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Coming dissertations at Uppsala university

  • Stelar Performance Under Drought : Regulation of Developmental Robustness and Plasticity of the Arabidopsis Root Xylem Author: Prashanth Ramachandran Link: Publication date: 2020-02-06 08:14

    Plants have evolved genetic mechanisms to sense, modulate and modify developmental programs in response to the changing environment. This brings forth challenges in stably generating tissue patterns while simultaneously allowing amenability. Gene perturbation studies have identified molecular regulators that control fate specification and differentiation of various tissues. However, we lack a complete understanding of how these processes are influenced by the environment. In this thesis, using Arabidopsis xylem as a model, I show that developmental regulators that function in maintaining a stable growth pattern are also involved in the manifestation of phenotypic plasticity. We found that the generation of a robust xylem developmental program is dependent on a feed forward loop between components of the auxin signalling pathway and the master regulators of xylem development, class III Homeodomain Leucine-Zipper (HD-ZIP III) transcription factors (TFs). By directly activating an auxin signalling activator (MP) and repressor (IAA20), the HD-ZIP III TFs facilitate stable xylem patterning and development. We also show that alterations to the HD-ZIP III mediated xylem developmental program were caused non-cell autonomously by changes in levels and signalling of a key regulator of abiotic stress response, abscisic acid (ABA). The suppression and enhancement of ABA signalling resulted in lower and higher levels respectively of mir165, a known post transcriptional regulator of HD-ZIP III levels. Under conditions of enhanced ABA signalling we found that ABA also acts cell autonomously through master regulators of xylem differentiation, VASCULAR RELATED NAC-DOMAIN (VND) transcription factors. Furthermore, we show that both cell autonomous and non-cell autonomous pathways are employed during water deficit conditions to alter xylem morphology and differentiation rate, likely to enhance water uptake. Taken together, our results show that ABA’s influence on evolutionarily conserved development regulators is important for xylem developmental plasticity. The identification of genetic regulators that control plant phenotypic alterations to limited water availability such as those identified in this thesis will be important to develop tolerant varieties that can survive the extended periods of drought caused by the alarming rise in global temperatures.

  • Solid-state nanopores : fabrication and applications Author: Shuangshuang Zeng Link: Publication date: 2020-01-31 10:13

    Nanopores are of great interest in study of DNA sequencing, protein profiling and power generation. Among them, solid-state nanopores show obvious advantages over their biological counterparts in terms of high chemical stability and reusability as well as compatibility with the existing CMOS fabrication techniques. Nanopore sensing is most frequently based on measuring ionic current through a nanopore while applying a voltage across it. When an analyte passes through the pore, the ionic current temporarily changes, providing information of the analyte such as its size, shape and surface charge. Although many magnificent reports on using solid-state nanopores have appeared in the literature, several challenges still remain for their wider applications, which include improvement of fabrication reproducibility for mass production of ultra-small nanopores and minimization of measurement instability as well as control of translocation speed and reduction of background noise. This thesis work explores different techniques to achieve robust and high throughput fabrication of sub-10 nm nanopores for different applications.

    The thesis starts with presenting various fabrication techniques explored during my PhD studies. Focused ion beam method was firstly employed to drill nanopores in free-standing SiNx membranes. Sub-10 nm nanopores could be obtained with a focused helium ion beam. But the fabrication throughput was limited with this technique. A new fabrication process combing electron beam lithography (EBL) with reactive ion etching/ion beam etching, which is compatible with the existing CMOS fabrication technology, was developed to realize a high throughput, mass production of nanopores in free-standing SiNx membranes. However, the smallest size that could be controllably achieved with this process was around 40 nm, which is still far from sub-10 nm in size required for, e.g., DNA sequencing. Finally, by using anisotropic etching of single-crystal silicon in KOH solution, sub-5 nm truncated pyramidal nanopores were mass produced with good process controllability in a silicon-on-insulator (SOI) substrate. In addition, nanopore arrays were also successfully fabricated using a modified EBL based fabrication process.

    Then, several sensing application examples using either single nanopores or nanopore arrays were investigated. Translocation of nanoparticles, DNA and proteins were demonstrated using the fabricated single nanopores or nanopore arrays in a single freestanding membrane. Moreover, the kinetics and mechanism of the lipid bilayer formation in nanopore array, aiming to prevent non-specific adsorption, were studied using ionic current measurements. In addition, individual addressability of a solid-state nanopore array on separated freestanding membranes was realized by integrating microfluidics and a customized multiplexer.

  • Too close for comfort : The role of Contact-Dependent growth Inhibition (CDI) in interbacterial competition and cooperation Author: Marcus Wäneskog Link: Publication date: 2020-01-31 06:21

    Contact-Dependent growth inhibition (CDI) was discovered in 2005 in the E. coli isolate EC93. Since then our knowledge of CDI systems and their impact on bacterial communities have increased exponentially. Yet many biological aspects of CDI systems are still unknown and their impact on complex microbial communities have only just begun to be studied. CDI systems require the function of three proteins; CdiBAI. The outer-membrane transport protein, CdiB, allows for the transportation of the toxin delivery protein CdiA to the cell surface of an inhibitor cell. Through a contact- and receptor-dependent interaction with a target cell the toxic C-terminal domain of CdiA is cleaved off and delivered into the target cell were it mediates a growth arrest. Different CdiA-CT domains encodes for diverse toxic activities, such as nucleases and membrane ionophore toxins. Each unique CdiA-CT toxin has a cognate immunity protein (CdiI) that binds and neutralize against its toxic activity, thus preventing a possible self-inhibition.

    In this thesis I have studied the effect of CDI system(s) on both single cell and population level, within both intra- and interspecies bacterial communities. The findings presented here shows that multiple class I cdiBAI loci within a cell can function in a synergetic manner and act as versatile interbacterial warfare systems able to inhibit the growth of rival bacteria, even when CdiA expression is low. We also show that class II CdiA receptor-binding domains can mediate broad-range cross-species toxin delivery and growth inhibition, even when a non-optimal target cell receptor is expressed at a low level. Additionally, we show that the cdiA gene supports the expression of two separate proteins. The full-length CdiA protein, able to mediate an extracellular toxin delivery, but also the discrete CdiA-CT toxin domain. This stand-alone CdiA-CT expression was stress-dependent and together with its cognate CdiI immunity protein functioned as a selfish-genetic element. Moreover, we show that CDI systems can increase bacterial stress tolerance via an extracellular toxin delivery between kin-cells. This stress tolerance phenotype only occurred under conditions when we also observed a selective degradation of the CdiI immunity protein. Therefore, we suggest that a selective CdiI degradation allows for a sub-population of cells to self-intoxicate, thereby becoming transiently dormant, which confers an increase in stress tolerance. The findings presented in this thesis collectively suggest that CDI systems could function as a pseudo-quorum sensing system able to mediate behavioral changes and stress tolerance within a sub-population of cells in a bacterial community.