The placenta and fetal intestines are two key nutrient transport organs that sustain and nurture growing fetus. Insufficient placental development and consequently inadequate fetal nutrient supply can lead to IUGR resulting in low birth weight offspring. Our experimental objectives were to investigate the effects of elevated maternal nutrition, IUGR, and E2 supplementation during mid-gestation (in an attempt to rescue IUGR offspring) on placental and fetal intestinal cell proliferation, angiogenic gene expression, and vascularity. Limited responsiveness in placental development and vascularization to E2 supplementation was observed, likely due to inappropriate timing or dose of E2. However, maternal E2 supplementation increased fetal small intestinal length and GUCY1b3 mRNA expression, suggesting that E2 supplementation has positive effects on IUGR fetal intestinal growth. In conclusion, understanding molecular mechanisms associated with IUGR and possible effects of E2 supplementation in rescuing IUGR may lead to enhanced human health and livestock production efficiency.

Despite progress in prenatal diagnosis, fetal invasive autopsy remains crucial for diagnosis and counselling for recurrence risk in a subsequent pregnancy. However, a decline in parental consent rate has been repeatedly reported in recent years, mainly due to concerns about body disfigurement. Consequently, non-invasive postmortem imaging technologies have been studied as a possible alternative or adjunct to invasive autopsy.However, the accuracy of the most studied 1.5-T magnetic resonance imaging (MRI) was poor for small fetuses and cardiac examination. Furthermore, only one large study had been published at the beginning of the present thesis, and other data were from small series or case reports.Therefore, the objectives of this thesis were to investigate the performance of postmortem MRI (PMMRI) using 1.5-T and 3-T magnets, to evaluate the contribution of postmortem ultrasound (PMUS) and to explore specific alternatives for small fetuses, such as microfocus computed tomography (micro-CT). We began by investigating the comparative accuracy of 1.5-T and 3-T PMMRI with autopsy in a prospectively acquired sample of 135 fetuses. 3-T PMMRI presented better image quality with increased tissue contrast and provided higher accuracy and diagnostic rate, particularly for fetuses ≤20 weeks of gestational age (GA) and for cardiac examination. We demonstrated that training influenced the performance of PMMRI and a diagnostic accuracy similar to that obtained by an expert radiologist could be achieved after a period of self-directed learning.To increase access to postmortem imaging, we prospectively evaluated the diagnostic accuracy of 2D ultrasound using high-frequency probes in 2 studies including 163 and 160 fetuses. 2D PMUS provided acceptable accuracy of 78% when performed by operators blinded to prenatal diagnosis. In direct comparison with 3-T PMMRI, PMUS was more often non-diagnostic, although it presented comparable accuracy when both techniques were diagnostic. To assess the factors increasing the non-diagnostic rate of PMUS, we retrospectively analysed the data of all fetuses terminated ≥20 weeks GA. We identified several factors that were associated with an increased risk of a non-diagnostic PMUS. Longer delays between fetocide and delivery adversely affected the diagnostic rates for the brain, and both intracardiac injection and advancing gestational age were related to non-diagnostic cardiac PMUS.However, the examination of fetuses ≤20 weeks of GA remained unsatisfactory using 3-T PMMRI or PMUS. Therefore, we explored the feasibility of micro-CT for whole-body postmortem imaging in early gestational fetuses. This technology, initially used in industry, was recently applied in human pathology. We demonstrated a 97% agreement with invasive autopsy in a group of 20 fetuses between 11 and 21 weeks of GA. Micro-CT also produced images comparable to low-power histology in a 7-week human embryo. Unfortunately, access to micro-CT machines for human studies, is currently limited to a few specialist centres. We therefore explored the effect of gadolinium staining before 3-T PMMRI in 5 fetuses ≤16 weeks, however, this method did not