semen at the first insemination (SAGE) and number of spermper insemination dose (AIDOSE).  With stepwise regression analysis,it was evident that FR was associated with semen volume, MOR and SAGE. Meanwhile, TB and TBA were associated with SAGE, AIDOSE and total numberof spermatozoa/ejaculate.  In conclusion, the assessment of semencharacteristics may not necessarily delineate fertility between boars.<P>

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 147-165). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Maximal oxygen uptake (VO2 max) is the best criterion measure for aerobic fitness and the prescription of exercise intensity for programs designed to enhance cardiorespiratory fitness. There are two ways of obtaining VO2 max: maximal tests, which require subjects to exercise to the point of volitional exhaustion and provide the most accurate measure; and submaximal tests, which are less physically strenuous but have lower accuracy. A popular submaximal protocol is the YMCA bike test. Steady state heart rate (HR) is measured at multiple submaximal workloads and extrapolated to the subject's estimated maximal HR (220-age). The VO2 corresponding to the estimated maximal HR is accepted as the estimated VO2 max. The accuracy of this submaximal testing protocol effects the ability to estimate a subject's actual aerobic capacity. To help better investigate the YMCA protocol, submaximal measures (HR, VO2) were utilized at specific workloads in an attempt to improve the accuracy of the prediction. The standard YMCA protocol was completed and then extended to actual maximal exertion. Submaximal measures (HR, VO2, etc.) were used to develop a regression equation predicting VO2 max. T-tests were used to compare VO2 data between protocols. Multiple regression analyses were performed to generate regression equations to enhance the accuracy of VO2 max estimations from the YMCA submaximal protocol. Results were considered to have no significant difference in the new regression equation and the actual measured VO2 max. Because submaximal measures (HR, VO2) could not be utilized to improve the accuracy of the prediction of the YMCA protocol, the original purpose was deemphasized and redirected. Considering the apparent utility of anthropometric measures in estimating VO2 max, this study sought to improve the accuracy of the YMCA protocol by adding anthropometric measures (BMI, Skinfolds) to develop two separate regression models. Results were significantly different (p < 0.05) between Measured V02 (MV02) and V02 estimated from the YMCA protocol (YV02). Additionally, results were significantly different (p = 0.003) between the Houston nonexercise test and MVO2. In conclusion, although a significant correlation resulted between MVO2 and YVO2, it was not stronger than other submaximal estimations. Also, it was not a strong predictor because of a significant difference between the Houston nonexercise test and MVO2. Therefore, by adding BMI and Skinfolds to the popular YMCA formula, r-values were increased (r = 0.817 and r = 0.822) and can therefore better estimate a subject's VO2 max versus solely using graphic plots of steady state HR responses at protocol-determined workloads.

Maximum oxygen consumption (VC^max) represents the highest rate at which oxygen can be consumed and utilized to produce energy sustaining aerobic activity. V02max is regarded as the gold standard for assessing aerobic fitness and is essential for prescribing appropriate exercise intensities. Therefore, accurate determination of VC>2max is vital. Usually, VC>2max is obtained when an individual reaches volitional exhaustion during a Graded Exercise Test (GXT). Previous studies show V02max during standing cycle ergometry protocols and treadmill protocols to be similar, while seated cycle ergometry V02tnax values are lower. Conversely, other studies show seated and standing cycle ergometry V02max to be comparable. Because previous studies are equivocal, the purpose of the current study was to compare V02max between seated and standing cycle ergometry protocols. In a counterbalanced order, male (n=14) and female (n=22) average fit volunteers completed a maximal exertion seated cycle ergometry protocol (SIT) and a maximal exertion standing cycle ergometry protocol (STD). Cadence for each protocol was 60 revolutions per minute (rpm), with resistance being increased 30 Watts each minute until volitional exhaustion. SIT required individuals to perform a maximal exertion test and remain seated until volitional exhaustion. For STD, subjects completed the same protocol; however, when the subjects felt they could no longer continue in a seated position, they were required to stand and perform "standing cycling" to volitional exhaustion. V02max (ml/kg/min), heart rate (HR) (b/min), respiratory exchange ratio (RER), and ventilation (VE) (L/min) were compared between SIT and STD using a Multivariate Analysis of Variance (MANOVA). Results were considered significant at p < 0.05. V02maxsTD (37.93 + 8.01) was significantly greater than V02maxSIT (36.82 + 6.63), while HRSTD (189.7 } 9.5) was significantly greater than HRSIT (187.3 + 9.6). V02maxsTD was on average 1.95% greater than V02maxsrr with a range of -16.93 to +17.43%, while HRSTD was on average 1.23% greater than HRS IT with values ranging from -5.59 to +7.43%. VES TD (86.02 }31.64) was not significantly greater than VESrr (82.64 } 26.77), while RERs i t (1.23 } 0.065) was significantly greater than R E R S T D (1-21 } 0.096). Results show a standing cycle ergometry protocol permits significantly higher V02max and HR values compared to a seated protocol. Twenty out of thirty-six subjects (55.6%) achieved a higher V02max and 25/36 (69.4%) recorded a higher peak HR during STD. The current results suggest a standing cycle ergometry protocol should be considered for implementation when cycle ergometry is the selected mode. However, future research should seek to determine characteristics of subjects who do and do not benefit from a standing versus seated protocol.