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Molecular authentication and phylogenetic studies of Chinese herbs.January 2009 (has links)
Wang, Yanli. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 90-104). / In English with some Chinese characters; abstract also in Chinese. / Acknowledgement --- p.I / Abstract --- p.III / 摘要 --- p.V / Table of Content --- p.VII / List of Figures --- p.XIII / List of Tables --- p.XV / Abbreviations --- p.XVI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Traditional Chinese Medicine (TCM) --- p.1 / Chapter 1.2. --- The development history and present situation of Traditional Chinese Medicine --- p.2 / Chapter 1.3. --- Modernization of Traditional Chinese Medicine --- p.3 / Chapter 1.4. --- Authentication of Traditional Chinese Medicines --- p.4 / Chapter 1.5. --- Methods for authentication of Traditional Chinese Medicine --- p.5 / Chapter 1.5.1. --- Morphological and histological methods --- p.5 / Chapter 1.5.2. --- Chemical methods --- p.6 / Chapter 1.5.3. --- Molecular methods --- p.6 / Chapter 1.6. --- DNA regions suitable for molecular authentication of Traditional Chinese Medicine --- p.8 / Chapter 1.6.1. --- The chloroplast genome --- p.8 / Chapter 1.6.2. --- Nuclear sequences --- p.9 / Chapter 1.6.3. --- Mitochondrial genome --- p.12 / Chapter 1.7. --- Herb Tu Si Zi --- p.12 / Chapter 1.7.1. --- The identity of Traditional Chinese Medicine Tu Si Zi --- p.12 / Chapter 1.7.2. --- The medicinal values of Tu Si Zi --- p.13 / Chapter 1.7.3. --- Local substitutes of Tu Si Zi --- p.14 / Chapter 1.7.4. --- The need for molecular authentication of Tu Si Zi --- p.15 / Chapter 1.8. --- Traditional Chinese Medicinal herbs from Isodon --- p.15 / Chapter 1.8.1. --- The genus Isodon --- p.15 / Chapter 1.8.2. --- Xi Huang Cao --- p.16 / Chapter 1.8.2.1. --- Identity of Xi Huang Cao --- p.16 / Chapter 1.8.2.2. --- Medicinal values of Xi Huang Cao --- p.17 / Chapter 1.8.2.3. --- Confusions of herb Xi Huang Cao --- p.17 / Chapter 1.8.3. --- Dong Ling Cao --- p.18 / Chapter 1.8.3.1. --- Identity of Dong Ling Cao --- p.18 / Chapter 1.8.3.2. --- Medicinal values of Dong Ling Cao --- p.18 / Chapter 1.8.4. --- The molecular authentication of two Isodon herbs --- p.19 / Chapter 1.9. --- Fagaropsis and Luvunga --- p.20 / Chapter 1.9.1. --- The classification of Rutaceae --- p.20 / Chapter 1.9.2. --- Controversial taxonomic issues with Fagaropsis and Luvunga --- p.21 / Chapter 1.9.3. --- The need of phylogenetic studies of genus Fagaropsis and Luvunga --- p.23 / Chapter Chapter 2. --- Objectives --- p.24 / Chapter Chapter 3. --- Materials and Methods --- p.25 / Chapter 3.1. --- Samples used in this study --- p.25 / Chapter 3.1.1. --- Tu Si Zi (Dodder seeds) --- p.25 / Chapter 3.1.2. --- Isodon herbs --- p.28 / Chapter 3.1.3. --- Fagaropsis and Luvunga --- p.31 / Chapter 3.2. --- Methods --- p.34 / Chapter 3.2.1. --- Sample preparation --- p.34 / Chapter 3.2.2. --- Total DNA extraction --- p.34 / Chapter 3.2.2.1. --- Cetyltriethylammonium bromide extraction --- p.34 / Chapter 3.2.2.2. --- Commercial kit extraction --- p.36 / Chapter 3.2.3. --- DNA amplification --- p.38 / Chapter 3.2.3.1. --- psbA-trnH intergenic spacer --- p.39 / Chapter 3.2.3.2. --- trnL-trnF region --- p.39 / Chapter 3.2.3.3. --- ITS region --- p.42 / Chapter 3.2.4. --- Agarose gel electrophoresis --- p.43 / Chapter 3.2.5. --- Purification of PCR product --- p.44 / Chapter 3.2.6. --- Cloning --- p.46 / Chapter 3.2.6.1. --- Ligation --- p.46 / Chapter 3.2.6.2. --- Transformation --- p.46 / Chapter 3.2.6.3. --- Cell cultivation --- p.47 / Chapter 3.2.6.4. --- Plasmid extraction --- p.47 / Chapter 3.2.6.5. --- Insert confirmation --- p.49 / Chapter 3.2.7. --- DNA sequencing --- p.49 / Chapter 