improve the image contrast.Finally, in order to initiate the research on the clinical implementation of minimally invasive autopsy, including postmortem imaging and biopsies, we evaluated the parental acceptance rate of such an approach and demonstrated a 20% increase compared to autopsy.In conclusion, our research has defined the diagnostic accuracy of different clinically accessible postmortem imaging technologies, suggesting the best clinical indications for each method. Further research is needed to assess the optimal diagnostic approach using all possible postmortem investigations in order to achieve the maximal diagnostic yield and as a consequence, improved parental counselling. / Malgré les progrès récents des techniques de diagnostic anténatal, l’autopsie fœtale invasive reste primordiale pour obtenir le diagnostic final et établir le risque de récurrence pour les grossesses suivantes. Le nombre d’autopsies fœtales ne cesse de diminuer ces dernières années en raison du refus des parents pour lesquels la défiguration corporelle que le geste entraîne est insupportable. C’est pourquoi, un intérêt croissant pour les technologies non-invasives par imagerie post-mortem a été rapporté dans la littérature, considérées comme des alternatives ou des compléments à l’autopsie invasive.L’imagerie par résonnance magnétique (IRM) à 1,5-T post-mortem est la technique la plus étudiée chez le fœtus. Cependant, la performance diagnostique est moindre pour les foetus ≤20 semaines d’aménorrhée et pour l’examen du coeur foetal. Quant à la littérature, seule une étude avec un large échantillon avait été publiée au début de ce travail, pour le reste il s’agit de description de cas clinique ou de petites séries. Dans ce contexte, la présente thèse a pour objectif d’investiguer les performances diagnostiques de l’IRM post-mortem à 1,5-T et à 3-T, d’évaluer la contribution de l’échographie à haute fréquence en post-mortem, et d’explorer des alternatives spécifiques pour les fœtus <20SA telles que la tomodensitométrie microfocale (micro-CT).Nous avons d’abord comparé la performance diagnostique de l’IRM post-mortem à 1,5-T et à 3-T avec l’autopsie classique sur 135 foetus recrutés prospectivement. Nous avons ainsi démontré que l’IRM 3-T offrait une meilleure qualité d’images en augmentant le contraste tissulaire, permettant plus fréquemment un examen diagnostic, particulièrement pour les fœtus <20 SA et dans les anomalies cardiaques fœtales. Par ailleurs, nous avons également montré que l’entrainement influençait la performance de l’IRM post-mortem: une performance diagnostique similaire à celle d’un expert pouvait être obtenue après une période de 18 mois d’auto-apprentissage.Afin de faciliter l’accès à l’imagerie post-mortem, nous avons évalué la performance diagnostique de l’échographie bidimensionnelle en utilisant des sondes à hautes fréquences dans 2 études prospectives incluant respectivement 163 et 160 fœtus. L’échographie post-mortem a présenté une précision diagnostique acceptable de 78% quand elle était réalisée par des opérateurs en aveugle. Mais quand elle est comparée à l’IRM post-mortem 3-T, nous avons pu déterminer que l’échographie était plus souvent non-diagnostique. Cependant, quand les deux techniques obtenaient un diagnostic, l’échographie offrait une sensibilité et une spécificité comparables à l’IRM. Ensuite, afin d’évaluer les facteurs influençant le taux d’examens non contributifs par l’échographie post-mortem, nous avons analysé rétrospectivement les données de fœtus issus d’interruptions médicales de grossesses après 20 semaines d’aménorrhée, chez qui un foeticide préalable était réalisé. Nous avons démontré d’une part qu’un long délai entre le foeticide et l’accouchement augmentait le taux d’examen non concluant pour le cerveau ;d’autre part un foeticide par injection intracardiaque et un âge gestationnel avancé conduisaient à des examens cardiaques non-diagnostiques.Quant à l’examen des fœtus ≤20 semaines d’aménorrhée, les résultats apportés par l’IRM post-mortem à 3-T ou l’échographie post-mortem restaient insuffisants. Dès lors, nous avons exploré la faisabilité du micro-CT pour l’imagerie du corps entier chez les fœtus à un âge gestationnel précoce. Cette technologie, qui était initialement