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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A mortalidade infantil afeta milhÃes de crianÃas em todo mundo e a desnutriÃÃo contribui com cerca de um terÃo dessa mortalidade. A vitamina A jà à utilizada no tratamento da desnutriÃÃo infantil. Contudo, poucos ou nenhum estudo fora realizado, ao nÃvel celular, para avaliar o impacto da suplementaÃÃo com a vitamina A em cÃlulas epiteliais intestinais com desnutriÃÃo proteica in vitro. Desse modo, objetivamos demonstrar os efeitos da desnutriÃÃo e do tratamento com retinoides nos processos de proliferaÃÃo, apoptose/necrose, ciclo celular e diferenciaÃÃo celular, assim como na ativaÃÃo das vias de sinalizaÃÃo em cÃlulas IEC-6 de rato com desnutriÃÃo proteica. Demonstramos que a desnutriÃÃo diminuiu a proliferaÃÃo celular nos tempos de 6, 12, 24 e 48 horas. Em adiÃÃo, nos tempos de 24 e 48 horas a suplementaÃÃo com palmitato de retinol intensificou esta reduÃÃo em comparaÃÃo ao grupo desnutrido. Contudo, a diminuiÃÃo da proliferaÃÃo nÃo estava relacionada à morte celular. Na verdade, o palmitato de retinol reduziu apoptose no tempo de 48 horas apÃs a induÃÃo de desnutriÃÃo. Adicionalmente, a desnutriÃÃo estimulou a quiescÃncia celular e a mesma foi intensificada pelo retinol identificada pelo aumento do percentual de cÃlulas na fase G0/G1 e diminuiÃÃo dos percentuais de cÃlulas nas fases S e G2/M, principalmente nos tempos entre 24 e 48 horas. Para verificar se a desnutriÃÃo, associada ou nÃo à suplementaÃÃo dos retinoides, estimulava a diferenciaÃÃo celular foi realizada a anÃlise da transcriÃÃo gÃnica dos marcadores especÃficos FABP e IAP no perÃodo de 42 horas. Os dois retinoides testados aumentaram em aproximadamente cinco vezes a expressÃo dos mRNAs de FABP e IAP. Visto que a diferenciaÃÃo fora estimulada, foram verificadas quais as vias de sinalizaÃÃo intracelular, acopladas à ativaÃÃo da proteÃna G, estavam ativadas. Os resultados deste trabalho demonstraram que a desnutriÃÃo proteica diminuiu a transcriÃÃo gÃnica de c-Jun, STAT3, MEF2C, e ATF2, mas aumentou a expressÃo dos mRNAs de GLI-3 e c-Fos em relaÃÃo ao grupo nutrido. Entretanto, os retinoides aumentaram a transcriÃÃo dos mRNAs de ELK-1, SRF, c-Jun, FOXO, STAT3, ATF2, GLI-3 e NF-κB em relaÃÃo com o grupo desnutrido. Deste modo, este trabalho mostrou que a desnutriÃÃo interferiu na proliferaÃÃo estimulando a quiescÃncia celular. PorÃm, o tratamento com os retinoides intensificou a quiescÃncia diminuindo a apoptose com estimulaÃÃo da diferenciaÃÃo celular associado com a ativaÃÃo das vias moleculares do ATF2, MAPK/ERK/JNK, IL-6, Hedgehog, PI3K/AKT e NF-κB. / Infant mortality affects millions of children around the world and malnutrition contributes about one third of this mortality. Vitamin A is already used in the treatment of childhood malnutrition. However, few or no study has been done, carried out at the cellular level, to assess the impact of vitamin A supplementation in intestinal epithelial cells under in vitro protein malnutrition. Thus, we aimed to demonstrate the effects of malnutrition and treatment with retinoids in the processes of proliferation, apoptosis/necrosis, cell cycle and cell differentiation as well as activation of signaling pathways in IEC-6 rat cells under protein malnutrition. We demonstrated that malnutrition decreased cell proliferation in periods of 6, 12, 24 and 48 hours. In addition, retinyl palmitate treatment intensified the decrease of cell proliferation when compared to the malnourished group in the periods of 24 and 48 hours after supplementation. However, the reduction in proliferation was not related to cell death. Indeed, retinyl palmitate reduced apoptosis in the period of 48 hours after induction of malnutrition. Additionally, malnutrition stimulated cell quiescence, and the same was enhanced by retinol, which was identified by the increase in the percentage of cells in G0/G1 phase and the reduction of the percentage of cells in S phase and G2/M, especially in periods of 24 and 48 hours. To check if malnutrition, with or without supplementation of retinoids, was stimulating cell differentiation, we evaluated the gene transcription of the specific markers FABP and IAP within 42 hours. Both retinoids tested increased approximately 5-fold the expression of FABP and IAP mRNAs. Knowing that differentiation had been stimulated, we decided to check which intracellular signaling pathways, coupled to the activation of G proteins, were activated. Our results demonstrated that protein malnutrition decreased gene transcription of c-Jun, STAT3, MEF2C and ATF2, but increased the expression of the GLI-3 and c-Fos mRNAs in relation to the nourish group. However, retinoids increased the transcription of ELK-1, SRF, c-Jun, FOXO, STAT3, ATF2, GLI-3, NF-kB in relation to the malnourished group. Thus, this study showed that cell malnutrition interfered with cell proliferation stimulating cell quiescence. However, retinoids treatment intensified cell quiescence, decreased IEC-6 apoptosis and stimulated cell differentiation associated with the activation of the molecular pathways of ATF2, MAPK/ERK/JNK, IL-6, Hedgehog, PI3K/AKT and NF-kB.

