The Drosophila neuroblast undergoes repeated asymmetric cell divisions that produce one daughter cell that assumes a neuronal fate and another that remains a neuroblast. During mitosis, the neuroblast polarizes the conserved Par polarity complex to the apical cortex, which is responsible for segregating fate determinants to the basal cell cortex. Polarity is accompanied by orientation of the mitotic spindle through the proteins Pins, Mud, and Dlg to ensure that the cleavage furrow properly segregates the fate determinants. The adaptor protein Inscuteable coordinates these two pathways. In my work, I have addressed how asymmetrically dividing cells are dynamically polarized during the cell cycle and how the resulting polarity is coupled to spindle position. To address how neuroblast polarity is dynamically controlled, I identified the protein Inscuteable as a continuously polarized cue for Par complex localization during mitosis. Inscuteable and Bazooka, a member of the Par complex, interact directly and form a complex that is regulated by the mitotic kinase Aurora A. Regulating this interaction allows for cell-cycle dependent establishment of polarity and for the subsequent loss of polarity after the cell divides. To investigate how Par complex directed polarity is connected to spindle position, I investigated the effect of Inscuteable binding on the spindle orientation ability of the protein Pins. When bound to Inscuteable, Pins' spindle orientation activity becomes repressed. Inscuteable competes with Mud for Pins binding and represses the Gai-Pins-Mud signaling pathway. Function of the parallel Pins-Dlg pathway remains unaffected. This repression behavior may allow differential timing of spindle attachment (through Dlg) and spindle shortening (through Mud) pathways that ensures correct alignment of the mitotic spindle. I was able to model the spindle orientation behavior of Pins using a synthetic protein containing activation sites that have different affinities for the activator. Changing the number and affinities of these activation sites leads to different response profiles that mimic the ultrasensitive behavior of Pins using a non-cooperative mechanism. Together, these regulatory mechanisms cooperate to allow for spatial and temporal control of polarity and for physical connection of polarity to the mitotic spindle. This dissertation includes previously published and unpublished co-authored material.

This work is concerned with the automatic understanding of evaluative text. We investigate sentence level opinion polarity prediction by assigning lexical polarities and deriving sentence polarity from these with the use of contextual valence shifters. A methodology for iterative failure analysis is developed and used to refine our lexicon and identify new contextual shifters. Algorithms are presented that employ these new shifters to improve sentence polarity prediction accuracy beyond that of a state-of-the-art existing algorithm in the domain of consumer product reviews. We then apply the best configuration of our algorithm to the domain of movie reviews. / Science, Faculty of / Computer Science, Department of / Graduate

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During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1

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This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex.

Polarized cell movements are essential to the cell rearrangements that occur during morphogenesis. In Xenopus, cell polarity is reflected in the directional cell intercalations that drive the morphogenetic movements characterizing gastrulation. While these cell behaviours are well described, the molecular mechanism underlying this cell polarity is unknown. PAR-3 is a multi-domain scaffolding protein and a key regulator of cell polarity. I have isolated a cDNA encoding Xenopus PAR-3 and generated several mutant constructs, each lacking a conserved domain. Initial characterization of GFP-tagged PAR-3 in A6 cells demonstrates localization to points of cell-cell contact in epithelial sheets, as well as at the leading edge of migrating cells. PAR-3 constructs lacking the CR1 or PDZ1 domain fail to compartmentalize properly and are found in the cytoplasm. Eliminating the PDZ3 domain resulted in a loss of contact inhibition. Mutation of the aPKC phosphorylation site created a membrane hyper-accumulation phenotype. Together these data suggest that the CR1 and PDZ1 domains mediate membrane compartmentalization that is modulated through aPKC phosphorylation, while the PDZ3 domain is required for contact inhibition. In embryos, PAR-3 is expressed throughout gastrulation and over-expression of PAR-3 inhibits blastopore closure indicating a requirement during gastrulation. Inhibition is relieved when the construct lacking the CR1 domain is over-expressed. PAR-3 was localized to the cell periphery in axial mesoderm. Localization was abolished with deletion of the CR1 domain indicating that membrane targeting of PAR-3 is required for gastrulation and this targeting is dependent on oligomerization of PAR-3. This investigation also suggests PAR-3 functions independent of the PAR complex in Xenopus embryos indicating involvement of a different PAR-3 signaling pathway.