The microtubule [MT] cytoskeleton of S. pombe is a highly dynamic network of filaments that facilitates intracellular transport, determines cell polarity and plays an essential role in chromosome separation during mitosis. In fission yeast, MTs are nucleated in a temporally and spatially regulated manner from sites called Microtubule Organising Centres [MTOCs], through the activity of both the g-tubulin complex [g-TuC] and the Mto1/2 complex. The Mto1/2 complex determines the localisation of the g-TuC at MTOCs, which change throughout the cell cycle. As cells enter mitosis the cytoplasmic array of MT bundles depolymerise. They are replaced by the intranuclear mitotic spindle and cytoplasmic spindle pole bodyderived astral MTs that in turn give way to the formation of the post-anaphase array. Although much is known about the properties of each type of MT array, the mechanism by which the timing of MT nucleation at different MTOCs is regulated over the cell cycle remains unclear. In the Mto1/2 complex, Mto1 is thought to provide the primary interaction with the g-TuC, and Mto2 functions by reinforcing this interaction. Due to the lack of structural information for the Mto1/2 complex, the molecular mechanism of Mto1/2- mediated assembly of the g-TuC at MTOCs is unknown. The aim of my study is to investigate the possibility that the Mto1/2 complex is able to promote g-TuC assembly by forming a direct template. In addition, I will attempt to determine the molecular role of Mto2 within the Mto1/2 complex and examine ways in which regulation of Mto2 may influence the function the Mto1/2 complex at specific MTOCs. As part of the investigation into the mechanism of Mto2 function, an in vitro analysis of recombinant protein demonstrated that in the absence of Mto1, purified Mto2 is able to self-interact as a tetramer. I have confirmed this interaction in vivo and have also shown that Mto2 forms a dimer as cells enter mitosis. However, in the context of an Mto1/2 complex the significance of the change in Mto2 oligomeric state remains unknown. Hydrodynamic analysis of a truncated form of the Mto1/2 complex suggests that it may form a heterotetramer, a hypothesis which is consistent with the equimolar levels of Mto2 and Mto1 protein within the cell. This information provides some structural insight as to how the Mto1/2 complex may interact with the g-TuC at MTOCs. Further analysis of the Mto1/2 complex revealed that in vivo, the Mto1-Mto2 interaction is disrupted during mitosis. This was found to correlate with the hyperphosphorylation of Mto2, which occurs as cells enter mitosis. Subsequently, an in vitro kinase assay demonstrated that phosphorylation of the Mto1/2 complex reduces the stability of the complex. Mass spectrometry techniques and sequence conservation were used to identify several phosphorylated residues within Mto2 and the ability of these mutants to bind to Mto1 was analysed in vivo and in vitro. In summary, in this study I have uncovered a mechanism which allows fission yeast cells to regulate the nucleation of cytoplasmic MT nucleation in a cell-cycle dependent manner, through a phosphorylation-dependent remodelling of the Mto1/2 complex.

The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 30-37).

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells. / text

Spinning, a task modern western society has eliminated from the list of household chores, was once a staple of every medieval woman’s life. This facet of medieval women’s work should not be neglected, since its shift appears to play a fundamental role in allowing industrialization through relocation of workload. When the new tool, the spinning wheel, was added to a woman’s possible ways of finishing this task, was it universally adopted? A look at the pervasive task from three perspectives shows that this new tool was slowly accepted and did not replace the original tool, the spindle. These perspectives are, first a literary review of how and when the term spinning wheel entered the the the cultural vocabulary. The second is a pictorial review of what type of spinning implements are shown in artwork and when the spinning wheel joins these pictures. An archaeological review of spindle whorls form York and Sweden to see if the introduction of the spinning wheel can be inferred from their inertial values is last. / Access to thesis permanently restricted to Ball State community only / Department of Anthropology

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [69]-76).

