Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20076号 / 医博第4169号 / 新制||医||1018(附属図書館) / 33192 / 京都大学大学院医学研究科医学専攻 / (主査)教授 影山 龍一郎, 教授 斎藤 通紀, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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CLIC1 is an intracellular membrane protein that is unusual in that it can exist in both a soluble and an integral membrane form. The manner in which this protein inserts into membranes is unknown although it is proposed to undergo a change in structure whereby it initially experiences a degree of unfolding and then refolds into its new membrane-bound conformation. This study focuses on the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary structure, the determination of its conformational stability at equilibrium and the establishment of its unfolding kinetics, all under conditions of varying pH, polarity, redox conditions, temperature and ionic strength. CLIC1 was found to be most stable at pH 7.0 / 20oC. The unfolding process is two-state and cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2 kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature to 37oC resulted in the stabilisation of an equilibrium intermediate species under mild denaturing conditions and a destabilisation of the native state. This was further evidenced by an increase in the rate of unfolding of CLIC1 from the native state to the denatured state under these conditions. A state with similar properties to the intermediate species was detected in the absence of urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either the native or denatured state; it is stabilised by salts, has a reduced secondary structure, increased flexibility and a buried Trp35 relative to the native state. The rate of formation of the intermediate species is a slow process which may involve an oligomerisation step. The results from this study provide an interpretation for the structure and mechanism of CLIC1 pore formation in vivo by comparing the effects of the environment on the structure and stability of the protein.

Microcephaly is a debilitating condition in which children are born with small brains. It can be caused by a variety of factors including maternal infection, harmful substance exposure, and genetic mutation. Cerebral cortical development is often severely disrupted in human microcephaly patients. In order to form the billions of neurons which exist in the cortex, efficient and correct neural progenitor division, differentiation, and migration are key. As the center of higher brain function in mammals, reduction in cortical mass is associated with the developmental delays that are symptomatic of microcephaly. Recently, a number of microcephaly causes have been linked to P53-mediated apoptosis of neural cells. The tumor suppressor protein P53 is upregulated in response to mitotic cycle stress, and its activation can trigger cell cycle arrest or apoptotic cell death. In microcephaly, P53 can become activated by mitotic stress and trigger apoptosis to cause the loss of cortical cell numbers that leads to microcephaly. Microcephaly has often been linked to mutations in mitotic proteins that alter neural progenitor division. However, the apical polarity complex Protein Associated with Lin-7 1 (PALS1) – known as membrane palmitoylated protein (MPP)5 in people – has recently been implicated in human microcephaly. PALS1 is integral to establishing polarity in neural progenitors. Deletion of Pals1 in mouse models has also resulted in microcephaly characterized by smaller brains and a global reduction in cortical cell numbers. Interestingly, a cellular phenomenon known as entosis can be caused by polarity disruptions in epithelial cells, and P53 activation has been shown to cause entosis in MDCK cell culture. While entosis is mainly associated with cancer cells, it is a form of competitive cell cannibalism that can eliminate unfit cells from a population. The loss of PALS1 from the developing cortex is known to result in apical polarity complex disruption and microcephaly in mouse models. However, the mechanism by which the loss of PALS1 results in cortical abrogation has yet to be determined. In Chapter 1 of this dissertation, I begin by reviewing cortical development. The normal progression of cortical cells from neural progenitors to fully differentiated neurons is explained in detail. Neural progenitor mitosis in particular is addressed in detail. Furthermore, an overview of microcephaly is provided to address the similarities between known causes of microcephaly. Next, I review the polarity complex proteins and their roles in cortical development. I compare and contrast the cortical phenotypes that have been described when each of the polarity complex proteins has been genetically deleted from the mouse cortex. I go on to review studies that have shown P53-mediated apoptosis in microcephaly in order to address the phenotypic features of microcephaly that are or are not caused by P53 activation. Finally, I provide a brief history of entosis. As a newly discovered cellular process in neural progenitors, the overview of entosis highlights what is known about cell cannibalism and the contexts in which it occurs. Following this background, I describe the experimental aims, hypotheses, and methods for this project in Chapter 2. In Chapter 3, I describe our investigation of three human patients with mutations in the Pals1 gene. One of the patients, possessing a heterozygous de novo nonsense mutation in Pals1 (or MPP5), was diagnosed with microcephaly. In order to model this patient’s phenotype, we generated a heterozygous conditional knockout of Pals1 from the entire mouse nervous system with Nestin-Cre. Through behavioral analysis of these mice, I demonstrate that they are hyperactive and blind, mimicking the microcephaly patient’s symptoms. Furthermore, via analysis of the mouse cortex, I show that heterozygous deletion of Pals1 results in severe microcephaly in mice with a global reduction in cortical cell numbers at both adult and embryonic stages. Importantly, I determine that Pals1 deletion does not result in proliferation or migration defects in the mouse cortex. Instead, loss of PALS1 results in massive apoptotic cell death that affects every cell type produced in the developing cortex. In Chapter 4, I detail our investigation into the mechanism underlying cell death in the PALS1-deficient cortex. By studying dividing neural progenitors at the apical surface in both Emx1-Cre and hGFAP-Cre drive Pals1 conditional knockout models, I demonstrate an as yet undescribed neural progenitor phenotype called entosis. As has been shown in cancer cells, neural progenitor entosis is dynamic and reliant on Rho-ROCK activity to occur. Furthermore, entosis produces observable cell-in-cell structures that persist through outer cell division and cause mitotic delay. I go on to demonstrate P53 activation in Pals1 deficient mouse cortices, and show that genetic deletion of Trp53 significantly rescues microcephaly. Trp53 deletion significantly restores all cortical cell types in addition to ameliorating entosis and mitotic length. This study suggests that P53 activation is a major mechanism by which PALS1 loss results in microcephaly. Overall, these studies show that deletion of Pals1 in mice can mimic microcephaly found in a human patient with a Pals1 mutation. Furthermore, PALS1 loss promotes P53-mediated cortical cell apoptosis. These studies provide the first description of entosis in neural progenitors, and suggest that entosis could be a mechanism for unfit cell removal in the developing cortex. Furthermore, I provide evidence that ROCK inhibition can fully rescue the presence of entosis in PALS1-deficient neural progenitors, and that genetic deletion of Trp53 significantly restores microcephaly pathology after PALS1 loss. These studies open up a field of research into the causes and effects of entosis in neural progenitors, and provide further evidence that apoptotic cell death in microcephaly is largely mediated by P53 activation. / Biomedical Sciences

