Natural Compounds As Selective Inducers of Apoptosis In Human Melanoma Cells

Melanoma is the deadliest form of skin cancer notorious for its aggressiveness and chemo-resistance to standard anti-cancer therapy. Most of the current chemotherapeutic agents e.g. doxorubicin, cisplatin, taxanes and etoposide are highly toxic to non-cancerous cells. In this thesis my objective was to evaluate the effects of a new wave of natural products: pancratistatin (Hymenocallis littorale), dandelion root extract (DRE, from Taraxacum Officinale) and curcumin (from Curcuma longa) on chemo-resistant melanoma cells. I also investigated the mitochondria-targeting potential of tamoxifen (an estrogen antagonist) in sensitizing melanoma cells to the effects of pancratistatin and curcumin in combinatorial treatments. All the natural products are efficient in inducing apoptosis in melanoma cells. Interestingly, pancratistatin and curcumin were very effective when combined with tamoxifen (eliciting a synergistic response), and are non-toxic to normal fibroblasts. Therefore, this thesis provides evidence of potential anti-cancer therapies and non-toxic combinations to this deadly cancer

Epithelial morphogenesis during mammary gland development and tumourigenesis

Epithelial cells are the functional building blocks of many tissues and organs, and the most frequent origin of human cancers. Epithelia have been extensively characterized with regards to architecture, localization of different cell types, specified cellular functions, gene and protein expression patterns that govern these functions, and changes to their properties during development and cancer.In this thesis I broaden the understanding of cell dynamics using the mouse mammary gland as an in vivo model as it pertains to epithelial morphogenesis and tumourigenesis. The pleiotropic protein Numb is known to regulate multiple proteins that are involved in various processes required for epithelial morphogenesis. We therefore hypothesized that Numb must also be important for epithelial morphogenesis. To test this, we developed a mouse in which Numb was conditionally deleted in the mammary gland to study epithelial morphogenesis during puberty. Numb-deficient mammary glands displayed significant delays in ductal elongation during puberty. Notably, we attributed this to changes in cell shape and size, which led to an increase in mammary ductal cell density compared to control wild-type ducts. To ascertain how an increase in cell density correlated with reduced ductal elongation we performed mathematical modelling and laser ablation to examine the effect of cell tension. We demonstrated that Numb-depleted cells had reduced cortical tension that impacted luminal cell packing, resulting in reduced ductal elongation during pubertal mammary gland development. To further support a direct role for Numb and test the importance of different Numb domains in pubertal mammary development, we used a new technique involving intraductal injection with electroporation to deliver cDNAs of various Numb mutants into the mammary gland to rescue the Numb-null phenotype.To understand epithelial morphogenesis in the context of cancer, we examined the cell dynamics between normal and tumour cells during the mammary cancer initiation. While breast tumourigenic cells have been widely characterized, there is still a blackbox regarding the interactions that occur between transformed breast cells and normal cells during tumour onset, and how that influences cell processes in both cell types. Combining in vivo studies with new image analysis tools, we investigated cell competition between normal and oncogene-expressing cells using a doxycycline-inducible MMTV-PyMT model of breast cancer. We discovered that the presence of normal cells restricts the tumour progression to solid ducts, and that normal cells decline as tumourigenesis continues. We mapped proliferation and apoptosis profiles during early mammary tumour initiation. Global apoptosis within normal-PyMT populations was highest at tumour onset when lesions are still duct-like, and decreased over the course of tumour progression. Furthermore, global proliferation within normal-tumour populations showed an inverse trend as it increased as lesions become more solid.We found that the cellular context and interactions between normal and cancer cells influences their survival and proliferative properties. By using cell segmentation to map the interactions between normal and tumour cells we discovered that tumour cells are twice as likely to undergo apoptosis when surrounded by other tumour cells than when surrounded by other normal cells; but tumour cells were most proliferative in the presence of normal neighbours. Overall, this highlights previously unappreciated dynamics between normal cell and incipient cancer cells at the normal-tumour interface during mammary cancer initiation.Dans la présente thèse, j'élargis la compréhension de la dynamique cellulaire en utilisant comme modèle la glande mammaire de souris pour l’étude de la morphogenèse épithéliale et de la tumorigenèse.La protéine pléiotropique Numb régule de nombreuses voies de signalisation cellulaires impliquées dans la morphogenèse épithéliale. Nous avons donc émis l'hypothèse qu’elle serait également importante dans la morphogenèse épithéliale.Pour évaluer cela, nous avons développé un modèle de souris transgénique dans lequel Numb était supprimée spécifiquement dans la glande mammaire afin d'étudier la morphogenèse épithéliale pendant la puberté.Les glandes mammaires déficientes ont présenté un retard significatif dans l'élongation des canaux lors de la puberté. Nous avons attribué ce phénomène à des modifications de la forme et de la taille des cellules, qui ont conduit à une augmentation de la densité cellulaire des canaux mammaires par rapport aux canaux témoins. Pour déterminer la corrélation entre l'augmentation de la densité cellulaire et l'allongement canalaire réduit, nous avons procédé à une modélisation mathématique avec ablation au laser pour mesurer l'effet de la tension cellulaire. Nous avons démontré que les cellules appauvries en Numb ont une tension corticale réduite, ce qui a un impact direct sur le resserrement des cellules luminales, réduisant ainsi l'allongement des canaux lors du développement de la glande mammaire pubertaire. Afin de fortifier davantage le rôle direct de Numb et de vérifier l’importance de ces différents domaines protéiques dans le développement mammaire pubertaire, nous avons utilisé une nouvelle technique consistant en l’injection par voie intra-canale avec électroporation pour livrer des ADNc de divers mutants de Numb dans la glande mammaire afin de restituer le phénotype d’appauvrissement de Numb. Pour comprendre la morphogenèse épithéliale dans le contexte du cancer, nous avons examiné la dynamique entre les cellules normales et tumorales au cours de l’initiation du cancer mammaire. Bien que les cellules tumorales du sein aient été largement caractérisées, les interactions entre les cellules du sein transformées et normales lors de l'apparition de la tumeur, et comment cela influence les processus biologiques dans les deux types de cellules demeure un mystère. En combinant des études in vivo avec de nouveaux outils d'analyse d'images, nous avons étudié la compétition cellulaire entre les cellules normales et les cellules exprimant un oncogène à l'aide du modèle inductible de cancer du sein MMTV-PyMT. Nous avons découvert que les cellules cancéreuses concurrencent avec les cellules normales au cours de leur progression vers des canaux solides. Nous avons également cartographié les profils de prolifération et d’apoptose lors de l’initiation précoce de tumeurs mammaires. L'apoptose globale dans les populations normales de PyMT était la plus élevée au tout début de la formation de la tumeur, alors que les lésions ressemblaient encore à des conduits, et diminuait au cours de l'évolution de la celle-ci. De plus, la prolifération globale dans les populations de tumeurs normales montre une tendance inverse, puisqu'elle augmente à mesure que les lésions deviennent plus solides. Nous avons aussi constaté que le contexte cellulaire et les interactions entre les cellules normales et cancéreuses influaient leur survie et leurs propriétés prolifératives. En utilisant la segmentation cellulaire pour cartographier les interactions entre les cellules normales et les cellules tumorales, nous avons découvert que les cellules tumorales sont deux fois plus susceptibles de subir l'apoptose lorsqu'elles sont entourées de cellules tumorales que lorsqu'elles sont entourées d'autres cellules normales, mais, ne proliféreront qu'en présence de voisins normaux.Globalement, cela met en évidence une dynamique jusqu'alors inconnue entre les cellules normales et les cellules cancéreuses lors de l'initiation du cancer mammaire

