No description available.

Includes list of articles published or accepted for publication during the period of PhD candidature. / "July, 2004" / Includes bibliographical references (leaves 255-293) / xxviii, 293 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 2004?

Relationships between the steroid hormones estradiol (E2), testosterone (T), cortisol (CRT) and dehydroepiandrosterone-sulfate (DHEAS), memory and mood were investigated in men, in women estrogen-users and non-users, and in patients with Alzheimer's Disease (AD). In Study 1, 72 year-old healthy men and women estrogen-users performed better than estrogen non-users on Forward and Total Digit Span, which test attention and short-term memory, concomitant with their higher E2 levels. The estrogen-users performed better than the men and the non-users on Delayed Selective Reminding, a test of explicit verbal memory. Men and women with higher CRT levels performed worse on several explicit verbal memory tests compared to those with lower endogenous, CRT levels. In Study 2, male patients with AD performed better than estrogen non-using women with AD on several everyday memory tests, and women estrogen-users with AD performed similarly to the men. Both the men and estrogen-users had higher levels of E2 than the non-users. AD patients with higher endogenous levels of DHEAS performed better than those with lower levels on several everyday memory tests, and AD patients with higher CRT levels were impaired on one aspect of everyday spatial memory, Route Recall. In Study 3, no differences in hormone levels between AD patients and age-matched healthy elderly controls were found. The AD patients were most severely impaired on tasks involving explicit verbal recall compared to healthy controls, and least impaired on short-term memory and concentration tasks. The AD patients reported more dysphoric mood and mental dulling symptoms than healthy age-matched controls, but they did not report feeling less positive about the future. Taken together, these results suggest that higher levels of DHEAS and E2 are related to better memory performance in both healthy elderly men and women and in patients with AD, and higher CRT levels are associated with poorer explicit verbal memory performanc

Eczema or dermatitis is the most common dermatological condition accounting for one-third of all diagnoses in the total population surveyed in South Africa. The prevalence of seborrhoeic dermatitis, extreme photodermatitis and severe psoriasis has increased markedly over the last decade and this increase may be ascribed to the HIV epidemic, first diagnosed in South Africa in 1982. Potent innovator corticosteroids, such as clobetasol 17-propionate (CP) that are used to treat skin disorders, are expensive and there is therefore a need for the production of generic topical corticosteroid products. Formulation and manufacturing processes can be challenging aspects for formulation scientists to produce a robust product that will elicit an appropriate and desirable pharmacokinetic-pharmacodynamic profile. Laboratory scale CP creams were manufactured using different concentrations of Gelot® 64 and propylene glycol in order to establish a composition that would produce a formulation, with similar physical and chemical characteristics and in vitro release profile as an innovator product, Dermovate®. These formulations were assessed in terms of their viscosity, spreadability, pH, content uniformity and in vitro release characteristics using a Franz diffusion cell apparatus. A formulation containing 3% w/w Gelot® 64 and 46% v/v propylene glycol (CPLS-02) was found to exhibit similar viscosity and spreadability characteristics and released CP in a manner similar to Dermovate®. The mechanism of drug release was evaluated using mathematical models such as zero order, first order and Higuchi models. In addition, the in vitro release profiles were characterised by use of difference (f1) and similarity (f2 and Sd) factors. A scale-up formulation with the same % w/w composition as the laboratory scale was also investigated following manufacture using a Wintech® cream/ointment mixer. A Central Composite Design approach was used to investigate the effect of process variables on the performance of the scale-up cream formulations. The homogenisation speed, anchor speed, homogenisation time and cooling time were the process variables investigated. Thirty scale-up batches were manufactured and analysed in terms of their viscosity, spreadability, pH, % drug content and cumulative % drug released per unit area over 72 hours. Model fitting using Design-Expert® software was undertaken and revealed that a correlation between the process variables and the cream responses was most suitably described by quadratic polynomial relationships. The homogenisation speed had the most significant effect on the quality of the scale-up formulations, whereas the anchor speed had a secondary effect on the measured responses, for the formulations investigated. The qualitative interpretation and statistical analysis of the in vitro release data from the scale-up formulations using ANOVA and the f1, f2 and Sd factors revealed that one scale-up batch (CPSU-04), for which the process variables were a homogenisation speed of 1900 rpm, an anchor speed of 35 rpm, a homogenisation time of 100 minutes and a cooling time of 100 minutes, released CP at a similar rate and extent to Dermovate®. A diffusion-controlled mechanism appeared to be predominant in these formulations. A human skin blanching study, using both visual and chromameter assessments, was performed to establish whether batch CPSU-04 was bioequivalent to Dermovate®. The bioequivalence of the selected scale-up formulation to Dermovate® was confirmed, following the calculation of a 90% CI.