3.2.7.1. --- Cycle sequencing --- p.49 / Chapter 3.2.7.2. --- Purification of cycle sequencing product --- p.50 / Chapter 3.2.7.3. --- DNA analysis --- p.50 / Chapter 3.2.8. --- Sequence analysis and phylogeny construction --- p.51 / Chapter Chapter 4. --- Tu Si Zi (Dodder Seeds) - Results and Discussion --- p.52 / Chapter 4.1. --- Results --- p.52 / Chapter 4.1.1. --- Dendrogram constructed using psbA-trnH intergenic spacer --- p.52 / Chapter 4.1.2. --- Dendrogram constructed using trnL-trnF region --- p.53 / Chapter 4.1.3. --- Dendrogram constructed with the combination of psbA-trnH and trnL-trnF region --- p.59 / Chapter 4.2 --- Discussion --- p.60 / Chapter 4.2.1. --- Identification of DNA markers for Cuscuta species --- p.60 / Chapter 4.2.2. --- Molecular authentication of dodder seeds --- p.60 / Chapter Chapter 5. --- Isodon herbs - Results and Discussion --- p.64 / Chapter 5.1. --- Results --- p.64 / Chapter 5.1.1. --- Dendrogram constructed with internal transcribed spacer 1 --- p.64 / Chapter 5.1.2. --- Dendrogram established with internal transcribed spacer 2 --- p.65 / Chapter 5.1.3. --- Dendrogram established with the whole internal transcribed spacer region --- p.66 / Chapter 5.2. --- Discussion --- p.73 / Chapter 5.2.1. --- ITS region performing as DNA marker for Dong Ling Cao --- p.73 / Chapter 5.2.2. --- The identify of TCM materials of Xi Huang Cao --- p.73 / Chapter Chapter 6. --- Fagaropsis and Luvunga - Results and Discussion --- p.75 / Chapter 6.1. --- Results --- p.75 / Chapter 6.1.1. --- Phylogenetic tree constructed with internal transcribed spacer 1 --- p.76 / Chapter 6.1.2. --- Phylogenetic tree constructed with trnL-trnF region --- p.76 / Chapter 6.1.3. --- Phylogenetic tree constructed with combined of trnL-trnF region and ITS-1 region --- p.77 / Chapter 6.1.4. --- The location of Fagaropsis and Luvunga in 3 different phylogenetic trees --- p.78 / Chapter 6.2. --- Discussion --- p.85 / Chapter 6.2.1. --- Fagaropsis 一 a member of the ´بProto-Rutaceae´ة group --- p.85 / Chapter 6.2.2. --- Luvunga 一 a member of Aurantioideae --- p.86 / Chapter 6.2.3. --- DNA sequencing providing a useful methodology in plant phylogenetic studies --- p.87 / Chapter Chapter 7. --- Conclusions --- p.89 / References --- p.90 / Appendix 1. Sequence alignment ofpsbA-trnH intergenic spacer of dodder --- p.105 / Appendix 2. Sequence alignment of trnL-trnF region of dodder samples --- p.108 / Appendix 3. Sequence alignment of ITS region of Isodon herbs and specimens --- p.117 / Appendix 4. Sequence alignment of ITS-1 region of Rutaceae species --- p.124 / Appendix 5. Sequence alignment of trnL-trnF region of Rutaceae species --- p.129
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The Role of Type-2 Cannabinoid Receptors in Calcification of Atherosclerotic Lesions.Hinshaw, Kaitlyn 01 May 2013 (has links)
Introduction: Atherosclerosis is a chronic inflammatory disease characterized by the buildup of cholesterol, fat and other debris within arterial walls. Atherosclerotic lesions undergo a calcification process with similarities to bone remodeling. In mice, the type-2 cannabinoid receptor (CB2) is known to regulate bone remodeling processes and has also been shown to alter atherosclerotic lesion characteristics. However, the role of CB2 in lesion calcification is still unclear. CB2 modulates bone remodeling by affecting differentiation of osteogenic precursor cells; thus we hypothesize that CB2 alters lesion calcification by altering osteoblastogenesis and osteoclastogenesis of precursor cells of vascular origin. To test this hypothesis, we studied the role of CB2 receptor in mediating osteoclastogenesis and osteoblastogenesis from murine monocyte/macrophage and smooth muscle cell lines in vitro.