Οι υπνικές άτρακτοι είναι χαρακτηριστικές κυματομορφές του ηλεκτροεγκεφαλογραφήματος (ΗΕΓ) του σταδίου 2 του ήσυχου (NREM) ύπνου, που έχουν υπναγωγικό ρόλο και σχετίζονται με διαδικασίες μνήμης και μάθησης. Μια υπνική άτρακτος είναι ομάδα ρυθμικά επαναλαμβανόμενων κυρίως αρνητικών κυμάτων με πλάτος που σταδιακά αυξάνεται και στη συνέχεια, σταδιακά μειώνεται, εντός της ζώνης συχνοτήτων 12-15 Hz (ζώνη συχνοτήτων ρυθμού σίγμα) και με διάρκεια μεταξύ 0.5 και 2 δευτερολέπτων. Η απεικόνιση, για μια κορυφή της ατράκτου, της κατανομής καποιας ηλεκτροεγκεφαλογραφικής μεταβλητής (π.χ. του ηλεκτρικού δυναμικού) στο πεδίο των ηλεκτροδίων καλείται τοπογραφία της κορυφής αυτής. Σκοπός της μελέτης ήταν να διερευνηθεί η τοπογραφία των ταχέων υπνικών ατράκτων κατά μέσο όρο, έτσι από ένα σύνολο 1449 γρήγορων ατράκτων επιλέχθηκαν προς ανάλυση 843 οι οποίες εμφανίζονταν σποραδικά και τουλάχιστον 3 δευτερόλεπτα μακριά από άλλα εμφανή εγκεφαλογραφικά συμβάντα που θα μπορούσαν να τις επηρεάσουν. Οι υπνικές άτρακτοι σημαδεύτηκαν πάνω στο ΗΕΓ οπτικά στην πρώτη, στην μεσαία και στην τελευταία αρνητική κορυφή τους, ακολούθησε προ-επεξεργασία, αποθορυβοποίηση των δεδομένων και εν συνεχεία μεσοποίηση τους στο πεδίο του χρόνου και των συχνοτήτων. Από την ανάλυση προκύπτει ότι η μέση τοπογραφία των ατράκτων στην αρχή, στη μέση και στο τέλος μεγιστοποιείται κεντρικά της κεφαλής και είναι σχετικά σταθερή και στις τρεις περιπτώσεις με μικρή τάση μετατόπισης προς τα εμπρός καθώς το φαινόμενο εξελίσσεται. Ομως, οι μεμονωμένες άτρακτοι εμφανίζουν μεγάλη ποικιλία προτύπων κίνησης του σημείου μεγίστης ισχύος η οποία δεν αποτυπώνεται στο μέσο όρο αλλά γίνεται αντιληπτή εξετάζοντας την τοπογραφία ενός μεγάλου αριθμού ατράκτων πριν τη μεσοποίηση. / Sleep spindles are characteristic waveforms of NREM sleep stage 2 electroencephalogram (EEG) which are involved in the generation and preservation of sleep as well as learning and memory processes. A sleep spindle is a group of rhythmic waves with gradually increasing and then decreasing amplitude (crescendo decrescendo morphology) within the frequency band 11-15 Hz and duration from 0.5 to 2 seconds. The voltage map of all electrodes for a spindle’s peak is called topography of the peak (power distribution visualization). The purpose of this study was to investigate the topography of fast spindles average thus, from a set of 1449 fast spindles, 843 were at least 3 seconds away from other EEG notable events which could affect them and were chosen for the average study. Sleep spindles were marked on EEG signal visually at first, middle and last negative peak, the next steps were preprocessing and denoising and finally averaging in time and in frequency domains. After the analysis we conclude that the average topography of the spindles at their start, middle and end is relatively stable and maximizes at the center of the head with a little tension to move forward as the phenomenon evolves. However, individual spindles show a great variety of movement patterns of the point of maximal electrical potential which patterns are not depicted in their average but can be understood by examining a large number of spindles before averaging.

xiv, 81 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Protein-protein interaction networks translate environmental inputs into specific physiological outputs. The signaling proteins in these networks require regulatory mechanisms to ensure proper molecular function. Two common regulatory features of signaling proteins are autoinhibition and ultrasensitivity. Autoinhibition locks the protein in an inactive state through cis interactions with a regulatory module until it is activated by a specific input signal. Ultrasensitivity, defined as steep activation after a threshold, allows cells to convert graded inputs into more switch-like outputs and can lead to complex decision making behaviors such as bistability. Although these mechanisms are common features of signaling proteins, their molecular origins are poorly understood. I used the Drosophila Pins protein, a regulator of spindle positioning in neuroblast cells, as a model to study the molecular origin and function of autoinhibition and ultrasensitivity. Pins and its binding partners. Gαi and Mud, form a signaling pathway required for coordinating spindle positioning with cellular polarity in Drosophila neuroblasts. I found Pins switches from an autoinhibited to an activate state by modular allostery. Gαi binding to the third of three GoLoco (GL) domains allows Pins to interact with the microtubule binding protein Mud. The GL3 region is required for autoinhibitoon, as amino acids upstream and within GL3 constitute this regulatory behavior. This autoinhibitory module is conserved in LGN, the mammalian Pins orthologue. I also demonstrated that Gαi activation of Pins is ultrasensitive. A Pins protein containing inactivating point mutations to GLs l and 2 exhibits non-ultrasensitive (graded) activation. Ultrasensitivity is required for Pins function in vivo as the graded Pins mutant fails to robustly orient the mitotic spindle. I considered two models for the source of ultrasensitivity in this pathway: cooperative or "decoy" Gai binding. I found ultrasensitivity arises from a decoy mechanism in which GLs 1 and 2 compete with the activating GL3 for the input, Gai. These findings suggest that molecular ultrasensitivity can be generated without cooperativity. This decoy mechanism is relatively simple, suggesting ultrasensitive responses can be evolved by the inclusion of domain repeats, a common feature observed in signaling proteins. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Kenneth Prehoda, Member, Chemistry; Christopher Doe, Member, Biology; Peter von Hippel, Member, Chemistry; Karen Guillemin, Outside Member, Biology