utilisée en industrie, a trouvé récemment des applications en anatomo-pathologie humaine. Nous avons obtenu par micro-CT une concordance diagnostique de 97% avec l’autopsie invasive pour un groupe de 20 fœtus âgés de 11 à 21 semaines d’aménorrhée. Le micro-CT était également capable de produire des images de qualité comparable à des coupes histologiques à faible grossissement pour un embryon humain de 7 semaines d’aménorrhée. Cependant, l’accès aux machines de micro-CT reste limité actuellement à certains centres spécialisés. C’est pourquoi, nous avons investigué l’effet d’une immersion dans une solution de gadolinium avant une IRM post-mortem 3-T sur 5 fœtus ≤16 semaines d’aménorrhée. Cette méthode n’a pas pu démontrer une amélioration du contraste des images.Enfin, pour initier la recherche sur une future application clinique des autopsies mini-invasives, incluant une imagerie post-mortem et des biopsies, nous avons demandé l’avis des parents faisant face à une perte fœtale. Nous avons montré une augmentation de 20% du consentement pour l’autopsie mini-invasive comparée à celui de l’autopsie classique.En conclusion, nos recherches ont décrit la performance diagnostique de différentes technologies d’imagerie post-mortem accessibles en clinique, en suggérant les meilleures indications cliniques pour chaque méthode. Des recherches ultérieures sont nécessaires pour investiguer la meilleure approche diagnostique possible en utilisant toutes les technologies post-mortem accessibles afin d’obtenir un rendement diagnostique maximal, pour un meilleur conseil de récurrence aux parents endeuillés. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Rennet extract is an important commercial enzyme preparation which is indispensable to the cheese making industry, The term "rennet" is used to denote the enzyme system extracted from the fourth stomach (abomasum) of a suckling calf. Most of the enzymatic material in the extract is rennin. It also contains a small amount of pepsin and some other proteolytic enzymes. The amount of pepsin in rennet extract appears to depend upon the age of the calves at the time of slaughter. It has been suggested that some proteolytic enzymes from higher plants might be useful in cheese manufacture. Krishnaswamy et al. reported that Cheddar cheese made with ficin quickly developed a bitter flavor which decreased in intensity during curing. Cheddar cheese made with an extract from the flower petals of Cynara cardunculus developed extreme bitterness and a pasty body during 30 days curing at 10 C. An extract from the fruit of Withania coagulans was used by Kothavalla and Khubchandani as a coagulating agent in the manufacture of Surati and Cheddar cheese. No unusual flavors were reported, but they experienced rather high fat losses.

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Using the chemically defined medium designed by Smibert (1963), the stimulatory or inhibitory effect of test compounds on the growth and the production of acid precipitable material by seven strains of vibrio fetus was determined. The test compounds were Krebs cycle intermediates, lactate, pyruvate, fatty acid, amines, surface active agents, aminobutyrate, hydroxybutyrate, glutathione, asparagine, phenylacetyl chloride, and sodium bicarbonate. Experimental media consisted of the chemically defined medium containing various concentrations of the test compounds. An exact number of cells of each strain of V. fetus was inoculated into tubes of experimental broth media. The cultures were incubated at 37 °C in desiccator jars in an atmosphere containing 85% nitrogen, 10% carbon dioxide, and 5% oxygen. Cultures were observed daily for visible growth. After five days incubation, the optical density of each culture was read at 5/+0 mμ; in a spectrophotometer. The concentration of APM produced by each strain of V. fetus grown in experimental media was also determined. Five day old cultures of each strain were centrifuged, and the APM present in the supernatant fluid was precipitated with trichloroacetic acid. The optical density of the mixture was read at 380 mμ, in the spectrophotometer. Lactate increased the growth of four of five strains of V. fetus tested, and did not inhibit the growth of any strains. Lactate