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Fimose, definida como a dificuldade em retrair o prepúcio para expor a glande, devido à presença de um anel fibroso, tem sido tradicionalmente tratada por meio da circuncisão. Durante a última década, no entanto, a corticoterapia foi proposta como tratamento clínico da fimose. Apresentamos um estudo duplo cego, para tratamento clínico de fimose em crianças, comparando um creme de furoato de mometasona 0,1%, aplicado topicamente, com um creme hidratante. Crianças de 2 a 13 anos de idade (n=110) apresentando fimose grau 5 de acordo com a escala de retratibilidade de Kikiros et al, todas previamente agendadas para cirurgia, foram incluídas neste trabalho. Após oito semanas de tratamento tópico, tanto com o creme hidratante quanto com o corticóide, os pacientes foram reavaliados e os que não responderam ao tratamento, antes da abertura do estudo duplo cego, foram submetidos a um adicional tratamento com corticóide, por mais oito semanas. No grupo dos pacientes tratados com corticóide, o anel fimótico desapareceu e a glande foi exposta em quarenta e nove pacientes (88%). No grupo placebo, o mesmo aconteceu 28 pacientes (52%) (*p <0,05). Dos 7 pacientes que não responderam ao tratamento inicial, com corticóide, 5 apresentaram boa resposta, ao tratamento adicional, enquanto que dos 26 pacientes do grupo placebo, 22 apresentaram exposição da glande, após o tratamento adicional (*p <0,05). Dois meninos, do grupo tratado com corticóide e quatro do grupo placebo, ainda continuaram com o anel fimótico, mesmo após o tratamento adicional com corticóide, e foram submetidos à postectomia. Concluímos que o uso de corticóide tópico, durante oito semanas, representa uma boa opção para o tratamento da fimose, principalmente na idade pré-escolar. / Phimosis, defined as the inability to retract the prepuce in order to expose the glans, due to the presence of a preputial fibrotic ring, is surgically treated in 1% of children. During the last decade, however, topical steroid treatment was proposed for phimosis. We herein present a double blind study comparing 0,1% mometasone furoate topical cream vs moisturizing cream (placebo) treatment for phimosis. Children aged 2 to 13 year old (n=110) presenting with phimosis (retractability grade 5 Kikiro´s classification) and scheduled for circumcision were included in this trial. The patients were evaluated after eight weeks topical treatment with moisturizing cream (n=54) or steroid cream (n=56). In the steroid group, the ring disappeared and glans was exposure obtained in forty nine patients (87,5%) vs twenty eight patients (52%) in the placebo group (*p<0,05). Non responders from both groups received an additional eight weeks steroid cream treatment. In the steroid group, five of seven patients were finally cured vs twenty two out of twenty six in the placebo group (*p<0,05). Two children with persisting phimosis (Kikiro´s retractability grade 5 and appearance grade 3 in the steroid group (4%) vs four children in the placebo group (7%) ended up receiving circumcision. We conclude that eight weeks topical steroid cream treatment represents a very good option for phimosis management in this age group.