Cervical cancer is reported by the World Health Organisation to be the second most common type of cancer to affect women in poorer socioeconomic countries. Treatment of this pathology remains sub-optimal at advanced stages and continues to be of importance on the research agenda. Previous studies have reported cytotoxic and antiproliferative effects of 2-methoxyestradiol (2-ME) in vitro on a HeLa cervical cancer cell line. These results were promising but use of 2-ME itself is limited due to pharmacodynamic constraints. In an attempt to overcome these, a sulphamoylated analogue of 2-ME, namely 2-ethyl-3-O-sulphamoyl-estra- 1,3,5(10)16-tetraene or compound 19 (C19), was synthesised. In this in vitro study, the induction of a block in mitosis with subsequent culmination of apoptosis and autophagy as types of cells death was investigated after HeLa cells were exposed for 24 hours to a 0.5 μM C19 solution. This was achieved by morphological assessment (fluorescent, Polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) and transmission electron microscopy (TEM)) and flow cytometry (cell cycle progression, cyclin B1 analysis, phosphatidylserine (PS) flip and aggresome formation). Spectrophotometric quantification of the apoptotic initiator and executioner caspases 8 and 3 respectively was done to determine their involvement in the crosstalk between apoptosis and autophagy. Results included the following: (i) PlasDIC microscopy illustrated the appearance of an increased number of cells blocked in metaphase, stress signaling, premature cell shrinkage, hypercondensed chromatin and the presence of apoptotic bodies after C19 exposure. The presence of ghost cells, cell debris and decreased cell density of the treated cells correlated with the autophagy control. (ii) Fluorescence microscopy employing triple staining highlighted an increased lysosomal activity and staining of C19-exposed cells when compared to the control, as well as evidence of apoptotic and metaphase-blocked cells. This is indicative of both the autophagic and apoptotic cell death process. (iii) TEM allowed for examination of the ultrastructure of the intracellular processes, and revealed that apoptotic cells have hallmarks of both autophagy and apoptosis, confirming the results of light microscopy. (iv) Cell cycle analysis demonstrated more cells present in the sub-G1 and G2/M populations, indicating the induction of apoptosis (confirmed with PS fip flow cytometric quantification) and a metaphase block (corroborated by an increased cyclin B1 fluorescence). (v) The increase in autophagosome formation seen on fluorescence- and transmission electron microscopy was confirmed by flow cytometry demonstrating an upregulation of aggresome formation in C19-exposed cells. This investigation demonstrated induction of both types of cells death by this novel compound. (vi) The upregulation of caspases 8 and 3 was demonstrated in the C19-treated cells, indicating apoptosis induction via the extrinsic pathway. (vii) Confocal microscopy demonstrated complete microtubule disintegration in the C19-exposed HeLa cells. Both apoptotic and autophagic cell death mechanisms were induced in C19-treated HeLa cells after spindle abrogation kept the cells in metaphase block. Insight gained into the molecular effect of C19 on HeLa cells may be used as a springboard for in vivo studies, furthering the development of this promising anticancer agent toward clinical application. / Dissertation (MSc)--University of Pretoria, 2012. / Physiology / MSc / Unrestricted

The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal genes that are mutated in the developmental disorder Primary Microcephaly, where affected individuals have smaller brains than expected for the age- and sex-adjusted mean. Orthologues of CDK5RAP2 in the fruit fly (Centrosomin/Cnn) and in fission yeast (Mod20p) have been well characterised and are known to have important roles in maintaining centrosome structure and in regulating microtubule nucleation. CDK5RAP2 shares two evolutionarily conserved domains with Cnn, known as CNN motif 1 and 2. Using the chicken B-cell line, DT40, I have used gene-targeting methods to disrupt both of these domains in CDK5RAP2. This revealed a function for CDK5RAP2 in attaching centrosomes to mitotic spindle poles. Centrosome attachment to spindle poles is mediated by a binding partner of CDK5RAP2, AKAP450. AKAP450 also localises to centrosomes and provides anchorage sites for spindle poles in the centrosome. Disruption of the CNN1 and CNN2 domains of CDK5RAP2 causes mislocalisation of AKAP450 from the centrosome and detachment of centrosomes from spindle poles. My studies in DT40 and in human cell lines revealed that CDK5RAP2 and AKAP450 also cooperate during interphase to maintain the two centrioles in the centrosome as a pair. In addition to a structural role in the centrosome, I also find that CNN motif 1 of CDK5RAP2 plays a role in the cellular response to DNA damage. In the absence of CNN motif 1, cells no longer efficiently arrest the cell cycle in response to damage. Centrosome-mediated mitotic spindle alignment and the DNA damage response have both been implicated in microcephaly. Therefore, defects in these functions of CDK5RAP2 may explain how mutations in cdk5rap2 may lead to microcephaly.