Dilemmas in considering β-thalassemia status in subjects with borderline HbA2 values: a pilot study in Eastern India

Interpreting hemoglobin disorders by high performance liquid chromatography can sometimes deceptive, especially with borderline HbA2 values. It is often problematic, especially in antenatal cases if the partner is a known thalassemia trait. We tested for underlying β-thalassemia mutations in 24 subjects with borderline HbA2 values (between 3.0%-4.0%). Amplification refractory mutation system-polymerase chain reaction was used to detect the five common Indian β-thalassemia mutations: [IVS-I-5 (G>C), Cod 15 (G-A), Cod 8/9 (+G), Fr. 41/42 (-TTCT) and Cod 26 (G-A)]. β-globin gene sequencing was performed if no mutation was detected. β-globin gene defect was not identified in any of the samples. There was no presence of any of the five common mutations in the small cohort. The average value of HbA2 in 24 normal samples was found to be 3.96. The average values for mean cell volume and mean cell hemoglobin (MCH) were found to be 82 and 28.8 pg respectively. Among these 24 normal samples, 13 had MCH below 27 pg and 11 had MCH above 27 pg. On the contrary, one thalassemic family was screened, in which the father of an HbE-β thalassemia patient was found to have HbA2 3.1, being a β-thalassemia carrier. Mutation analysis should be offered to all at-risk couples with borderline HbA2, especially those with values between 3.5% and 4.0% and microcytic hypochromic indices. As, cases with some specific mutational background or clinical condition shows abnormally low HbA2, so mutation screening should be performed in other partner if one partner found to be carrier or patient of thalassemia