Methods: RAW264.7 cells are a murine monocyte/macrophage cell line known to undergo osteoclastogenesis in response to receptor activator of nuclear factor kappa B ligand (RANKL). RAW264.7 cells were cultured in media containing RANKL and supplemented with either CB2 agonists or antagonists. Effects on RANKL-induced osteoclastogenesis were then evaluated by measuring the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP) activity and further verified by microscopic quantitation of multi-nucleate TRAP-positive osteoclasts. MOVAS-1 cells are a murine vascular aortic smooth muscle cell line known to differentiation into osteoblasts when cultured in osteogenic media. MOVAS-1 cells were cultured in osteogenic media supplemented with CB2 agonists or antagonists. Effects on osteoblastogenesis were evaluated by measuring marker enzyme activity. Alizarin red staining was performed to visualize and quantitate effects on calcium deposition.
Results: RAW264.7 cells treated with Win55, 212-2, a nonselective CB agonist, or HU-308, a selective CB2 agonist, displayed a dose-dependent decrease in RANKL-induced TRAP activity. Co-administration of a CB2-selective antagonist (SR144528), but not a CB1-selective antagonist (AM251), blocked this effect. Visual quantitation of multinucleated TRAP-positive cells confirmed Win55,212-2 treatment reduced osteoclastogenesis in RANKL-treated RAW264.7 cells. Induction of osteoblastic differentiation of MOVAS-1 cells, as determined by ALP activity, was enhanced by supplementation with Win55, 212-2 or 2-archidonyl glycerol. Co-administration of SR144528, but not AM251, reduced the induction of ALP activity in MOVAS-1 cells by Win55,212-2 and 2-AG. Alizarin red staining revealed increased calcium deposition in cultures of MOVAS-1 cells treated with Win55,212-2 compared to those cultured in osteogenic medium without Win55,212-2.
Conclusions: These results demonstrate that CB2 activation can affect osteogenic differentiation of vascular cells in vitro. These results support the hypothesis that CB2 signaling promotes lesion calcification by altering the balance of osteoclastic and osteoblastic differentiation of vascular precursors.
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Desenvolvimento e caracterização de comprimidos matriciais de dupla camada contendo ParacetamolLiberal, José Pedro Machado 07 April 2009 (has links)
Mestrado em Controlo de Qualidade / MSc in Quality Control
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Estudo do Envolvimento da Bioactivação Metabólica no Efeito Hiponatrémico da 3,4 - Metilenodioximetanfetamina (Ecstasy)Silva, Daniel Gomes Esteves da 24 September 2008 (has links)
Mestrado em Controlo de Qualidade / MSc in Quality Control / A 3,4-metilenodioximetanfetamina (MDMA; ecstasy ), tal como outras
anfetaminas, tem sido considerada por muitos como sendo uma droga segura . No
entanto estão descritas na literatura muitas respostas de toxicidade, reacções adversas e
mortes relacionadas com a sua ingestão recreativa.