Orientador: Lúcia Helena Simões da Costa Paiva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T17:10:16Z (GMT). No. of bitstreams: 1 Leao_RogeriodeBarrosFerreira_M.pdf: 2600347 bytes, checksum: 3c70f4a08f6a98df16d66375a85ccd5f (MD5) Previous issue date: 2012 / Resumo: Introdução: O tamoxifeno é utilizado no tratamento do câncer de mama receptor de estrogênio positivo. Seu mecanismo de ação está na inibição do crescimento das células malignas por antagonismo competitivo com estrógenos pelos receptores estrogênicos. A ação do tamoxifeno nestes receptores é variável e, dependendo do tecido, pode ter ação antagonista ou agonista. Em mulheres menopausadas usuárias de tamoxifeno, observa-se uma maior incidência de patologias endometriais, sendo o pólipo endometrial a alteração mais frequente. Sua patogênese não está bem estabelecida, mas parece estar relacionada a fatores hormonais. Objetivos: Comparar a expressão de receptor de estrógeno (RE) e de receptor de progesterona (RP) em pólipos endometriais de usuárias de tamoxifeno com pólipos endometriais e endométrio atrófico de não usuárias na pós-menopausa. Material e métodos: Entre mulheres submetidas à histeroscopia cirúrgica no Hospital da Mulher Prof. Dr. Aristodemo Pinotti - CAISM/UNICAMP de janeiro de 1998 a dezembro de 2008, foram selecionadas 84 mulheres na pós-menopausa usuárias de tamoxifeno, com pólipo endometrial benigno. Esse grupo foi comparado a 84 amostras de endométrio atrófico e 252 pólipos benignos de mulheres na pós-menopausa não usuárias de tamoxifeno e sem antecedente de terapia hormonal. As expressões de RE e RP foram avaliadas através de imuno-histoquímica segundo a porcentagem de células coradas, intensidade da coloração nuclear e escore final. A expressão de RE e RP no epitélio glandular e no estroma dos pólipos de usuárias de tamoxifeno foi comparada com o endométrio atrófico e os pólipos de não usuárias separadamente, utilizando o Teste de Mann-Whitney, corrigido pelo método de Bonferroni, teste exato de Fisher ou Qui-quadrado. Resultados: os pólipos de usuárias de tamoxifeno apresentaram maior expressão de RE e RP no epitélio glandular e estroma, em relação ao endométrio atrófico (p<0,0001). Em relação aos pólipos de não usuárias, as usuárias apresentaram maior expressão de RP no epitélio glandular (p=0,0014) e estroma (p=0,0056), não apresentando diferença significativa em relação ao RE. A maioria dos pólipos das usuárias e não usuárias de tamoxifeno apresentavam RE/RP positivos enquanto a maioria dos endométrios atróficos era RE/RP negativos. Conclusões: Os pólipos apresentam aumento frequente de RE, independentemente do uso do tamoxifeno. Por outro lado, os altos níveis de RP parecem consistentes com os efeitos agonistas da droga / Abstract: Introduction: Tamoxifen has been used for the treatment of strogen receptor-positive breast cancer. The effect of tamoxifen in breast cancer is the it inhibition cancer cell growth by competitive antagonism with strogen for strogen receptor (ER). The mechanism of action of tamoxifen in this receptor varies among different tissues, with antagonist effect (e.g. in breast) ou agonist (e.g. in endometrium). Thus, in menopausal women who use tamoxifen, a higher incidence of endometrial alterations is observed and endometrial polyps are the most common. The pathophysiology of endometrial polyp is still not definitely established but it seems to be related to hormone influence. Objectives: To compare the expression of estrogen receptors (ER) and progesterone receptors (PR) in endometrial polyps of tamoxifen users to atrophic endometrium and endometrial polyps of postmenopausal nonusers of tamoxifen. Material and methods: Among women undergoing surgical hysteroscopy in Hospital da Mulher Prof. Dr. Aristodemo Pinotti - CAISM/UNICAMP, from January 1998 to December 2008, 84 tamoxifen users with benign endometrial polyp were selected. This group was compared to 84 samples of atrophic endometrium and 252 benign polyps of postmenopausal women who were non-users of tamoxifen and no previous history of hormone therapy use. ER/PR expression was assessed by immunohistochemistry study according to the percentage of stained cells, intensity of nuclear color and final score. The expression ER and PR in the glandular epithelium and stroma of polyp tissue from tamoxifen users was compared to atrophic endometrium and polyps from non-users separately, using the Mann-Whitney test corrected by the Bonferroni method, Fisher's exct test or Chi-square test. Results: Polyps of tamoxifen users had a higher expression of ER and PR in the glandular epithelium and stroma, in relation to the atrophic endometrium (p<0.0001). Regarding polyps of women not treated with tamoxifen, users had a higher PR expression in the epithelium (p=0.0014) and stroma (p=0.0056), without any difference in ER expression. Most of polipys expressed ER/PR positives while atrophic endometrium were ER/PR negatives. Conclusions: Polyps frequently exhibit increase in ER expression, independent of the use of tamoxifen. High levels of PR seem to be consistent with agonist effects of the drug / Mestrado / Fisiopatologia Ginecológica / Mestre em Ciências da Saúde