One of the primary contributing factors to the escalating costs of health care is the high cost of innovator pharmaceutical products. As a consequence, health authorities in various countries and in particular in the developing world have identified generic prescribing and generic substitution as possible strategies to contain the escalating costs of health care provision. There is therefore a need for formulation scientists in developing countries to invest more time in the research and development of generic formulations. Clobetasol 17-propionate (CP) generic cream formulations containing 0.05% w/w of the drug were manufactured and characterized using in vitro testing. Formulation development studies were preceded by the development and validation of an RP-HPLC with UV detection for the quantitation and characterization of CP in innovator and generic cream formulations during formulation development and assessment studies. Furthermore the in vitro release ates of CP release from innovator and generic cream formulations were monitored using a validated in vitro release test method developed in these studies. The formulation of CP cream products was accomplished using a variety of commercially available mixed primary emulsifiers, such as Estol® 1474, Ritapro® 200, Emulcire® 61 WL and Gelot® 64. Successful formulations were selected based on their ability to remain physically stable immediately after manufacture and for 24 hours after storage at room temperature (22°C). Estol® 1474 was found to produce an unstable cream and was therefore not investigated further. The other three emulgents produced stable creams, but only the in vitro release profile of CP from a cream manufactured to contain Gelot® 64 was found to be statistically similar to that of the innovator formulation. Therefore the cream containing Gelot® 64 was selected as the most appropriate prototype generic cream formulation and was characterized in vitro in terms of CP content, viscosity, pH and in vitro release rate. Data generated from these studies were compared to those of the innovator product, Dermovate® cream, using statistical methods. The CP content, pH and in vitro release rate data of the CP formulation were similar to those of the innovator product, however the intrinsic viscosity of Dermovate® cream was almost three (3) times greater than the intrinsic viscosity of the test formulation developed using Gelot® 64. The CP cream formulation developed in these studies was stored for 4 weeks at 40 ± 2°C and 25 ± 5% RH in an incubator and the formulation was found to be stable. A formulation has been developed and assessed and found to be suitable for use as a topical semi-solid dosage form for CP.

The human skin blanching assay (HSBA) is a well-researched and validated method for the bioequivalence assessment of topical corticosteroids. Traditionally, visual assessment of skin blanching has been used. Such testing methods are not conducive for interlaboratory comparisons. Regulatory bodies prefer less subjective methods of analysis. The FDA released guidelines on the assessment of bioequivalence for topical corticosteroids that recommends the use of a chromameter as a reliable method to measure skin blanching although the use of visual assessment with acceptable validation is also provided for. However, the FDA does not elucidate on the manipulation and handling of the chromameter during skin blanching measurements. The purpose of this project was several fold, which included investigations to standardize the manipulation and handling of a chromameter. In particular, measures to avoid skin whitening resulting from the effects of pressure on the skin during chromameter use were investigated. Other methods of analysis should surpass or at least be comparable to the HSBA if such methods are to be used for the assessment of topical corticosteroids. Microdialysis is a relatively new technique for assessing the rate at which drug penetrates the skin. The advantage of using this method is that there are fewer restrictions for selection of an appropriate study population unlike those required for the HSBA where one has to be both a ‘responder’ and a ‘detector’ for their results to be used in data analysis. Microdialysis was investigated by initially conducting experiments in which microdialysis probes were embedded into topical formulations containing mometasone furoate (MF) and the initial results revealed that relatively low drug was released from the formulations. These results indicated that should microdialysis be applied to measure the in vivo release of MF from such topical formulations following application to the skin, even lower concentrations of MF would likely result in the dialysate, necessitating the need for ultra-high sensitive methods of analysis. Typically, the availability of an appropriate analytical technique such as liquid chromatography coupled with mass spectrometry (LCMS) would be a pre-requisite for such in vivo studies. However, only high-pressure liquid chromatography (HPLC) and other less sensitive equipment was available in the laboratories. The study objectives were therefore focussed on in vitro assessment of the release of MF from topical formulations using microdialysis and Franz cells. In addition, the in vivo release of MF was also studied using the HSBA. Data obtained from the microdialysis experiments were compared with the data obtained from the Franz cell diffusion studies in order to provide information on the pharmaceutical availability of MF from the various topical MF dosage forms. Subsequently, pharmaceutical equivalence was investigated from the comparative pharmaceutical availability data using statistical analysis. An additional objective was to attempt to correlate in vitro with in vivo data (IVIVC) to establish a model that could be used to assess safety and efficacy of generic topical drug products. The in vivo data obtained from the HSBA were processed according to the FDA requirements and these pharmacodynamic data were subsequently compared with the microdialysis and Franz cell results. In summary the objectives of this project were: 1. To develop a system to improve the reproducibility of the use of a Minolta® chromameter and compare this with the standard/normal manipulation and handling of such instruments. 2. To develop and validate an HPLC method for the analysis of MF for use with in vitro diffusion studies using microdialysis and Franz cells. 3. To conduct a comparative HSBA on proprietary MF topical creams from two different countries in accordance with the FDA guidance. 4. To assess the pharmaceutical equivalence of topical formulations containing MF using Franz diffusion cells and in vitro microdialysis. 5. To compare the in vivo data obtained from the HSBA with those obtained in vitro using microdialysis and Franz cells.

Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM). These conditions have for a long time been associated with infertility. Obesity is characterized by high levels of circulating leptin and cytokines as well as insulin resistance. Type I DM is associated with low or no insulin whereas, Type II DM is characterised by insulin resistance. As the prevalence of obesity and DM continues to rise, it is likely that the incidence of infertility associated with these pathological conditions will likewise increase. The effects of insulin and leptin on male reproductive function have been reported on the endocrine and spermatogenesis level, but their effects on cellular level of human ejaculated spermatozoa are yet to be elucidated. This study presents data on the role of insulin and leptin on human ejaculated spermatozoa and their interaction with cytokines and nitric oxide. In the first part of the study, we established the suitable concentrations of glucose, insulin and leptin that could be administered to human spermatozoa in vitro. Glucose concentration of 5.6 mM was chosen as the suitable concentration to be administered to human spermatozoa because it has previously been reported in the literature; furthermore, it is within the range of the physiological glucose levels found in the blood of fasting humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen respectively because they gave much improved sperm function and this was within the range of insulin and leptin levels previously measured in human ejaculated spermatozoa. This was followed by investigating the signalling pathway of insulin and its beneficial effects on human spermatozoa function. Endogenous insulin secretion from human ejaculated spermatozoa was blocked by nifedipine and its receptor tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol 3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous insulin administration significantly increased human sperm motility parameters as well as the sperm ability to acrosome react. The inhibition of endogenous insulin release from spermatozoa as well as the inhibition of the insulin receptor substrate (IRS) tyrosine phosphorylation significantly decreased motility parameters and the ability of spermatozoa to acrosome react. The study also investigated the effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin significantly increased sperm motility parameters, acrosome reaction and NO production. The NO production induced by insulin and leptin was via PI3K signalling as evidenced by a reduction in NO levels when PI3K activity was inhibited by wortmannin. To investigate whether insulin and leptin could improve motility parameters of asthernozoospermic and teratozoospermic spermatozoa, the spermatozoa were separated into two fractions by means of a double density gradient technique. The gradient system was able to separate spermatozoa into high morphologically abnormal and less motile spermatozoa similar to that of asthernozoospermic and teratozoospermic patients as well as a more motile fraction. Insulin and leptin significantly increased the motility parameters of spermatozoa from the immature and less motile fraction. The fourth part of the study was aimed at investigating the effects of the cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm motility, viability, acrosome reaction and NO production. The study shows that TNF-α and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4 and time-dependent manner. These cytokines were also shown to significantly increase NO production from human spermatozoa. The decreased motility parameters induced by these cytokines could be attributed to their ability to induce excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo the acrosome reaction. The fifth part of the study was to investigate the expression and localization of glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8 is constitutively expressed and located in the midpiece region of the human spermatozoa. The study also showed that stimulating spermatozoa with insulin led to an increase in GLUT8 expression as well as translocation to the acrosomal region. In the last part of the study we wanted to investigate why the increase in NO generation by spermatozoa due to insulin and leptin stimulation is accompanied with increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation is accompanied with decreased sperm function. We observed that TNF-α and IL-6 not only increased NO production but also ROS production. This study speculates that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6 were administered was due to the