Epithelial polarity is established by polarity proteins and maintained by polarized trafficking through intricate interdependent mechanisms. In the Drosophila ventral neuroectoderm (VNE), Cdc42/Par complex regulates the retromer retrieval complex to promote apical recycling and maintain epithelial polarity. I have further characterized the retromer by generating antibodies against subunits Vps35 and Vps5. In wild-type VNE, both proteins partially colocalized with early endosomes, sorting endosomes (SEs), and the Golgi network. In the VNE of dominant-negative Cdc42-expressing embryos, Vps35 becomes enriched at SEs, where apical cargo was previously shown to accumulate abnormally. This suggests that in the absence of Cdc42 function, the retromer recognizes cargo in SEs, but recycling is blocked. I have also performed a deficiency screen for genes that cooperate with Cdc42 in the VNE. I identified the ESCRT machinery, which promotes protein degradation, and the exocyst complex, which regulates vesicle secretion at the plasma membrane, as functional interactors of Cdc42.

Epithelial polarity is established by polarity proteins and maintained by polarized trafficking through intricate interdependent mechanisms. In the Drosophila ventral neuroectoderm (VNE), Cdc42/Par complex regulates the retromer retrieval complex to promote apical recycling and maintain epithelial polarity. I have further characterized the retromer by generating antibodies against subunits Vps35 and Vps5. In wild-type VNE, both proteins partially colocalized with early endosomes, sorting endosomes (SEs), and the Golgi network. In the VNE of dominant-negative Cdc42-expressing embryos, Vps35 becomes enriched at SEs, where apical cargo was previously shown to accumulate abnormally. This suggests that in the absence of Cdc42 function, the retromer recognizes cargo in SEs, but recycling is blocked. I have also performed a deficiency screen for genes that cooperate with Cdc42 in the VNE. I identified the ESCRT machinery, which promotes protein degradation, and the exocyst complex, which regulates vesicle secretion at the plasma membrane, as functional interactors of Cdc42.

Retroviral infection requires an initial interaction between the host cell and the virion. This interaction is predominantly mediated by an envelope (env) protein exposed on the external face of the virion. For gammaretroviruses, such as feline leukemia virus (FeLV), the receptor-binding domain (RBD) is located in the N terminus of env. The RBD forms a distinct domain that is sufficient for binding to the host receptor, but is inefficient in the absence of the corresponding C terminal env, Cdomain, sequence in viral infection studies. I developed a series of hybrid constructs between subgroup C, A and T FeLVs that use distinct receptors for infection to determine the role of Cdom in FeLV binding and infection. Using this approach, I have shown that the C domain (Cdom) of FeLV-C env forms a second receptor-binding domain, distinct from its RBD, which is critical for efficient binding and infection of FeLV-C to host cells expressing FLVCR1. I propose that this mechanism of interaction is conserved for all gammaretroviruses. My results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells. Upon infection with FeLV-C virus, cats develop a disease known as pure red cell aplasia (PRCA). This disease is characterized by a defect in erythropoeisis that results in a decreased number of mature erythroid cells. PRCA has been suggested to be caused by the FeLV-C env binding to and disrupting the host receptor, FLVCR1. Interestingly, feline PRCA is clinically identical to Diamond Blackfan Anemia (DBA), a fatal congenital anemia characterized by a specific disruption in erythroid progenitor cellular development. I show that erythroid cells from five DBA patients exhibit low levels of total FLVCR1 transcript expression. In addition, the DBA patients express unique alternatively spliced FLVCR1 transcripts. These alternatively spliced transcripts encode FLVCR1 proteins that are defective in their cellular expression, cell surface localization, and receptor function. Taken together, I propose that the specific anemia observed in DBA is caused by decreased levels of functional FLVCR1 protein due to lowered and alternative splicing of FLVCR1 transcript.

Retroviral infection requires an initial interaction between the host cell and the virion. This interaction is predominantly mediated by an envelope (env) protein exposed on the external face of the virion. For gammaretroviruses, such as feline leukemia virus (FeLV), the receptor-binding domain (RBD) is located in the N terminus of env. The RBD forms a distinct domain that is sufficient for binding to the host receptor, but is inefficient in the absence of the corresponding C terminal env, Cdomain, sequence in viral infection studies. I developed a series of hybrid constructs between subgroup C, A and T FeLVs that use distinct receptors for infection to determine the role of Cdom in FeLV binding and infection. Using this approach, I have shown that the C domain (Cdom) of FeLV-C env forms a second receptor-binding domain, distinct from its RBD, which is critical for efficient binding and infection of FeLV-C to host cells expressing FLVCR1. I propose that this mechanism of interaction is conserved for all gammaretroviruses. My results could have important implications for designing gammaretrovirus vectors that can efficiently infect specific target cells. Upon infection with FeLV-C virus, cats develop a disease known as pure red cell aplasia (PRCA). This disease is characterized by a defect in erythropoeisis that results in a decreased number of mature erythroid cells. PRCA has been suggested to be caused by the FeLV-C env binding to and disrupting the host receptor, FLVCR1. Interestingly, feline PRCA is clinically identical to Diamond Blackfan Anemia (DBA), a fatal congenital anemia characterized by a specific disruption in erythroid progenitor cellular development. I show that erythroid cells from five DBA patients exhibit low levels of total FLVCR1 transcript expression. In addition, the DBA patients express unique alternatively spliced FLVCR1 transcripts. These alternatively spliced transcripts encode FLVCR1 proteins that are defective in their cellular expression, cell surface localization, and receptor function. Taken together, I propose that the specific anemia observed in DBA is caused by decreased levels of functional FLVCR1 protein due to lowered and alternative splicing of FLVCR1 transcript.