Les stéroïdes anabolisants androgènes peu acides ou basiques ne permettent pas d’obtenir des signaux importants lors de leur analyse par spectrométrie de masse électrospray (ESI-MS), rendant ainsi leur détection compliquée par l’utilisation de cette technique. Bien que certains stéroïdes puissent être analysés de manière adéquate avec des méthodes d’analyse antidopage classiques comme la chromatographie en phase gazeuse (GC) couplée à la MS avec une source d’ionisation électronique (EI), il reste un besoin pour le développement d’une méthode qui soit capable de détecter efficacement les composés plus difficiles, c’est-à-dire ceux qui sont moins susceptibles de s’ioniser en ESI. Pour résoudre ce problème l’approche envisagée se base sur l’addition d’anions (sous forme de sel d’ammonium) à des stéroïdes anabolisants comme principe pour le développement d’une méthode analytique de détection de ces composés. Il a été confirmé que les limites de détection instrumentales obtenues avec ces anions sont plus basses qu’en comparaison des mêmes analyses sans additif en ESI positif ou négatif. Les étapes de développement de cette méthode basée sur la chromatographie en phase liquide (LC) couplée à la MS en tandem sont également présentées. Les résultats obtenus sur des échantillons en matrice urinaire démontrent que l’attachement anionique apporte une nouvelle approche prometteuse pour la détection des stéroïdes en contrôle antidopage. De plus, des expériences sur des stéroïdes bifonctionnels comme la 5-α-pregnan-3α,20β-diol ou la d4-17,21,21,21-pregnenolone réduite ont permis de mieux appréhender les facteurs qui permettent que l’attachement préférentiel d’un anion donné puisse se faire sur une région spécifique de la molécule, i.e. que l’attachement anionique soit « régiosélectif ». La fragmentation des adduits anioniques de stéroïdes bifonctionnels en MS en tandem a montré des voies de décomposition produisant des ions produits caractéristiques de chaque anion. La détermination d’acidités en phase gazeuse obtenues par des calculs de chimie théorique a également permis d’apporter une nouvelle lumière sur le comportement des adduits anion-stéroïde. / Androgenic anabolic steroids that are neither acidic or basic do not provide strong signals for their analysis by electrospray mass spectrometry (ESI-MS), making their detection difficult with the use of this technique. Although some steroids can be adequately analysed by standard antidoping analytical methods, such as gas-phase chromatography mass spectrometry (GC-MS) with an electron ionization source (EI), there is nonetheless a need for the development of the method that could efficiently detect more difficult compounds, i.e. the least likely to ionize by ESI. In order to solve this problem, the chosen approach is based on addition of anions (found as ammonium salts) to anabolic steroids as principle for the development of an analytical method for the detection of those compounds. It was confirmed that the instrumental limits of detection obtained with the anions were lower compared to the same analyses without additive in positive or negative ESI. The steps for this method development based on liquid-phase chromatography tandem mass spectrometry (LC-MS) are also presented. The results obtained on samples in urine matrice have shown that anion attachment brings a new promising approach for steroid detection in antidoping control. Furthermore, experiments on bifonctionnal steroids such as 5-α-pregnan-3α,20β-diol or reducted d4-17,21,21,21-pregnenolone have brought light on the factors that dictate preferential attachment of a given anion.

Abstract The hormonally active form of vitamin D, 1,25(OH)2D3, is involved in many biological functions throughout the body, such as regulation of calcium and phosphate homeostasis, bone remodeling and controlling cell proliferation and differentiation. Vitamin D receptor (VDR), a member of the nuclear hormone receptor (NHR) super family, mediates those genomic actions of 1,25 (OH)2D3 by actively repressing or activating its target genes. In the present study recombinant human nuclear VDR and its ligand binding domain (LBD) were expressed in Spodoptera frugiperda (Sf9) insect cells and in E.coli. Recombinant proteins were purified and their biochemical and biophysical properties were characterized. Recombinant VDR was shown to bind to the vitamin D response element (VDRE) of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor (RXR)-αΔAB. Full-length VDR and its LBD were demonstrated to bind natural ligand 1,25 (OH)2D3 with high affinity. The binding affinities of several vitamin D analogs were also determined. Ligand binding induced conformational change within the receptor was studied using several methods such as partial proteolytic digestion, small angle neutron scattering (SANS), native gel electrophoresis and circular dichroism (CD) spectroscopy. Results indicate that ligand binding induces conformational change within VDR and different 1,25(OH)2D3 analogs might induce a somewhat different conformation within the receptor. This is seen as an unequal capacity of analogs to stabilize receptor against proteases or heat and as differences in the promotion of receptor homodimerization. Compared to other nuclear hormone receptors, VDR presents a large insertion region at the N-terminal part of the LBD between helices H1 and H3, encoded by an additional exon. In the present study this additional exon was deleted and the properties of mutated LBD were compared to the wild type LBD. Biochemical analyses indicated that the mutant protein exhibits the same ligand binding, dimerization with RXR and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Furthermore, solution studies by small angle X-ray scattering indicated that the insertion region in the VDR locates on the surface of molecule and it is not structurally well ordered.

