<p> Arteriogenesis, the expansion of collateral arteries, is vital process for compensatory blood flow to tissues in response to vascular occlusions. In patients with peripheral arterial disease (PAD), arteriogenesis is crucial for overcoming limb ischemia, but for many, further treatment is required. In the United States alone, approximately 80,000 individuals lose limbs to this disease each year. Thus, it is critical to understand the mechanisms regulating arteriogenesis. Indirect evidence suggests that mitogen-activated protein kinase kinases 1 and 2 (MEK1 and 2) are involved in arteriogenesis, but this has not been directly tested. To do this, we used a mouse model of hindlimb ischemia, femoral artery ligation (FAL), treated with a specific allosteric MEK1 and 2 inhibitor (PD0325901). Whereas control mice showed increased myogenesis, angiogenesis, and arteriogenesis, mice dosed daily with PD0325901 failed to recover. In order to examine for any temporal regulation, mice were treated days 8 to 28 post surgery. Interestingly, mean arterial luminal area increased. In a complimentary experiment, mice were treated out to 7 days post surgery, treatment was stopped, and tissues were collected at day 28 post surgery. Although muscle tissue had recovered by this time, mean arterial luminal area remained low relative to controls, suggesting a critical window of MEK1 and 2 signaling being necessary for recovery. Surprisingly, results from MEK2^{&ndash;/&ndash;} mice also fail to undergo arteriogenesis after surgery, indicating this effect may be specific to MEK2 alone. BrDU-injected mice co-stained with either CD31 or &alpha;SMA show that loss of MEK2 predominantly affects endothelial cells within the arteries. Lastly, equivalent results are shown in mice lacking tumor endothelial marker 8 (TEM8). These results indicate MEK2 activity is required for arteriogenesis, and show the first known physiological role for TEM8. In addition, the results have implications in the current use of MEK1 and 2 inhibitors for anti-cancer therapy, as these drugs may affect remodeling arteries. Our results also have potential implications for future therapies for PAD, as MEK2 activation after blockage could stimulate arterial growth, preventing the need for amputation.</p><p>

There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

<p> Integrins are trans-membrane receptors that form focal contacts and mediate the attachment of the smooth muscle cell cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) regulates the recruitment and assembly of focal adhesion proteins. It has been established that integrin proteins and FAK play important roles in cell adhesion, tension generation, and mechanotransduction. Integrins and FAK are abundant in smooth muscles; however, the function of FAK in gastric smooth muscle cell contractile regulation remains unclear. To better understand the role of FAK in gastric fundus smooth muscle contractile regulation, we performed two major projects: (1) addressing gastric smooth muscles contractile regulation in cholinergic neurotransmission by FAK phosphorylation (Chapter 2) and (2) characterizing the change in Ca²⁺ sensitization proteins distribution at focal adhesion sites under electric field stimulation induced cholinergic neurotransmission and FAK phosphorylation regulation (Chapter 4). In carrying out the first project, the contractile responses of murine gastric smooth muscles were determined using standard myobath &ndash; isometric force transducer techniques and phosphorylated or total proteins determined by automated capillary electrophoresis and immunoblotting by Wes Simple Western. For the second project, we developed smooth muscle tissue <i>in situ </i> proximity ligation assay (PLA) for the quantitative PLA analysis of protein-protein interaction and protein phosphorylation (Chapter 3). From these studies, we revealed a novel role of FAK in gastric fundus smooth muscle contractile regulation by cholinergic stimulation. We also demonstrate quantifiable tissue level PLA that can be extended and applied to studies of protein-protein interaction, and protein phosphorylation in various tissues and signaling pathways.</p><p>

