I. The function, structure and response to environmental factors of a cytoplasmic protrusion found in rat leukemia cells IRC 741 were investigated. A greater rigidity and adhesiveness of the protrusions, as compared to the main cell body, was demonstrated by time-lapse cinematography, and this functional difference was correlated with localized ultrastructural differences in the cytoplasm and on the cell surface, and with higher negative surface-charge density, as shown by cell electrophoresis. The formation or maintenance of the cytoplasmic protrusions depended on adequate nutritional conditions, and was interfered with by diminished intercellular contact, by environmental temperatures below 37°C, by alkaline pH and by calcium-ion depletion. The protrusion appears to represent a type of adhesive organelle not previously described in either cancer cells or normal cells. II. In the course of the above work, a method was developed whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde-fixation. / Science, Faculty of / Zoology, Department of / Graduate

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A Thesis Submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy, / The cell type exclusively dealt with in this thesis is the human blood neutrophil, which is also referred to in the text as polymorphonuclear leukocyte (PMN). The experimental work in this thesis has been accomplished using one immunological and a number of biochemical investigative techniques. The former is the Boyden technique (Boyden, 1962) for the quantitative assessment of leucocyte motility. / IT2018

A number of viruses exhibit microtubule-based bidirectional transport following cell entry. This behavior raises three questions: First, what mediates their transport along microtubules? Second, how do viruses recruit the motor proteins? Finally, how do they go to the right place by bidirectional transport in a variety of cell types with different microtubule organizations? We studied these questions with Adenovirus 5 (Ad5), a virus with well characterized, dynein-mediated minus transport mechanism. One form of plus end directed motor, Kif5C, has been reported to disrupt Ad5 capsids at the Nuclear Pore Complexes(NPC), but the mechanisms and roles of microtubule plus end-directed Ad5 transport prior to this stage are largely unknown. Here we performed a RNAi screen of 38 microtuble plus end-directed kinesins, which implicated Kif5B (kinesin-1 family) in plus-end directed Ad5 transport, along with several other forms of kinesin. Kif5B knockdown caused an accumulation of Ad5 particles near the centrosomes in human pulmonary epithelial A549 cells. This effect was strongly enhanced by blocking Ad5 nuclear pore targeting with Leptomycin B and supports a role for Kif5B in Ad5 transport prior to NPC docking. Kif5B RNAi was rescued by expression of any of the three Kif5 orthologues. We also found that Ad5 directly interacts with kinesin-1 via the capsid subunit Penton Base in a PH-independent manner. Together with our earlier studies, these findings reveal that Ad5 has evolved distinct recruitment mechanisms for cytoplasmic dynein and at least one form of kinesin-1 during early infection. Despite clear evidence for short-range linear microtubule-associated Ad5 transport, we found the overall behavior of most Ad5 particles to be stochastic at a larger time scale, by mean-square-displacement (MSD) analysis. We named this behavior "assisted diffusion''. In consistent with this mechanism, Ad5 was able to maintain a normal nuclear targeting after we displaced centrosomes away from the nucleus by inhibiting CDK1 in late G2 cells. We also directly observed Ad5 switching from microtubule based transport to nuclear targeting from a microtubule near the nucleus. Kif5B RNAi dramatically inhibited this novel microtubule-based random-walk/“assisted-diffusion” mechanism. By super resolution microscopy, we found a more local distribution of NPC attached Ad5 over the entire nuclear surface under conditions of Kif5B knock down. We propose that adenovirus uses independently-recruited kinesin and dynein to fully explore the cytoplasm to search for and dock at the nucleus, a mechanism of potential importance for physiological cargoes as well.

One-half million cases of septicemia are reported annually with a mortality of around 35%. Diagnosis depends in part on blood cultures which require one to two weeks. It would be advantageous if an early sign of septicemia were available. Cytoplasmic vacuolation of polymorphonuclear neutropnils is occasionally seen in the peripheral blood smears of patients who have infections. The object of this work was to determine the cause and significance of cytoplasmic vacuolation with the goal of using the occurrence of vacuolation as an early indicator of infection.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...