increased APM production of V. fetus due to the increased number of cells produced in its presence. Therefore, the addition of lactate to the 8 chemically defined mediums used for growth or APM production would be advantageous. Citrate and succinate, which increased the growth of V. fetus without affecting APM production, could be added to the chemically defined medium used for obtaining maximal growth of vibrios. Studies requiring a large cell crop of vibrios would be facilitated by the use of the chemically defined medium to which lactate, citrate, and/or succinate had been added. A large cell crop of V. fetus would be useful in preparation of V. fetus cellular antigen. Any study requiring large cell crops cf vibrios would be facilitated by the use of a medium which increased the growth of V. fetus. Alpha-ketoglutarate and tween 80 increased APM production of V. fetus, although these compounds neither increased nor decreased the growth of the vibrios. Therefore, the addition of α-ketoglutarate and tween 80 to the medium used for maximal production of APM would be advantageous. An increased production of APM by V. fetus would aid the study of the biosynthesis of APM. Also, the antigenic nature of APM could be studied more successfully by using a medium which increased the production of APM by vibrios. Compounds, such as glutathione and tergitol 7, which inhibited the growth and the production of APM of vibrios should not be added to the chemically defined medium. Glutamine and asparagine inhibited the growth of V. fetus and should also be avoided as additions to the medium. / Master of Science

Hypoxia and reperfusion can result in many pathological complications in the fetus including retinopathy, ischemic encephalopathy and even stillbirth. The adverse effects are due to excess production of free radicals that attack vital bio-molecules such as DNA and enzymes. Antioxidant treatment may be a way to alleviate oxidative stress. Green tea is a source of antioxidants. It contains polyphenols mainly catechins, that possess high reducing power and low toxicity. Major catechin compounds in green tea are (+)-catechin (C), (-)-epicatechin (EC, (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG) and (-)-epigallocatechin gallate (EGCG). Accordingly, catechins may be ideal agents for antioxidant treatment of the fetus exposed to hypoxia during pregnancy. / In the animal experiments, rat mothers, at the 15.5th gestation day, were intragastrically administrated a single dose of green tea extract. The pharmacokinetic profiles of catechins in maternal plasma, whole embryos and embryonic organs were investigated. The catechins GC, ECG, C, EC, were found to exhibit non-linear capacity limited pharmacokinetic behaviour implying their metabolism or absorption was saturated. Catechin gallates, EGCG and ECG, appeared to exhibit enterohepatic re-circulation behaviour. Peak time was about 1 hour for both groups of catechins; the half life of the catechin group was about 1 hour while that of EGCG and ECG was about 3.7 hours. EC, EGC and EGCG were the dominant compounds present in plasma. All catechins exhibited a consecutive one-compartment model in the embryo, where EGCG, ECG, EGC and EC were dominant compounds and ECG had the highest penetrability. (Abstract shortened by UMI.) / In this study, pregnant rat dams were fed green tea extract in an attempt to raise catechin levels in the rat embryo in order to scavenge free radicals. To test this hypothetical application, we first established analytical methods to evaluate oxidative stress and catechins levels of the fetus in vivo. The methodologies included assaying F2-isoprostanes in cord blood and determining catechin levels in biological fluids and tissues. We further utilized these new sensitive analytical methods to investigate the pharmacokinetics of the catechins in maternal rat plasma, whole embryos and embryonic organs. Since no data has been previously reported on the toxic effects of catechins on embryos, we also tested the toxic effects of various concentrations of catechins on the developing embryonic features in embryo culture. / Chu Kai On. / "April 2005." / Advisers: Michael Scott Rogers; Chi Pui Pang. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0244. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 208-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Leung, Chun Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-169). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.viii / List of Graphs --- p.x / List of Tables x --- p.iv / Abbreviations --- p.xvii / Abstract --- p.xviii / Abstract (Chinese) --- p.xx / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Introduction to retinoids --- p.2 / Chapter 1.2 --- Role of endogenous retinoic acid in embryonic development --- p.3 / Chapter 1.3 --- Regulation of retinoic acid in embryonic development --- p.5 / Chapter 1.3.1 --- Retinoic acid synthesis and degradation --- p.5 / Chapter 1.3.2 --- Retinoic acid signaling --- p.8 / Chapter 1.4 --- Effect of excess vitamin AJ RA on embryogenesis --- p.8 / Chapter 1.4.1 --- Examples of human animal studies --- p.9 / Chapter 1.4.2 --- Mechanisms of retinoid teratogenesis --- p.11 / Chapter 1.4.2.1 --- Apoptosis --- p.11 / Chapter 1.4.2.2 --- Altered proliferation --- p.12 / Chapter 1.4.2.3 --- Altered cell migration --- p.12 / Chapter 1.4.2.4 --- Altered differentiation --- p.13 / Chapter 1.4.3 --- Critical period of RA administration caused specific Malformations --- p.14 / Chapter 1.5 --- Effect of vitamin A/ RA deficiency on embryogenesis --- p.15 / Chapter 1.6 --- Excess and deficiency of RA cause similar types of malformations --- p.17 / Chapter 1.6.1 --- Retinoic acid-induced renal malformations mouse model --- p.18 / Chapter 1.7 --- Strategy of thesis --- p.19 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Mouse maintenance and mating methods --- p.23 / Chapter 2.2 --- All-trans retinoic acid preparation and injection --- p.23 / Chapter 2.3 --- Whole mount in situ hybridization --- p.24 / Chapter 2.3.1 --- Riboprobe synthesis --- p.24 / Chapter 2.3.1.1 --- Bacterial culture --- p.24 / Chapter 2.3.1.2 --- DNA plasmids extraction --- p.24 / Chapter 2.3.1.3 --- Linearization of plasmid --- p.25 / Chapter 2.3.1.4 --- Purification of linearized plasmid --- p.26 / Chapter 2.3.1.5 --- In vitro transcription and labeling --- p.26 / Chapter 2.3.2 --- Sample collection --- p.27 / Chapter 2.3.3 --- Hybridization --- p.28 / Chapter 2.3.4 --- Post hybridization wash and antibody development --- p.29 / Chapter 2.3.4.1 --- Embryo powder preparation --- p.30 / Chapter 2.3.4.2 --- Pre-absorption of antibody --- p.30 / Chapter 2.3.5 --- Post-antibody and staining --- p.31 / Chapter 2.4 --- Real-time quantitative reverse transcription -polymerase chain reaction (RT-PCR) --- p.32 / Chapter 2.4.1 --- Sample collection --- p.32 / Chapter 2.4.2 --- RNA extraction --- p.32 / Chapter 2.4.3 --- Reverse transcription into cDNA --- p.33 / Chapter 2.4.4 --- Quantitative real-time PCR --- p.33 / Chapter 2.4.5 --- Preparation of cDNA standards --- p.34 / Chapter 2.5 --- High pressure liquid chromatography (HPLC) --- p.35 / Chapter 2.5.1 --- Chromatographic system --- p.35 / Chapter 2.5.2 --- Standards preparation --- p.35 / Chapter 2.5.3 --- Embryo sample collection and preparation --- p.36 / Chapter 2.5.4 --- HPLC conditions --- p.36 / Chapter 2.5.5 --- Sample recovery --- p.37 / Chapter 2.5.6 --- Bradford assay --- p.38 / Chapter 2.6 --- RA-responsive cell line --- p.38 / Chapter 2.6.1 --- Cell culture --- p.39 / Chapter 2.6.2 --- Seeding and loading sample to 96-well plate --- p.40 / Chapter 2.6.3 --- X-gal staining --- p.41 / Chapter Chapter 3: --- Time and Dose Responses to RA / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Time response to RA --- p.43 / Chapter 3.1.2 --- Dose response to RA --- p.45 / Chapter 3.1.3 --- Other factors affecting susceptibilities to RA --- p.46 / Chapter 3.2 --- Experimental design --- p.48 / Chapter 3.3 --- Materials and methods --- p.50 / Chapter 3.3.1 --- Time response to RA --- p.50 / Chapter 3.3.2 --- Dose response to RA --- p.50 / Chapter 3.3.3 --- Examination of fetuses --- p.51 / Chapter 3.3.4 --- Statistical analysis --- p.51 / Chapter 3.4 --- Results --- p.53 / Chapter 3.4.1 --- Time response --- p.53 / Chapter 3.4.1.1 --- Time response to RA-induced resorption --- p.53 / Chapter 3.4.1.2 --- Time response to RA-induced renal malformations --- p.54 / Chapter 3.4.1.3 --- Time response to RA-induced