Central Nervous System (CNS) vasculitis is the most common life-threatening complication of coccidioidal meningitis. It is manifested by cerebral ischemia, hemorrhage, and infarction. We report a case of CNS vasculitis in a patient receiving chemotherapy and review of the literature on coccidioidal meningitis. The patient was treated with combination antifungal therapy and a short course of high dose corticosteroids with a modest improvement in her neurological examination after initiation of steroids.

PROJECT ONE Introduction HPV is detected in about 99.7% of cervical cancers. However, the HPV type distribution in South African women is unknown. Objectives To determine HPV-type distribution among women with cervical dysplasia in relation to oral contraceptive usage. Methods Prospective cross-sectional study of four groups of patients according to oral contraceptive usage: non-users, users of less than five years duration, users of between five years and ten years and users of more than ten years duration. Swabs of the cervix were analysed for HPV DNA using polymerase chain reaction method. Results A total of 124 women were recruited for the study. There were 75 HIV-infected patients (seroprevalence 61%). Of the 102(82%) HPV-positive patients, 79 patients had high-risk HPV DNA (78%). In terms of the four oral contraceptive groups, high-risk HPV DNA was detected in 70% (n=21), 79% (n=22), 90% (n=21) and 71% (n=15) of patients, respectively. The odds of having HPV DNA was six times higher for the combination of contraceptive users of less than 5 years duration/non-users (OR 5.9, 95% CI: 1.87 - 18.77). There was no change when adjustment was made for age (OR 6.1, 95% CI: 1.9 - 19.4). HPV DNA types 16 and or 18 was present in a total of 21 patients (49%) (non-contraceptive users and users < 5years duration) versus 15 patients (42%) who used oral contraceptives of more than 5 years duration (p=0.524). HPV type 16 was the commonest HPV type detected (20.2%) and HPV type 58 was the next commonest high-risk HPV type (16.1%). HPV types 58 and 33 was detected in a much greater percentage of our population and HPV 16 in a much smaller percentage of our population compared with a non-South African population. Conclusion The findings of this study demonstrate an interesting distribution of HPV types in a South African population. PROJECT TWO Introduction Various risk factors have been implicated in the causation of cervical cancer including human papillomavirus (HPV), the early genes (E6 and E7) of which encode the main transforming proteins. Studies have suggested that steroid hormones may enhance the expression of these genes leading to loss of p53 gene-mediated cell apoptosis. Methods A total of 120 cervical tissue samples were obtained from patients with proven cervical cancer. Patients who used depo-medroxyprogesterone acetate steroid contraception were recruited as part of the study arm. Only HPV DNA type 16 samples were used for the study. Controls included three cell lines (CaSki, SiHa,&C33A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal housekeeping gene. Of 120 patients, there were 111 patients with HPV type 16 identified. Of this number, RNA was present in 63 samples. There were 30 women (30/63) who used steroid contraception. In relation to patients who used contraception, HPV 16 E6 gene expression was present in 79% (n = 23) and 88% (n = 30) of steroid users compared to nonusers, respectively. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. There were 57% of steroid users (n = 17) who had expression of the E6*I/E6*II gene, compared to 52% (n = 17) of nonusers (P = 0.800). Conclusion From a molecular level, this study reflects almost similar distribution of the HPV 16 E6/E6*1 and E6*11 and does not confirm the role of injectable progesterones in cervical carcinogenesis. Further studies with larger patient numbers are needed. / Thesis (PhD)--University of Pretoria, 2011. / Obstetrics and Gynaecology / unrestricted

Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.