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Three approaches were made to study the interrelations between thyroid function and vitamin A in avian metabolism. These were a histological assessment of the activity of the thyroid gland as it is affected by hypervitaminosis A, the effect of hypervitaminosis A on the incubation time of eggs injected with different levels of vitamin A alcohol and palmitate, and finally the effects of hypervitaminosis A on thyroidal uptake and release of radioiodine by chickens. These studies yielded the following observations. CI) Histological -measurements indicated that dietary hypervitaminosis A can depress the secretion of thyroid stimulating hormone. (2) The effect of hypervitaminosis A on thyroid activity as affected by environmental temperature. (3) Vitamin A palmitate and alcohol prolong incubation time. The alcohol form has, in addition, an adverse effect on embryonic development which is manifested in hemorrhaging and malformation of the embryo. (4) Vitamin A in excess may either depress or stimulate thyroidal uptake of radioiodine depending upon an unidentified component of the environment. (5) Vitamin A in excess affects the endocrine system. This is indicated by an increase in size of the thyroid and adrenal glands and a decrease in size of combs and testes. / Land and Food Systems, Faculty of / Graduate

Cervical cancer is the fourth most common female malignancy worldwide and is substantively higher in low-income and middle-income countries. In South Africa, cervical cancer is a leading cause of mortality amongst women. The anti-cancer actions of the vitamin D and its numerous metabolites are an active field of research. The family of vitamin D metabolites regulate numerous cellular pathways which are implicated in tumorigenesis. Pre-clinical studies and clinical studies have yielded promising, although conflicting results in various cancers. Some healthy and cancerous tissue express an autocrine vitamin D metabolising system (VDMS) which is capable of tightly regulating intracellular metabolism and growth. The VDMS expresses activating and inactivating enzymes and a vitamin D receptor (VDR). At the cellular level, the VDMS can activate and inactivate vitamin D precursors and transduce signals to the nucleus to regulate various cell health genes, including cell growth, metabolism and survival. Healthy and cancerous cervical tissue express a VDMS. The anti-cancer actions of cholecalciferol, an early precursor of activated vitamin D, is poorly studied in cervical cancer. This study aimed to characterise cholecalciferol’s action on cell growth, cell death and the VDMS in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Cell proliferation was enumerated by Ki67 nuclear antigen and the cell cycle profile analysed by flow cytometry. Apoptotic cell death was investigated by measuring mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and evaluation of DNA damage markers by flow cytometric analysis. The biochemical markers microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II) and lactate dehydrogenase (LDH) were also measured by flow cytometry and spectrophotometric analysis to identify autophagic cell death and necrosis, respectively. In addition, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis, autophagic cell death and necrosis. The VDMS in SiHa control and experimental cultures were characterised by the investigation of intracellular gene and protein expression of the cholecalciferol activating (CYP2R1 and CYP27A1) and inactivating (CYP24A1) enzymes, and the VDR. Qualitative microscopical analysis evaluated classical characteristics of cell death and semi-quantitative analysis of apoptosis was performed. Data were analysed using a one-way ANOVA and Bonferroni post-hoc test. p < 0.05 was considered statistically significant. A significant decrease in cell count and cell viability was identified in SiHa cell cultures treated with 2600 nM cholecalciferol. Furthermore, significant increase in biochemical markers of apoptosis were identified including, decreased ∆Ψm; PS exposure; terminal caspase activation; and nuclear damage at 2600 nM cholecalciferol treatment of SiHa cell cultures. Moreover, the biochemical findings were supported by brightfield microscopy and TEM, which observed classical apoptotic features viz. membrane blebbing, apoptotic bodies and nuclear fragmentation. Also, a significantly increased number of apoptotic cells were enumerated. There was no evidence of autophagic cell death and necrosis. Additionally, a significant increase in 25-hydroxylase (CYP2R1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Conversely, a significant decrease in 1α-hydroxylase (CYP27B1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, significant increase in both 24-hydroxylase (CYP24A1) and VDR expression at gene and protein levels were observed in 2600 nM experimental SiHa cultures. In conclusion, cholecalciferol exerts growth inhibition and apoptosis in SiHa cells at 2600 nM. This is accompanied by CYP2R1 and VDR upregulation which suggests autocrine activation to calcidiol and intracellular nuclear signalling, respectively. It is therefore hypothesised that calcidiol synthesised de novo binds to VDR and induces apoptosis in SiHa cell line. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020. / Chemical Pathology / MSc (Chemical Pathology) / Restricted

Solar ultraviolet-B (UVB) radiation is capable of converting 7-dehydrocholesterol (7-DHC) to previtamin D3 (preD3), which undergoes thermal isomerization to produce vitamin D3. Further ultraviolet irradiation of preD3 will produce other photoproducts, including lumisterol3, tachysterol3, and 7-DHC. Continued exposure to UVB results in a photoequilibrium of these photoproducts. During the winter months, people living at latitudes greater than 32° north or south are incapable of converting cutaneous 7-DHC to preD3. Because an increased zenith angle creates a longer path-length for UVB radiation to traverse through the atmosphere, ozone can absorb a much greater proportion of this radiation. Given the absorption spectrum of tachysterol3 which absorbs UV radiation up to 340nm, it was hypothesized that winter sunlight which contains UV radiation between 315nm and 340nm would be able to convert tachysterol3 to preD3. Each hour between sunrise and sunset, ampules containing 50g/mL tachysterol3, lumisterol3, and 7-DHC in 100% ethanol were exposed to solar radiation. These samples were chromatographed on a normal phase chromatographic column. Results revealed that tachysterol3 was efficiently converted to preD3 from sunrise to sunset, whereas as 7-DHC and lumisterol3 were not. Exposure of tachysterol3 to sunlight throughout the day revealed that tachysterol3 began converting to preD3 at sunrise at 8am and the peak conversion occurred between 10:00 and 13:00. PreD3 was generated from tachysterol3 until sunset. No preD3 was observed when 7-DHC or lumisterol3 were exposed at the same time. From this data, it is feasible to use tachysterol3 to produce preD3 in a topical preparation during winter.