We investigated the capacity of human umbilical cord perivascular cells (HUCPVCs) to repair tendon damage in a collagenase tendon injury model in rats. Our hypothesis was that HUCPVCs would be able to produce an organized extracellular matrix and facilitate tissue regeneration in this rat model. HUCPVCs were injected, in collagenase, into the Achilles tendon of nude rats. Harvested tendons showed the presence of HUCPVCs at the site of injury, whose morphology changed from rounded to elongated over the post-injury time of 30 days. Human genes for collagen type 1 and -actin were expressed at all time points and there was also a signi cant increase in the tensile strength and sti ness values at 30 day post-injury in the experimental group. We conclude that HUCPVCs facilitated regeneration in our model, and thus may be a putative cell source for the treatment of tendon and ligament injuries.

We investigated the capacity of human umbilical cord perivascular cells (HUCPVCs) to repair tendon damage in a collagenase tendon injury model in rats. Our hypothesis was that HUCPVCs would be able to produce an organized extracellular matrix and facilitate tissue regeneration in this rat model. HUCPVCs were injected, in collagenase, into the Achilles tendon of nude rats. Harvested tendons showed the presence of HUCPVCs at the site of injury, whose morphology changed from rounded to elongated over the post-injury time of 30 days. Human genes for collagen type 1 and -actin were expressed at all time points and there was also a signi cant increase in the tensile strength and sti ness values at 30 day post-injury in the experimental group. We conclude that HUCPVCs facilitated regeneration in our model, and thus may be a putative cell source for the treatment of tendon and ligament injuries.

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. To better understand the mechanisms of sensitivity and resistance to mitochondrial translation inhibition, we treated TEX leukemia cells with increasing concentrations of tigecycline over 4 months, and selected a population of cells resistant to tigecycline (RTEX+TIG). Compared to their wild type counterparts, the resistant RTEX+TIG cells had an altered metabolic profile with diminished oxidative phosphorylation and a greater reliance on glycolysis. Upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation to wild type levels. At the molecular level, these cells had increased levels of HIF1a. Strikingly, tigecycline-resistant cells had decreased expression of CD34 and CD117, clonogenic growth potential and engraftment capabilities in vivo. Thus, chronic inhibition of mitochondrial translation leads to the establishment of rho-zero-like metabolic phenotype, and is associated with differentiation of leukemia cells.

Recent advances in the field of bionanotechnology have enabled researchers to design a variety of tools to detect, image and monitor biological process in cells. Despite this progress, the limited understanding of nanomaterial-cellular interactions has hindered the widespread use of these nanomaterials in biological systems. In this thesis, we examined the potential effects of metallic nanoparticle geometry on important cellular processes such as membrane trafficking, intracellular transport and subcellular signalling. We found that the size of nanoparticles plays an important role on their ability to interact with the cell surface receptors thus dictating their subsequent ability to activate intracellular signalling cascades. Interestingly, trafficking of these nanoparticles was dependent on their size due to biochemical and thermodynamical constraints. These findings suggest that nanomaterials actively interact with biological systems, thus, directly modulating vital cellular processes. In addition, by utilizing various physical and chemical properties of nanomaterials, we developed a novel class of hybrid nanoscaled carrier systems capable of delivering semiconductor quantum dots (QDs) into live cells without inducing membrane damage. Using biodegradable polymeric nanoparticles, bioconjugated QDs were encapsulated and delivered into trafficking vesicles of live cells. The environmentally sensitive surface charge of the polymeric nanoparticles exhibited positive zeta potential inside acidic endo-lysosomes, thus enabling their escape from the vesicular sequestration into the cytosol. Hydrolytic-induced degradation then releases the bioconjugate QDs for active labelling of subcellular structures for real-time studies. Unlike previously described intracellular QD delivery methods, the proposed system offers an efficient way to non-invasively deliver bioconjugated QDs without inducing cell damage, enabling researchers to accurately monitor cellular processes in real-time. The understanding of both physical and chemical properties of nanomaterials is crucial to the design of biocompatible nanosystems to study fundamental processes in biological systems. Here, we demonstrated that both the size and surface chemistry of nanoparticles can be modified to obtain desired biological responses. Future experimental efforts to study other physical and chemical properties could allow the development of more sophisticated and effective platforms for biological applications.