Recent reports in the literature suggest that oral contraceptive steroid therapy may be implicated in the development of benign hepatic adenomas in women. Since estrogens and progestins are known to affect liver function, we studied effects of chronic administration of the oral contraceptive agents mestranol and norethindrone on various indices of hepatic integrity. Several hepatic mixed function oxidase activities were measured: benzo(a)pyrene hydroxylase, epoxide hydrase, aniline hydroxylase (Appendix II) and aminopyrlne N-demethylase (Appendix II). In addition, benzo(a)pyrene hydroxylase activity in lung tissue was measured. As an indication of whether metabolites of the contraceptive steroids were bound to liver macromolecules, irreversible binding of [³H]-benzopyrene was measured. Hepatic histopathology (light microscopy using hematoxylin and eosin stain and oil red 0 stain) was carried out to determine if functional alterations correlated with pathological changes in the liver. Comparisons were also made between ethanol treated and non-ethanol treated groups to determine if contraceptive steroid-associated hepatotoxiclty was enhanced by or would enhance, the hepatotoxiclty of ethanol administration. Female and male Wistar rats were pair-fed a nutritional liquid diet, Sustacal[sup R] (Mead Johnson) to which was added either sucrose or ethanol as 40% of calories. Oral contraceptive steroids were administered daily in the liquid, diet in the following doses: mestranol, 0.6 mg/kg per day, alone or in combination with norethindrone, 5.0 mg/kg per day. Initial short-term studies showed that the ethanol plus Sustacal[sup R] diet generally caused enzyme induction compared to the plain Sustacal[sup R] diet or the sucrose plus Sustacal[sup R] diet in animals treated for up to 6 weeks. Animals that were administered the contraceptive steroids for a similar time period also demonstrated hepatic microsomal enzyme induction. Enzyme activity in animals that received ethanol plus the contraceptive steroids was increased above that seen for each agent alone. Chronic studies showed that ethanol administration for 6 months produced hepatotoxiclty in both male and female rats. Hepatotoxiclty was observed functionally as decreased hepatic benzo(a)pyrene hydroxylase activity and histopathologically as increased fat accumulation in zone 3 of the liver lobules. It was found that administration of the contraceptive steroids to female rats tended to protect against ethanol-associated hepatotoxiclty. The protective effect was observed functionally as maintenance of control levels of hepatic benzo(a)pyrene hydroxylase activity and morphologically as lesser amounts of fat accumulation ln the liver. That is, there tended to be a correlation between the level of hepatic benzo(a)pyrene hydroxylase activity and histological fat accumulation as an indication of ethanol-associated hepatotoxiclty. A Sustacal associated phenomenon was evident in all animals in which hlstopathology was carried out. The "Sustacal effect" was observed, as mlcrodroplet fat accumulation ln zone 1 of the liver lobule. Contraceptive steroid treated females showed the least "Sustacal effect". Microsomal enzyme activity did not appear to be affected by the "Sustacal effect". It was concluded that the contraceptive steroids administered did not increase ethanol hepatotoxicity. Instead, it appeared that female sex steroids tended to attenuate ethanol-assoclated hepatotoxicity. There was no evidence to suggest that the oral contraceptive steroids were directly associated with overt hepatotoxicity. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Urine samples are routinely used in human and animal patients to diagnose health problems; often to investigate or monitor specific health-related problems that essentially may remain silent for extended periods. However, not much work has been performed on crocodilian urine for diagnostics. In general, crocodilian species lack a bladder as a separate storage organ (as found in mammals), possess metanephric kidneys (unable to concentrate urine) and have functional salt excreting glands. Collection of urine from the Nile crocodile (Crocodylus niloticus) is a simple and atraumatic procedure where a dog urinary catheter is used to collect relatively clean urine from the urinary chamber in the crocodile’s cloaca. Unfortunately, in-depth investigations of urine variables, and establishing baseline concentrations, have not been performed on Nile crocodile urine samples before. The specific focus areas of this research project were: (1) determination of urine and plasma biochemical concentrations by means of a standard veterinary clinical pathology profile and the establishment of the ratio between urine and blood biochemical parameters; (2) the validation of a gas chromatographic–mass spectrometric (GC/MS) method for the determination of steroid metabolite concentrations in urine; and (3) using this established analytical method to determine the presence (identify) and concentrations of steroid metabolites in the urine of individual crocodilians. / Urine and plasma samples collected at Izintaba Crocodile Farm during