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Digital archive of theses discussed at the University of Pisa


Thesis etd-02272015-130800

Thesis type
Tesi di dottorato di ricerca
Thesis title
In vivo effects of natural compounds on stem cells using a simple animal model
Academic discipline
Course of study
tutor Prof.ssa Batistoni, Renata
  • alkaloids
  • Berberine
  • Chelidonine
  • Chelidonium majus
  • Planarian
  • Regeneration
  • Sanguinarine
  • Stem cells
Graduation session start date
Because of the presence of a unique stem cell system and amazing regenerative capabilities, planarian flatworms represent an experimental model suitable to assess in vivo the effects of different molecules on stem cell and regeneration dynamics, as well as to identify the molecular mechanisms involved. Furthermore the discovery that planarian stem cells use evolutionarily conserved mechanisms that are surprisingly similar to the mechanisms we observe in mammalian stem cells, makes these invertebrates suitable to screen in vivo the effects of new drugs involved in various aspects of human biology and disease.
My work focused on the analysis of the effects produced on planarians by compounds of natural origin, particularly some major alkaloids (chelidonine, berberine and sanguinarine) contained in the herb Chelidonium majus. The analysis of chelidonine effects provides a basis for preclinical evaluation in vivo of the effects of this compound on physiologically proliferating stem cells. My experimental data demonstrate in fact that chelidonine inhibits regeneration producing significant anti-proliferative effects on planarian stem cells. Conversely, no significant cytotoxic effects on differentiated cells have been detected, supporting the possibility that this alkaloid acts in vivo on cell cycle progression mainly by inhibition of tubulin polymerization.
About berberine, my results demonstrate that this alkaloid perturbs planarian regenerative pattern. Although berberine does not influence cell proliferation/apoptosis, the experiments provide evidence that this compound causes abnormal regeneration of the planarian structures, visualized as visual system abnormalities. On the whole these findings, sustained by RNAi-based investigations, support the possibility that berberine effects in planarians are critically linked to anomalous ECM remodeling.
Finally sanguinarine causes abnormal anterior regeneration, as well as anomalous remodelling of the stump. Real time RT-PCR shows that sanguinarine treatment does not affect proliferation of stem cells while increases the expression levels of markers involved in their maintenance/differentiation and also promotes apoptotosis. I demonstrated that the expression level of H+,K+-ATPase pump, a gene with an important role during head regeneration in planarians, is affected by sanguinarine treatment. H+,K+-ATPase mediates tissue remodelling during regeneration through regulation of apoptosis. These results support the possibility that the sanguinarine-mediated alterations in the membrane polarization, through regulation of ATPase ion channels, are fundamental in determining anatomical polarity, shape and allometric organs scaling during planarian regeneration.
To the best of my knowledge, this study is the first to report the effects of natural alkaloids on stem cells in vivo. I believe that the understanding of molecular mechanisms underlying drugs effects and regeneration processes can be best achieved using a multidisciplinary approach. Development and application of pharmacological approaches to stem cells will undoubtedly lead to identification of additional active drugs potentially useful for therapeutic purpose. Such an approach also strengthens the possible utilization of planarians in the analysis of the effects of drugs and, at the same time, can help to better understand the process of planarian regeneration itself by providing novel information about how proliferation, differentiation and/or morphogenesis and patterning are regulated during this amazing process.