changes in growth parameters --- p.57 / Chapter 3.4.1.4 --- Time response to RA-induced non-renal malformations --- p.60 / Chapter 3.4.2 --- Dose response --- p.64 / Chapter 3.4.2.1 --- Dose response to RA-induced resorption --- p.64 / Chapter 3.4.2.2 --- Dose response to RA-induced renal malformations --- p.65 / Chapter 3.4.2.3 --- Dose response to RA-induced changes in growth parameters --- p.68 / Chapter 3.4.2.4 --- Dose response to RA-induced non-renal malformations --- p.71 / Chapter 3.5 --- Discussion --- p.74 / Chapter Chapter 4: --- Effect of Teratogenic Dose of RA on RA Synthesis and Endogenous RA Levels in the Embryo / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.1.1 --- RA synthesis in embryo --- p.79 / Chapter 4.1.2 --- Detection of endogenous RA in embryo --- p.81 / Chapter 4.2 --- Experimental design --- p.83 / Chapter 4.3 --- Materials and methods --- p.84 / Chapter 4.3.1 --- Localization of mRNA transcripts in whole embryo by in situ hybridization --- p.84 / Chapter 4.3.2 --- Vibratome sectioning --- p.85 / Chapter 4.3.2.1 --- Preparation of Gloop --- p.85 / Chapter 4.3.2.2 --- Sample preparation and sectioning --- p.85 / Chapter 4.3.3 --- Quantification of mRNA expression levels in whole embryo and in metanephros by real-time RT-PCR --- p.86 / Chapter 4.3.4 --- Detection of RA levels in whole embryo by HPLC --- p.87 / Chapter 4.3.5 --- Detection of RA levels in metanephros by RA-responsive cell line --- p.87 / Chapter 4.3.6 --- Statistical analysis --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.89 / Chapter 4.4.2 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between metanephroi of embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.93 / Chapter 4.4.3 --- Comparison of the in situ hybridization pattern of different iso forms of Raldh between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.95 / Chapter 4.4.3.1 --- In situ hybridization pattern of Raldh 1 --- p.96 / Chapter 4.4.3.2 --- In situ hybridization pattern of Raldh2 --- p.97 / Chapter 4.4.3.3 --- In situ hybridization pattern of Raldh3 --- p.100 / Chapter 4.4.4 --- Comparison of the in situ hybridization pattern of Cyp26al and Cyp26bl between embryos of RA-treated and vehicletreated control mice at different time points after treatment --- p.101 / Chapter 4.4.4.1 --- In situ hybridization pattern of Cyp26al --- p.101 / Chapter 4.4.4.2 --- In situ hybridization pattern of Cyp26bl --- p.102 / Chapter 4.4.5 --- Comparison of RA levels between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.103 / Chapter 4.4.6 --- Comparison of RA levels between metanephroi of embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.105 / Chapter 4.5 --- Discussion --- p.106 / Chapter Chapter 5: --- Effect of Supplementation with Low Doses of RA on RA Teratogenesis / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.1.1 --- RA supplementation --- p.111 / Chapter 5.1.2 --- Wilms' tumor suppressor gene Wtl --- p.112 / Chapter 5.1.3 --- Apoptosis --- p.113 / Chapter 5.2 --- Experimental design --- p.115 / Chapter 5.3 --- Materials and methods --- p.117 / Chapter 5.3.1 --- Oral gavage of low dose of RA --- p.117 / Chapter 5.3.2 --- Determination of Wtl expression level by real-time quantitative RT-PCR --- p.117 / Chapter 5.3.3 --- Preparation of paraffin sections and TUNEL staining --- p.118 / Chapter 5.3.3.1 --- Sample collection --- p.118 / Chapter 5.3.3.2 --- "Dehydration, embedding and sectioning" --- p.118 / Chapter 5.3.3.3 --- TUNEL staining --- p.119 / Chapter 5.3.4 --- Statistical analysis --- p.121 / Chapter 5.4 --- Results --- p.122 / Chapter 5.4.1 --- Time response to RA supplementation in rescuing kidney development --- p.122 / Chapter 5.4.2 --- Dose response to RA supplementation in rescuing kidney development --- p.127 / Chapter 5.4.3 --- RA supplementation restored various growth parameters --- p.132 / Chapter 5.4.4 --- RA supplementation rescued non-renal malformations --- p.134 / Chapter 5.4.5 --- Wtl expression in the metanephros after RA supplementation --- p.142 / Chapter 5.4.6 --- Apoptotic cell death in the metanephros after RA supplementation --- p.143 / Chapter 5.5 --- Discussion --- p.145 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.150 / References --- p.155 / Figures / Graphs