Um dos seus efeitos adversos, potencialmente fatal, é a hiponatrémia. Este efeito foi
relacionado com alterações na secreção da hormona antidiurética (ADH, AVP ou
arginina-vasopressina) desencadeadas pela MDMA. A hiponatrémia foi apontada como
causa possível para numerosas intoxicações severas e por vezes fatais decorrentes da
ingestão desta droga. Estudos recentes in vivo, em humanos saudáveis do sexo
masculino, e in vitro, em hipotálamo isolado de rato, demonstraram que a bioactivação
metabólica da MDMA, nomeadamente a desmetilenação seguida pela O-metilação do
catecol resultante, é crucial para a libertação da AVP quer in vivo quer in vitro.
Para a avaliação da contribuição desta via metabólica para a expressão in vivo do
efeito de hiponatrémia causado pela ingestão da MDMA, é crucial quantificar estes
metabolitos e relacionar o perfil metabólico com a magnitude do efeito hiponatrémico.
Com este objectivo, foi desenvolvido e validado um método de GC-MS/MS para a
quantificação da MDMA e dos seus principais metabolitos, metilenodioxianfetamina
(MDA), 4-hidroxi-3-metoxianfetamina (HMA) e 4-hidroxi-3-metoximetanfetamina
(HMMA), no plasma e na urina.
Para melhor compreender a influência da MDMA e da sua bioactivação na secreção
da AVP foram realizados estudos in vivo em ratos Wistar machos e fêmeas, aos quais
foi administrada MDMA na dose 20 mg/kg.
Nos estudos realizados 1 hora após a administração de MDMA foram avaliados os
níveis plasmáticos de AVP e as concentrações plasmáticas da MDMA e dos metabolitos
MDA, HMA e HMMA. Com estas quantificações foi possível observar, nos ratos de
ambos os géneros, o aumento estatisticamente significativo dos níveis de AVP em
relação aos animais controlo, ao mesmo tempo que não se detectaram correlações
estatisticamente significativas entre os níveis de AVP a MDMA e os metabolitos MDA,
HMA e HMMA.
Nos estudos realizados 24 horas após a administração de MDMA foram avaliados os
níveis plasmáticos e urinários de AVP e as concentrações urinárias de MDMA, MDA,
HMA e HMMA. Os resultados destas determinações demonstraram que apesar de não
se detectarem diferenças significativas nas concentrações plasmáticas de AVP entre os
animais tratados e os animais controlo, existem diferenças estatisticamente
significativas para as concentrações urinárias de AVP, verificando-se que os animais
tratados com MDMA apresentam concentrações urinárias de AVP superiores. Além
disso verificou-se também que os animais tratados excretaram menos urina
relativamente à água ingerida, mostrando o efeito anti-diurético desencadeado pela
AVP. Neste estudo foram também estabelecidas correlações positivas e estatisticamente
significativas entre os níveis de AVP e as concentrações de MDMA, MDA, HMA e
HMMA. A correlação com maior significado estatístico foi estabelecida com o
metabolito HMMA.
Estes resultados permitiram pela primeira vez demonstrar a secreção da AVP em
ratos após a administração da MDMA. Com estes estudos foi possível observar, in vivo,
não só as alterações da secreção da AVP induzidas pela MDMA mas também as
consequências dessas alterações nomeadamente na resposta antidiurética e o
envolvimento desta resposta no efeito de hiponatrémia. Finalmente foi possível observar
a contribuição da bioactivação metabólica para a secreção de AVP.
