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- Ilkhanizadeh, Shirin
(author)
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Microenvironmental control and molecular detection of neural stem cell state and fate
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Neural stem cell growth and specialization is dependent on microenvironmental cues, such as growth factors, oxygen levels and substrate stiffness. Together these cues define the genetic and epigenetic program of neural stem cells. In our studies we have manipulated these cues in order to achieve better control of neural stem cell state and fate. In addition, we introduce a novel way of detecting neural stem cells and cancer stem cells in culture. Luminescent conjugated oligothiophenes (LCO) are short polymers, which cross the cell membrane and generate luminescence in the range of wavelengths of ordinary fluorescent probes. In paper I we present a novel LCO, p- HTMI, for detection of live neural stem cells but not differentiated cells. p-HTMI represents a new generation of molecular probes for immediate and non-invasive live detection of specific cell types. In paper II we show that the NADH-sensitive transcriptional co-repressor, CtBP, controls a switch between neuronal and nonneuronal cell fates in oxygen and BMP rich environments. CtBP molecularly integrates microenvironmental levels of BMP and oxygen through Notch mediated regulation of anti- and pro-neural genes. Furthermore, we manipulated growth factor presentation in the stem cell microenvironment through inkjet printing technology. In paper III, we show that inkjet printed macromolecules remained biologically active when printed on hydrogels and control the differentiation of neural stem cells. Finally, in paper IV we demonstrate that soft substrates with brain-like stiffness promote the maturation of neurons. This defines substrate stiffness as a crucial parameter in neuronal differentiation of neural stem cells.
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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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