Comprehensive assessment of meta-analysis and contingent valuation technique for sustainable management of wetland of Middle Ganga Plain

In this study, the functions and threats of Suraha Tal Wetland are identified by the stated preference method and weightage is given according to their rank. The objective of the study is to determine the total economic value of Suraha Tal Wetland. The direct value can be drawn from the market price and from a survey of the stakeholders. Suraha Tal Wetland is also famous for the presence of the Jai Prakash Narayan Birds Sanctuary, which makes it a biodiversity-enriched area. The indirect value has been drawn from a review of the literature on Suraha Tal Wetland and the relevance of this literature is justified through the comprehensive meta-analysis (CMA) software. The total valuation of the wetland has been calculated. The paper concludes with suggestions for a few management strategies for better wetland management

A FACS Based Case Study on Two HbE-β Thalassaemia Members of a Family, Having Similar Mutational Background

In this report we have tried to explain the reasons behind the difference in the pattern of transfusion requirement between two members of a family with similar β-globin mutation. The father and younger son both are HbE-β, but the father never had transfusion, whereas the younger son takes transfusion monthly. Mother and the elder son are HbEE without any history of transfusion. β-globin mutations of all family members were determined by ARMS-PCR. These were reconfirmed by direct sequencing of β-globin gene. Father and younger son were found to be Cod 26 (G-A)/IVS 1-5 (G-C), whereas mother and elder son were found to be Cod 26 (G-A)/Cod 26 (G-A). XmnI sequencing also revealed that all members of the family were CC. Then, flow cytometry study of red blood cells (RBCs) was performed to measure the oxidative stress of the RBCs. This study was also done on the light and dense fractions of the RBC population of the father and younger son. It was seen that the younger son suffers severe oxidative stress, which can be explained by his higher transfusion requirement. From our work, we have established the importance of taking oxidative stress of RBCs into consideration to explain the clinical manifestation and progression of haemoglobin related diseases like thalassaemia

Differential regulation of plasma proteins between members of a family with homozygous HbE and HbEβ-thalassemia

In this report we’ve compared the plasma protein profiles of 4 individuals in a family. Father and the younger son both are hemoglobin (Hb) Eβ-thalassemic {Cod 26 (G-A)/IVS 1- 5 (G-C)}, but the father never requires transfusion, whereas the younger son requires monthly blood transfusion. Mother and the elder son are HbEE {Cod 26 (G-A)/Cod 26 (GA)} without any history of transfusion. Proteomic study was done on the plasma fraction of the blood following ammonium sulphate precipitation. Proteins were separated by 2D-gel electrophoresis, expression of proteins compared by densitometry and proteins identified by tandem MALDI mass spectrometry. Proteins responsible in hemolysis, hypercoagulation and hemoglobin scavenging have shown differential regulation, establishing the relation between the differences in the levels of plasma proteins with the progression of the disease phenotype, manifested in the extent of transfusion dependence of the patient

Benzaldehyde-induced developmental genotoxicity triggers both neural and non-neuronal cells including the cells of immunity in Drosophila melanogaster

Although benzaldehyde, an aromatic aldehyde, has been declared safe for uses in food, conflicting reports exist regarding its genotoxic and cytotoxic potentials in organisms. Our present study is the first attempt to evaluate the effects of exposure of benzaldehyde on the entire course of development of a eukaryote model organism, Drosophila melanogaster. Total time required for the initial appearance of the third instar larvae, pupae and adults increased dose dependently with the increasing dietary concentration of benzaldehyde. Exposure of flies to each concentration of benzaldehyde caused dose-dependent and significant reductions in the population of pupae and young adults of the fly. Developmental inhibition was associated with dose dependent and significant structural aberrations of larval polytene chromosomes like ectopic pairing, inversion, fusion, etc., and deformities of hemocytes and neuroblasts and death of hemocytes. As much as 34% (SD ± 1.76)-52% (SD ± 1.7) and 18% (SD ± 2.5)-40% (SD ± 3.38) hemocytes and neuroblasts, respectively, underwent nuclear deformations in response to dietary exposures of flies to BA 100–1000 mg/l. Moreover, 16% (SD ± 0.52)-31% (SD ± 1.97) and 19% (SD ± 0.3)-33% (SD ± 1.78) hemocyte mortalities in response to BA 100–1000 mg/l were determined by two cell viability assays. Thus our study revealed that benzaldehyde was genotoxic to Drosophila melanogaster larvae that might be responsible for larval cell death and their subsequent developmental retardation. As this fly possesses substantial genetic homology with human, possibility of developmental inhibition of the later due to exposure of this chemical during pregnancy may not be ruled out