concomitant high increase in NO and ROS they induced. In conclusion, this study has established in vitro beneficial effects of insulin and leptin in normozoospermic and asthernozoospermic human sperm function. These hormones influence sperm function via the PI3K signalling pathway in two ways. Firstly, by increasing GLUT8 expression and translocation thereby possibly increasing glucose uptake and metabolism and secondly, by increasing NO production. The study has also established that TNF-α and IL-6 have detrimental effects on human spermatozoa in a dose and time dependent manner. These effects are mediated via their ability to stimulate both NO and ROS production in human spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of human spermatozoa and that insulin stimulation upgrades its expression and leads to its translocation to the acrosomal region. / AFRIKAANSE OPSOMMING: Oor die afgelope jare was daar `n toename in obesiteit en diabetes mellitus (DM). Hierdie toestande word reeds vir ’n geruime tyd geassosieer met onvrugbaarheid. Obesiteit word gekenmerk deur verhoogde sirkulerende vlakke van leptiene en sitokiene sowel as insulien weerstandigheid. Tipe I DM word geassosieer met lae of geen insulien terwyl Tipe II DM gekenmerk word deur insulien weerstandigheid. Soos wat die voorkoms van obesiteit en DM toeneem, is dit waarskynlik dat die insidensie van onvrugbaarheid wat met hierdie patologiese toestande geassosieer word, gevolglik ook sal toeneem. Die effek van insulien en leptien op die manlike voortplantingsfunksie is alreeds aangetoon op endokriene en spermatogenese vlak, maar hul effekte op sellulêre vlak van menslike geëjakuleerde spermatosoë is nog onduidelik. Die studie vertoon data oor die rol van insulien en leptien op die menslike geëjakuleerde spermatosoë en hul interaksie met sitokiene en stikstofoksied (NO). In die eerste gedeelte van die studie, het ons ’n toepaslike konsentrasie van insulien en leptien bepaal wat aan menslike spermatosoë in vitro toegedien kan word. Glukose konsentrasies van 5,6 mM is bepaal as die gepaste konsentrasie om aan menslike spermatosoë toe te dien, omdat dit beter resultate tot gevolg het; verder is dit vergelykbaar met fisiologiese glukose vlakke in die bloed van `n vastende persoon. Insulien en leptien konsentrasies is op 10 μIU en 10 nm onderskeidelik vasgestel, aangesien dit tot beter resultate gelei het, en omdat dit vergelykbaar was met insulien en leptien vlakke wat reeds voorheen in menslike geëjakuleerde spermatosoë gemeet is. Dit was gevolg deur `n ondersoek na die insulien seintransduksie pad en sy voordelige effekte op menslike spermatosoë funksie. Endogene insulien afskeiding deur menslike geëjakuleerde spermatosoë was deur nifedipien geïnhibeer en sy reseptor tirosien fosforilasie effekte was deur erbstatin geïnhibeer terwyl fosfatidielinositol 3-kinase (PI3K) fosforilasie deur wortmannin geïnhibeer is. Eksogene insulien toediening het menslike sperm-motiliteit parameters betekenisvol laat toeneem asook die vermoë van sperme om die akrosoomreaksie te ondergaan. Die inhibisie van endogene insulien afskeiding deur spermatosoë sowel as die inhibisie van die insulien reseptor substraat (IRS) tirosien fosforilasie het die motiliteit parameters en die akrosoomreaksievermoë van spermatosoë verlaag. Die studie het ook die effekte van insulien en leptien op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie nagevors. Beide insulien en leptien het sperm-motiliteit parameters, -akrosoomreaksie en -NO produksie betekenisvol verhoog. NO produksie is deur insulien en leptien via PI3K seintransduksie geïnduseer, soos bewys deur die verlaging waargeneem in NO vlakke toe PI3K aktiwiteit deur wortmannin geïnhibeer was. Om vas te stel of insulien en leptien die motiliteit parameters van asthenozoospermiese en teratozoospermiese spermatosoë kon verbeter, het ons spermatosoë in twee fraksies met ’n dubbel digtheid gradiënt geskei. Die gradiënt sisteem was daartoe instaat om die spermatosoë in ’n onvolwasse, (morfologies abnormaal en minder motiel - soortgelyk aan dié van asthenozoospermiese en teratozoospermiese pasiënte), sowel as ’n volwasse meer motiele fraksie te skei. Insulien en leptien het die motiliteit parameters van spermatosoë van die onvolwasse en minder motiele fraksie verhoog. Die vierde gedeelte van die studie was daarop gemik om die effekte van die sitokiene tumor nekrose faktor alfa (TNF-α) en interleukin-6 (IL-6) op menslike sperm-motiliteit, -lewensvatbaarheid, -akrosoomreaksie en -NO produksie, te ondersoek. Die studie het getoon dat TNF-α en IL-6 motiliteit parameters en akrosoomreaksie in ’n tyd- en dosis-afhanklike wyse betekenisvol verlaag het. Hierdie sitokiene was ook in staat om NO produksie in menslike spermatosoë te verhoog. Die verlaging in motiliteit parameters wat deur hierdie sitokiene geïnduseer is, kan toegeskryf word aan hul vermoë om die produksie van oormatige hoeveelhede NO te stimuleer. Dit is nog nie duidelik hoe hulle die akrosoomreaksie in spermatosoë kan inhibeer nie. Die vyfde gedeelte van die studie het dit ten doel gehad om die uitdrukking en lokalisering van die glukose transporter 8 (GLUT8) in menslike spermatosoë te ondersoek. Hierdie studie kon aantoon dat GLUT8 konstitutief uitgedruk is en in die middelstuk van die menslike spermatosoë voorkom. Die studie bewys ook dat stimulering van die spermatosoë met insulien tot `n toename in GLUT8 uitdrukking sowel as translokasie na die akrosomale area, lei. In die finale gedeelte van die studie wou ons ondersoek waarom die toename in NO produksie in spermatosoë (as gevolg van insulien en leptien stimulasie) deur `n toename in spermfunksie gekenmerk word, terwyl die toename in NO produksie (as gevolg van TNF-α en IL-6 stimulasie) deur ’n afname in spermfunksie gekenmerk word. Ons het waargeneem dat TNF-α en IL-6 nie alleen NO produksie nie, maar ook reaktiewe suurstof spesies (ROS) produksie verhoog het. Ons vermoed dat die afname in sperm motiliteit en akrosoomreaksie met TNF-α en IL-6 toediening, die gevolg van die gelyktydige verhoging in NO en ROS was. In gevolgtrekking kan ons sê dat hierdie studie die voordelige in vitro effekte van insulien en leptien op asthenozoospermiese en teratozoospermiese menslike spermfunksie aangetoon het. Hierdie hormone beïnvloed spermfunksie via die PI3K seintransduksie pad op twee maniere. Eerstens, deur `n toename in GLUT8 uitdrukking en translokasie, met die gevolg dat glukose opname en metabolisme moontlik verhoog is, en tweedens, deur die toename in NO produksie. Die studie het ook vasgestel dat TNF-α en IL-6 nadelige effekte op menslike spermatosoë in `n dosis- en tyd-afhanklike wyse het. Hierdie effekte vind plaas a.g.v. hul vermoë om beide NO en ROS produksie in menslike spermatosoë te induseer. Die studie toon aan dat GLUT8 uitdrukking in die middelstuk van die menslike spermatosoon voorkom en dat insulien stimulasie GLUT8 uitdrukking opreguleer en tot translokasie na die akrosomale area lei.

Abstract not available

Postmenopausat women on hormone replacement therapy (HRT) have been reported to be stronger when compared to women who are not using HRT. The first goal of this study was to investigate whether muscle morphology was altered in women who use HRT when compared to women who do not use HAT. In addition, this study examined the combined effects of a 6-month moderate-intensity strength training (ST) routine and HRT on the neuromuscular system of early postmenopausal women. Because not all the women completed the ST, this study was separated into two separate analyses, baseline (n=17; 7 HRT, 10 non-HRT) and training (n=14; 6 HRT, 8 non-HRT). ST consisted of two exercises (squat and dead lift), two days a week, for 6-months. Vastus lateralis muscle biopsies were taken at baseline and 6-months after exercise training. Biopsy samples were sectioned and analyzed histochemically for muscle fiber type and fiber cross-sectional area (CSA). In addition, voluntary knee extension strength was assessed at 30��/sec using an isokinetic dynamometer at these two time points. At baseline there were no significant differences in knee extensor strength between groups (HRT: 443 �� 121 N, non-HRT: 490 �� 106 N). Regardless of hormone status, Type I fibers were significantly larger (p=.005) in GSA (Type I=3705 �� 877��m��; Type II=2790 �� 756��m��). However, there were significantly more Type II fibers (p<.0001) (61.5 �� 7.9% of total) and consequently, Type II fibers occupied significantly more total fiber area p=.00l2) (Type I=45.3 �� 7.4%; Type II=54.7 �� 7.4%). No significant differences were found in the fiber type distributions of the HAT (37.9 �� 2.5% Type I, 62.1 �� 2.5% Type II) and non-HRT (38.9 �� 2.9% Type I, 61.1 �� 2.9% Type II) groups. There were no significant differences in fiber CSA of Type I fibers (HRT: 3615 �� 886 ��m��, non-HRT: 3769 �� 912 ��m��) or Type II fibers (HRT: 2770 �� 722 ��m��, non-HRT: 2849 �� 804 ��m��) obtained from the two groups. Six months of ST had no effect on the strength, fiber CSA, and fiber type distribution for HRT and non-HRT subjects. These results suggest that HRT does not alter muscle strength, fiber type distribution, and fiber CSA in early postmenopausal women. / Graduation date: 2003