Estes resultados permitem assim compreender melhor o envolvimento da MDMA e
do seu metabolismo na resposta hiponatrémica. / Although considered as safe drugs by many, exaggerated responses and deaths
have been reported due to 3,4-methylenedioxymethamphetamine (MDMA; ecstasy)
abuse. One of the adverse effects associated with ecstasy intoxications is hyponatremia
that has been related with a disruption on the release of the antidiuretic hormone (ADH
or arginine-vasopressin) and pointed out as the possible cause of numerous severe and
fatal intoxications after intake of this drug. Recent in vivo studies with human healthy
volunteers and also in vitro studies performed with rat isolated hypothalamus have
shown that the metabolic bioactivation of MDMA, namely its demethylenation followed
by O-methylation of the resulting cathecol metabolite are crucial for the release of ADH
both in vivo and in vitro.
For the evaluation of the contribution of this metabolic pathway to the in vivo
expression of the hyponatremic effect of MDMA it is crucial to quantify these
metabolites, and to relate the metabolic profile with the magnitude of the hyponatremic
effect.
For this purpose, a GC-MS/MS method was developed to quantify MDMA and its
main metabolites: methylenedioxyamphetamine (MDA), 4-hydroxy-3-
methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA),
in plasma and urine.
To better understand the influence of MDMA and its metabolic bioactivation in the
secretion of AVP in vivo studies were performed with male and female Wistar rats, the
MDMA dose tested was 20 mg/kg.
In the studies preformed 1 hour after the MDMA administration the plasmatic levels
of AVP and the plasmatic concentrations of MDMA, MDA, HMA and HMMA were
evaluated. The plasmatic concentrations of AVP obtained with the treated animals were
compared with the concentrations obtained with the controls showing a statistically
significant increase of AVP levels in the animals treated with MDMA. Correlations
between the MDMA, MDA, HMA and HMMA and the AVP plasmatic levels were also
preformed. No significant correletions were obtained.
In the studies preformed 24 hours after the administration of MDMA the urinary and
plasmatic levels of AVP were evaluated. The concentration of MDMA, MDA, HMA
and HMMA were determined in plasma and urine. It was also established the ratio
between the volume of ingested water and the volume of excreted urine. The plasmatic
and urinary AVP concentrations obtained in the treated animals were compared with the
concentrations obtained from the controls. This compairison showed significant
increases of the urinary AVP levels in the treated animals. The evaluation of the
correlations between the urinary concentrations of AVP and the urinary concentrations
of MDMA, MDA, HMA and HMMA showed significant correlations between AVP and
MDMA, MDA, HMA and HMMA. The evaluation of the ratio between the volume of
ingested water and the volume of excreted urine showed that the treated animals
excreted less urine in comparison with the ingested water.
The studies performed with urines collected 24 hours after MDMA administration
have shown significant positive correlations between AVP and the concentrations of
MDMA, MDA, HMA and HMMA. The strongest correlation was established between
the concentrations of HMMA and AVP.
With this study it was possible to confirm the in vivo changes in the AVP secretion
profile and relate those changes with the levels of MDMA, MDA, HMA and HMMA.
It was also shown for the first time the induction of the secretion of AVP in male
and female rats, one hour after the administration of MDMA. The consequent
antidiuretic effect can be related with the hiponatremic effect.
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Controlo de Qualidade de PCR - Controlo interno e HACCPOliveira, Ana Elisabete Pereira Correia de 04 June 2009 (has links)
Mestrado em Controlo de Qualidade / MSc in Quality Control
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Avaliação da actividade antitumoral e investigação de potencial actividade estrogénica / antiestrogénica de xantonas e flavonasCamões, Ana Catarina Dias Gonçalves Sobral 09 January 2008 (has links)
Mestrado em Controlo de Qualidade / MSc in Quality Control / Aiming for new compounds with antitumor activity, the synthesis of prenylated xanthones and prenylated
and geranylated flavones was recently achieved on CEQOFFUP. In this work the potential antitumor
activity of these compounds in three tumor cell lines, namely MCF-7 (human breast cancer cells
expressing estrogen receptors (ER+)), MDA-MB-231 (human breast cancer cells non expressing estrogen
receptors (ER-)) and NCI-H460 (non-small cell lung cancer) was evaluated. Structure-activity relationships
were established highlighting the influence of prenylation and geranylation.