Der Zusammenhang zwischen Fettleibigkeit und Vitamin-D-Mangel sowie der Fettleibigkeit und der nicht-alkoholischen Fettlebererkrankung wird in der Literatur beschrieben, ist jedoch noch nicht gänzlich geklärt. In der Therapie der NAFLD wird als Ultima ratio eine bariatrische Operation erwogen. In der vorliegenden Arbeit wurde überprüft, welchen Einfluss eine bariatrische Operation auf den Outcome der Leberparameter, des NAFLD-Scores, des Ferritinwertes, des Vitamin-D-Spiegels sowie den Gewichtsverlauf bei Patienten mit NAFLD hat. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass die meisten statistisch ausgewerteten Parameter hinsichtlich einer Fettlebererkrankung postoperativ besser ausfielen. Es kann somit von einem positiven Einfluss der bariatrischen Operation auf die Fettlebererkrankung ausgegangen werden. In dieser Arbeit kam es zu einer Besserung der Vitamin-D-Spiegel (Gruppe 2 und 4) postoperativ, jedoch bedarf es weiterer größerer Studien um den Zusammenhang zwischen Vitamin-D-Spiegel, der NAFLD und der bariatrischen Operation zu klären. / There is a correlation between obesity and vitamin D deficiency and also between NAFLD and obesity. In this study the effect of bariatric surgery was tested on the outcome of liver parameters, the NAFLD-Score, Ferritin values, vitamin D values and the weight loss after bariatric surgery. In summary, this study was able to show that most of the statistically evaluated parameters regarding fatty liver disease turned out better postoperatively. Thus, a positive influence of bariatric surgery on fatty liver disease can be assumed. In this thesis an improvement of the vitamin D levels (group 2 and 4) postoperatively occurred, but further larger studies are needed to clarify the relationship between vitamin D levels, NAFLD and bariatric surgery.

The determination of total thiamin, free thiamin, thiamin di phosphate (TDP) plus thiamin triphosphate (TTP), and total α- hydroxyethylthiamin (HET) levels in rat brain in control, deficient, oxythiamin- (OTH) and pyrithiamin- (PTH) treated rats was accomplished. It was found that the TDP + TTP/thiamin ratio observed in the thiamin-deficient, OTH- and PTH-treated rats was constant and did not differ from the ratio observed in the control rat brains. The brain levels of TDP + TTP decreased to 39% and 12% of the control thiamin levels in deficient and PTH-treated rats respectively. The brain HET and TDP + TTP levels of the OTH-treated rats were not significantly different from the controls. The HET levels in the PTH rats decreased significantly ( α = 0.005) by treatment day 4 and decreased to one-seventh of the control values in the terminal stages. A significant drop in the HET levels from the control levels was interpreted to mean that the pyruvate utilization was significantly impaired in the brain.

Vitamin D deficiency (25(OH)D < 20 ng/mL) is likely to be present in about 40% of veterans and is associated with much higher health care costs and service use. The prevalence of vitamin D deficiency is likely to be higher in certain subgroups such as ethnic minorities, those who are chronically ill, and nursing home residents. The lack of adequate sunlight exposure and poor dietary intake are common contributors to this deficient state. Moreover, vitamin D deficiency has also been noted in individuals taking vitamin D supplements within the recommended daily intake. To achieve a 25(OH)D value in the normal range (30-100 ng/mL), many studies indicate a much higher daily oral intake than currently recommended is needed. Inadequate vitamin D dosing may account for failure of some studies to show a benefit. Testing for vitamin D insufficiency levels remains suboptimal and serial monitoring in veterans to assess if a vitamin D-replete state has been achieved also remains less than adequate. The lack of evidence-based guidelines for testing and monitoring has hampered optimal management of this very common condition. The cardiovascular, immunologic, anti-infective, and oncologic benefits of a vitamin D-replete state are becoming recognized. Achieving a vitamin D-replete state may prolong longevity. Achieving adequate vitamin D status in US veterans is an important health measure that should be undertaken.

Vitamin D and calcium play major roles in our bone health in addition to roles in tissues and cells. These roles in tissues and cells are associated with some cancers. The purpose of this study was to determine attitudes of cancer patients towards their dietary intakes of vitamin D and calcium. A questionnaire was completed by 128 volunteers (mean age=53.5 years±16.6, 94 women, 34 men) from the Montgomery Cancer Clinic in Montgomery, Alabama. Cancer-reporting participants (n=59) were likely to agree more (p=0.048) to the statement, “I try to eat healthy every day” compared to non-cancer reporting participants (n=69) on a 5-point Likert scale; similar results occurred for the statement, “I have a healthy diet” (p=0.050). Participants without cancer consumed more fish (salmon, tuna, halibut, etc.) than cancer-reporting participants (p=.035). Women and cancer-reporting participants were more concerned with eating healthy and obtaining vitamin D and calcium than non-cancer participants and men.