A Dialogical Narrative Analysis of Voice-Hearers and Emotions

This thesis is the result of a process of Dialogical Narrative Analysis with twelve participants who hear voices that other people cannot hear. It uses the socio-narratology of Arthur Frank to examine their stories about their experiences in the context of the complex and conceptually contested fields of knowledge that are considered relevant to voice-hearing. The 12 voice-hearers present a wide range of ideas and explanatory frameworks about their experiences. Emotions are deliberately part of this thesis and many stories are as moving as they are complex. The author’s own experiences of these stories is also acknowledged and explored. Whereas much research about hearing voices is biological, psychological or occasionally sociological, this research has many aspects of political exploration and the field is understood as one of conflicting vested interests and ideologies. Many ethical considerations come to light, especially concerning forms of power and knowledge within mental health professions and services in the UK, but also within the Hearing Voices Movement, which is seen as a vital movement with a variety of possible histories. One possibility suggested by this research is that voice-hearers are able to tolerate, and benefit from, more dialogical approaches and that those that do may find more successful ways to live with their experiences than those who are subject to the strongly monological emplotments of much professionalised knowledge. Another strong theme is the great influence of neoliberal politics over individuals and notions of ‘health’

Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres.

Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation.

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.Herchel Smith Fellowship, Funai Foundation scholarship, Austrian Science Fun

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways

Intra-tracheal multiplexed sensing of contact pressure and perfusion

Incorrect endotracheal tube (ETT) cuff inflation pressure causes significant problems for intubated patients. The technical development and first in vivo use of a smart ETT for measurements at the cuff-trachea interface during mechanical ventilation are described. The intra-tracheal multiplexed sensing (iTraXS) ETT contains integrated optical fibre sensors to measure contact pressure and blood perfusion. The device is tested during mechanical ventilation in a porcine model (N=6). For contact pressure, signals were obtained in all 30 measurements. For perfusion, data could be obtained in all 33 measurements. In the 3 cases where the cuff was inflated to an artificially high-level, blood occlusion is observed

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP–WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX–BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease–associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.J.L. Gallop is supported by a Wellcome Trust Research Career Development Fellowship (grant WT095829AIA). F.  Daste, A.  Walrant, J.R. Gadsby, and J. Mason are supported by an H2020 European Research Council Starting Grant (281971) awarded to J.L. Gallop. Gurdon Institute funding is provided by the Wellcome Trust (grant 092096) and Cancer Research UK (grant C6946/A14492). The Swedish Medical Research Council and the Swedish Foundation for Strategic Research supported the work of M.R. Holst and R. Lundmark. S.F. Lee is funded by a Royal Society University Research Fellowship (grant UF120277). M. Mettlen is funded by grant MH73125 to Sandra L. Schmid (University of Texas Southwestern Medical Center)

Microtubule Organisation in Normal and Colon Cancer Cells

Abstract+ ApicoNbasal" polarisation" of" epithelial" cells" is" essential" for" normal" function," with" the" rearrangement" of" the"microtubule" cytoskeleton" on" differentiation" critical" for" the" establishment" of" polarity." "During" this" process,"microtubules" reorganise" to" form"a"stabilised"array"of"apicoNbasal"bundles"via"a"release"and"capture"mechanism."" Disruption"of"this"array"can"cause"a"loss"of"epithelial"polarity,"which"can"contribute"to" diseases" such"as" cancer." "Microtubule" rearrangements" are"driven"by"their" inherent" dynamics,"which" are" regulated"by"+TIP"proteins." "This"project" aims" to"examine" the" mechanisms" behind" apicoNbasal" array" formation," and" how" they" may" be" compromised" in" colorectal" cancer." " It" has" focussed" on" two" +TIPs;" APC" (which" is" mutated" in" most" colorectal" cancers)," and" EB2" (which" has" also" been" observed" upregulated"in"cancer)." The"role"of"APC"at"the"cortex"was"examining"using"cell"lines"containing"wild" type"or"APC" truncation"mutations." "Microtubule"orientation" at" junctional" sites"was" determined,"with"Nocodazole"regrowth"assays"used"to"determine"cortical"approach."" Additionally,"the"angle"of"the"mitotic"spindles"was"analysed"in"the"different"cell"lines." The" impact" of" EB2" loss" on" microtubule" organisation" was" determined" by" immunolabelling" following" siRNA" treatment." "A" fluorescently" tagged" EB2" construct" was"created"and"used"to"rescue"depleted"phenotypes.""The"effect"of"EB2"depletion" on"microtubule"stability"was"assessed"by"analysing"live"microtubule"dynamics.""The" impact"of"EB2"overexpression"on"cell"migration"was"assessed"by"creating"a"stable"EB2" overexpressing" cell" line" and" live" imaging" sparsely" seeded" cells" and" scratch" assays."" Overexpressing"cells"were"grown"in"3D"culture"to"study"any"effects"on"polarisation." The" TC7" cell" line" containing" truncated" APC" possessed" fewer" microtubules" orientated"perpendicular"to"the"cortex"than"wildNtype"expressing"cells.""This"could"be" rescued"with"full"length"GFPNAPC.""Nocodazole"regrowth"showed"microtubules"in"TC7" cells"would"approach"the"cortex"perpendicularly,"and"then"curl"away.""TC7"cells"also" had"an"increased"proportion"of"misorientated"spindles." " EB2"depletion" caused" a"dramatic" increase" in" cell" size,"and" redeployment"of" EB1" and" the" microtubuleNactin" crosslinking" protein" ACF7" along" the" microtubule" lattice.""These"effects"could"be"rescued"by"transfection"with"mCherryNEB2.""Analysis" revealed" EB2" depleted" cells" possessed" less" dynamic" microtubules." " The" large" depleted"cells"were"found"capable"of"division,"although"some"appeared"to"fail"during" the"process,"with"cell"cycle"analysis"revealing"suggesting"the"potential"induction"of"a" cell"death"pathway." "Overexpressed"EB2" could"be" located" at" the"migratory" leading" edge,"and"was" found" to"promote" cell"migration,"with" cells"moving" at"an" increased" rate" and"wounds" healing"more" quickly." " There"was" also" evidence" of"compromised" tissue"integrity"in"overexpressing"cells"grown"in"3D"culture" The" results" gathered" identified" a" potential" role" for" full" length" APC" in" stabilising" cortical" microtubule" contacts," and" helped" identify" EB2" as" a" central" regulator" of" microtubule"dynamics"and"organisation"during"polarisation.""EB2"overexpression"was" found"to"increase"cell"migration,"and"may"be"able"to"induce"EMT." *[N.B.: Movie quicktime files were attached to this thesis at the time of its submission. Please refer to the author for further details.

Mental Health Nursing and Conscientious Objection to Forced Pharmaceutical Intervention

This paper attempts a critical discussion of the possibilities for mental health nurses to claim a particular right of conscientious objection to their involvement in enforced pharmaceutical interventions. We nest this within a more general critique of perceived shortcomings of psychiatric services, and injustices therein. Our intention is to consider philosophical and practical complexities of making demands for this conscientious objection before arriving at a speculative appraisal of the potential this may hold for broader aspirations for a transformed or alternative mental health care system, more grounded in consent than coercion. We consider a range of ethical and practical dimensions of how to realise this right to conscientious objection. We also rely upon an abolition democracy lens to move beyond individual ethical frameworks to consider a broader politics for framing these arguments