Orderliness, speed, and rhythm in biochemistry are vital for cellular function. In order to achieve this, cells implement compartmentalization via several methods, one of which is the formation of membrane-less compartments. These compartments, often referred to as “biomolecular condensates”, are understood to be formed by separation of proteins and other biomolecules into dense and dilute phases. While the formation of the resulting protein-rich condensates is fundamental for spatiotemporal organization of biochemistry within the cell, a vast majority of proteins found to phase separate in vitro do so at a concentration an order of magnitude above their endogenous expression levels. Recently, a theoretical study has shown that membrane binding of phase separating proteins can result in phase separation spatially occurring at the membrane well below bulk saturation concentrations. However, much remains unknown about the formation mechanism and function of these condensates. To that end, for my doctoral project, I used a synthetic system composed of supported lipid bilayers decorated with lipid-bound NTA(Ni) to allow for coordination and thus membrane binding of the well-characterized protein FUS via a C-terminal His-tag. Through this model system I found that 2D phase separation of FUS could occur an order of magnitude below the experimentally determined bulk saturation concentration. FUS was able to form dense and dilute phases in 2D and the transition point to form these phases could be controlled by modulating buffer conditions. Additionally, membrane-bound FUS condensates were able to further recruit FUS from the bulk to form a multilayer of protein through a prewetting transition. With this characterization of 2D phase separation of FUS, I then explored a physiologically relevant protein in the form of PAR-3, a fundamental protein of the PAR polarity system, which is necessary for the establishment of polarity in the C. elegans zygote. I found that full-length PAR-3 was able to phase separate under physiological salt conditions with a Csat of 100nM. Further, I identified a C-terminal predicted prion-like domain to act as a driver for phase separation. Additionally, I determined PAR-3’s affinity and specificity for PI(4,5)P2 and found that it could form 2D condensates upon binding to the membrane at physiological concentrations. Furthermore, these condensates were able to recruit PAR-6 alone and PAR-6 in complex with PKC-3 to the membrane, ultimately resulting the reconstitution of the anterior PAR complex which is known to exist in a condensed clustered form in vivo. Taken together, this work provides insight into a mechanism where phase separation can be locally triggered by membrane binding under sub-saturation concentration, offering a robust and potentially universal mechanism by which cells can spatially control phase separation and pattern cellular membranes.

The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis revealed that there was no difference in cell migration when cells were exposed to an exogenous Wnt5a source. However, this did not rule out the possibility that cells were responding in a more mild fashion and polarizing toward a Wnt5a source. Live cell imaging was performed to observe the movement and morphology of limb mesenchyme cell cultures in the presence or absence of a Wnt5a expressing cell bolus. It appears as though the cells orient and move in a random fashion regardless of Wnt5a. However, this in vitro method may not truly recapitulate in vivo events. Future studies aim to develop better methods of observing cell polarization in vitro, including developing better methods to tract the movement of cells and observe “PCP” events. Due to the lack of information gathered from our in vitro studies, an in vivo study was conducted to test if FGF is necessary to polarize limb mesenchyme cells. If FGF is turning on Wnt5a and Wnt/PCP signaling is directing cell polarization, then FGF mutant clones will not migrate toward the AER. Therefore, it is expected that these mutant clones would be unable to undergo directed cell movement and/or cell divisions. Early clonal analysis indicates that a response to FGFs appears to be necessary to direct polarized outgrowth of limb mesenchyme.

Opinion Mining is becoming the fastest growing area to extract useful and insightful information to support decision making. In the age of social media, user’s opinions and discussions have become a highly valuable source to look for users preferences, likes, and dislikes. The industry of wildlife parks (or zoos) is a competitive domain that requires careful analysis of visitor’s opinions to understand and cater for their preferences when it comes to wildlife. In this thesis, an opinion mining approach was proposed and applied on textual posts on the social media platform, Twitter, to extract the popularity, polarity (sentiment), and emotions toward birds and bird types such as owls, sparrows, etc. Then, the thesis provides recommendations based on popularity of birds and bird types and a ranked list of the most desired birds based on consumer emotions toward them. The findings of this thesis can help wildlife parks in the decision-making process on the types of birds to acquire.

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