the period November 2005 to July 2006, from captive bred, healthy young Nile crocodiles, were analysed for standard biochemistry variables. The urine samples (n = 101) were analysed for sodium, potassium, chloride, urea, creatinine, calcium, magnesium, phosphate, uric acid, osmolality, and ammonium ion, while the plasma samples (n = 101) were screened for total protein, glucose, sodium, potassium, chloride, urea, creatinine, total calcium, ionised calcium, magnesium, phosphate, uric acid and osmolality. Means, medians and standard deviations were statistically determined, as well as urine to plasma (U/P) ratios for corresponding variables. The value of this project is the establishment of reference concentrations for Nile crocodile urine samples that may become useful for interpretation of laboratory results, in future. / The clinical validation of a GC/MS method for the analysis of urinary steroids in the Nile crocodile was achieved using urine samples from two-year-old Nile crocodiles. The main objective of this investigation was to develop, optimize and validate the laboratory analysis of urinary steroid metabolites. Steroid profiling was performed on individual and pooled Nile crocodile urine samples. Ascending concentrations of representative steroid standards: androsterone, etiocholanolone, dehydroepiandrosterone, 11-OH androsterone, pregnanediol, pregnanetriol, 11-deoxytetrahydrocortisol, tetrahydrocortisone, tetrahydrocortisol and tetra-hydrocorticosterone, were spiked into aliquots of the pooled urine samples, to obtain calibration samples ranging from 0.2 to 20 μg. Sample preparation and analysis methodology were based on a well-established, validated GC/MS method for determination of human urinary steroid metabolites. The validation of the GC/MS method for Nile crocodile urine was successfully completed, by determining lower limits of quantitation and limits of detection for each analyte, obtaining linearity up to the highest calibration level, correlations exceeding 0.90, and recoveries of 82% and more. / Steroid profiling was performed on urine samples collected from a number of mature crocodilian species, namely Nile crocodile, American alligator (Alligator mississippiensis), spectacled caiman (Caiman crocodilus) and dwarf crocodile (Osteolaemus tetraspis). Steroid metabolites were identified and were quantitated and reported per urinary creatinine. Qualitative reporting was conducted in cases where creatinine concentrations were not available. Results included identification and quantitation of the steroid metabolites: androsterone, etiocholanolone, 11-hydroxy androsterone, pregnanediol, pregnanetriol, and the tetrahydro- metabolites of cortisone (THE), cortisol (THF), and corticosterone (THB). In some urinary steroid profiles, several prominent peaks were observed which could not be identified. The study findings confirmed that crocodile urine could successfully be used, as it is commonly used in humans, to determine steroid metabolite profiles. A follow-up study to identify the unknown peaks by structure elucidation with more sophisticated equipment is recommended - this could lead to valuable information about liver metabolism of steroids in crocodilians. / An adrenocorticotrophic hormone (ACTH) stimulation test was conducted on 18 captive Nile crocodiles. The experimental animals were temporarily housed in separate enclosures at Le Croc Crocodile Farm for four weeks, to ensure controlled conditions and easy and frequent access to the animals. Twenty-seven urine samples were collected both pre- and post-ACTH or saline injections. Steroid profiling was performed on 24 of the 27 urine samples to assess the corticosterone and tetrahydrocorticosterone concentrations following the ACTH treatment. Quantitation relative to urine creatinine levels was recorded following analyses with a standardised liquid chromatography/mass spectrometry (LC/MS) method, reporting the concentrations in nmol steroid/μmol creatinine. Unfortunately, a significant increase in urinary corticosterone concentrations 6 h after the injection of Synacthen® (5 μg/kg) was not observed. A possible explanation for this could be that the 6 h period was too short for a significant increase in urinary glucocorticoid metabolite excretion in the Nile crocodile. / In conclusion, this is the first in-depth study that focused, specifically, on Nile crocodile urine for analyses as diagnostic tools and for indices of health. The screening of the urine samples, collected from healthy Nile crocodiles, for a large array of biochemical variables contributed significantly to the database of “normal” concentrations. The establishment of a validated urinary steroid profiling method may significantly contribute to future validation and implementation of innovative diagnostic methods to monitor the health status and endocrine systems of wild Nile crocodiles in Africa. / Thesis (PhD)--University of Pretoria, 2017. / The Norwegian Council for Higher Education’s Programme for Development, Research and Education (NUFU) / Royal Netherlands Embassy in South Africa / Crocodile Specialist Group / SAVF / Paraclinical Sciences / PhD / Unrestricted