Concerning xanthones, prenylation of 3,4-dihydroxyxanthone (XXIX) furnished more potent and selective
derivates for MCF-7 (ER+) cells than the original oxygenated xanthone. Xanthone derivate XP13 showed
the strongest inhibitory effect on the growth of breast adenocarcinoma cell line ER+, MCF-7 (GI50 = 5,3
M). Thus, potential estrogenic/antiestrogenic properties were investigated for this compound. No
proliferative effect at low concentrations was observed for XP13 in experiments performed in steroid-free
medium (RPMI-SFM). However, when high concentrations of XP13 were used, this prenylated xanthone
inhibit cancer cell growth in a dose-dependent manner, being more active on MCF-7 (ER+) cell line than
on MDA-MB-231 (ER -) cell line. This antiproliferative effect was not influenced by the culture medium
(steroid (RPMI) or steroid-free medium (RPMI-SFM)). From these results it can be inferred that XP13 does
not directly act on the estrogen receptor, suggesting that it could interfere with other signaling pathways.
Moreover, XP13 enhanced the growth inhibitory action of 4-hydroxytamoxifen (4-OHT, XIV), a partial
antiestrogen in estrogen sensitive breast cancer cells.
Concerning flavone derivates, none of the six flavones investigated, that were resulted from prenylation
and geranylation of baicalein (BAIC, XIX), presented a higher cytotoxic effect on all tumor cellular lines
(MCF-7 (ER+), MDA-MB-231 (ER -) and NCI-H460) when compared to the original oxygenated flavone
BAIC (XIX). However, monoprenylation in C(7) conduced to a flavone (FP2) with a selective inhibitory
effect on the growth of MCF-7 (ER+) cells. Possible estrogenic/antiestrogenic properties were investigated
for this compound. It was verified that in steroid-free medium (RPMI-SFM) experiments, FP2 presented a
biphasic effect in vitro growth on the ER-positive MCF-7 cell line. Although at low concentrations this
flavone has stimulated cell growth, at high concentrations a cell growth inhibition was observed. Then, the
effect of FP2 in combination with E2 was examinated. Results showed that FP2 suppressed at low
concentrations, the mitogenic effect enhanced by estrogenic stimulation, suggesting a competition for
ERs. Also, the FP2 cancer cell growth inhibitory effect on MCF-7 (ER+) cells was stronger when assed in
steroid free medium, i.e., in the absence of estrogenic stimulation. These results suggest a direct
involvement of estrogen receptor in the proliferative/antiproliferative effect of flavone FP2. Moreover, FP2
enhanced the growth inhibitory action of partial (4-OHT, XIV) and pure (ICI 182,780, XII) antiestrogens in
estrogen sensitive breast cancer cells.
These results were consistent with previous reports of prenylated flavones and disclose for prenylated
xanthones effects compatible with antiestrogenic activity. Thus, the present work represents a promising
contribution for the prevention and treatment of hormone-dependent breast cancer.
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The Effect of Metals on Human Neutrophils.Freitas, Marisa Andreia Carvalho de 15 December 2010 (has links)
Doutoramento em Ciências Farmacêuticas / PhD in Pharmaceutical Sciences
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Design Synthesis and Evaluation of Xanthone Derivatives for Dual Activity: Antitumor and P-Glycoprotein InhibitionPalmeira, Andreia Filipa dos Santos 13 December 2011 (has links)
Doutoramento em Ciências Farmacêuticas / PhD in Pharmaceutical Sciences
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A biophysical approach to Phospholipase A2 activity and inhibition by non-steroidal anti-inflammatory drugsGaspar, Diana Maria Diez 02 December 2010 (has links)
Doutoramento em Ciências Farmacêuticas / PhD in Pharmaceutical Sciences
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Propriedades antioxidantes e biológicas de procianidinas oligoméricas. Caracterização antioxidante de pigmentos antociânicosFaria, Ana Isabel Gonçalves January 2005 (has links)
No description available.
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