<p> Celsr1 is an atypical cadherin central to the asymmetric cell-cell complexes that define planar cell polarity (PCP). Previous work has shown that Celsr1 undergoes bulk endocytosis during cell division in the basal layer of the mouse skin. Here, we report the unexpected finding that Celsr1-mediated intercellular complexes remain intact during mitotic internalization, resulting in uptake of Celsr1 and associated PCP components into dividing cells from their neighbors in a process known as trans-endocytosis. Our observations suggest that the bulk of this internalized pool of Celsr1 is targeted for degradation. Furthermore, Celsr1 internalized from neighboring cells carries with it additional core PCP proteins, including the posteriorly-enriched Fzd6 and anteriorly-enriched Vangl2. However surprisingly, Vangl2 originating from the dividing cell is excluded from mitotic endosomes and remains associated with the membrane. Overexpression of Vangl2 in vitro is sufficient to interfere with Celsr1 internalization, and mitotic internalization of Celsr1 within the skin is delayed at anterior cell surfaces. We propose that Vangl2 stabilizes Celsr1 at the membrane and its dissociation from Celsr1 is a prerequisite for Celsr1 turnover. Together our results indicate that mitotic turnover of Celsr1 depends on the displacement of Vangl2 from PCP complexes and results in the non-autonomous turnover of PCP proteins from neighboring cells.</p><p>

<p> <i>Sushi Domain Containing 2 (SUSD2)</i> encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. A clinically annotated HGSOC tissue microarray was stained with an anti-SUSD2 antibody. Patients with tumors that had weak SUSD2 staining had a shorter median survival (31.7 months) compared to patients that had tumors with strong SUSD2 staining (49.1 months; p value = 0.0083).</p><p> To investigate the role of SUSD2 in HGSOC, stable OVCAR3, OVSAHO and KURAMOCHI cell lines were established through transfection of shRNA targeted to <i>SUSD2</i> transcripts (SUSD2 knock-down [KD] cell lines) or non-targeting shRNA (SUSD2 NT) cell lines. Boyden chamber and wound healing assays demonstrated that OVCAR3, OVSAHO and KURAMOCHI SUSD2 KD cells migrated at significantly higher rates than their SUSD2 NT counterpart cell lines. RT-qPCR and western immunoblot analysis indicated an inverse relationship between SUSD2 and well characterized mesenchymal genes, such as <i>TWIST1, ZEB1</i> and <i>CHD2.</i> In addition, OVCAR3 and KURAMOCHI SUSD2 KD spheroids displayed increased mesothelial clearance ability compared to SUSD2 NT spheroids.</p><p> To explore the potential for SUSD2 to inhibit late-stage HGSOC metastasis, female athymic nude mice were injected with either OVCAR3 NT or OVCAR3 KD cells. Fewer nodules were observed in the pancreas and omentum of the OVCAR3 NT mice when compared to the OVCAR3 KD mice. Furthermore, OVCAR3 KD mice had a significantly shorter median survival compared to OVCAR3 NT mice (175 days compared to 185.5 days, respectively; p-value = 0.0047).</p><p> KURAMOCHI lysate was immunoprecipitated for SUSD2-associated immunocomplexes and subjected to liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis, yielding a list of candidate SUSD2-interacting proteins associated with RNA processing. Immunofluorescence analysis of OVCAR3, KURAMOCHI and SKBR3 cells and western immunoblot analysis of their subcellular extracts revealed SUSD2 to be present in cell nuclei, mitochondria and cytoplasm; however, SUSD2 was relatively less abundant in SKBR3 nuclei.</p><p> Our findings suggest that increased <i>SUSD2</i> expression in HGSOC impedes metastasis, consistent with prolonged survival observed in HGSOC patients with high <i>SUSD2</i>-expressing primary tumors. The differences in subcellular distribution between HGSOC cells and breast cancer cells may explain alternate functions of SUSD2 in different cancers.</p><p>

Pas de résumé en français / Pas de résumé en anglais

<p> Durotaxis is a mechanism of directed cell migration in which cells respond to gradients of extracellular stiffness. While durotaxis has been predominantly characterized in a subset of mesenchymal cells, the potential for cancer cells to durotax has not been well defined. Numerous studies have demonstrated the role of diffusible factors in cancer cell migration and metastasis. However, given the gradual stiffening of many tumor microenvironments, we hypothesized that a durotactic mechanism might also contribute to the migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing a stiffness gradient that mirrors the physiological stiffness encountered by cells throughout a variety of tissues. Customized MATLAB software permitted rapid acquisition of positional data for migrating cells. The automation of cell tracking allowed for large sample sizes and therefore a more robust statistical analysis than previously used to evaluate durotaxis. Durotaxis assays identified two glioblastoma lines, a metastatic breast cancer line, and fibrosarcoma line that are responsive to changes in extracellular stiffness. Of interest was our finding that cancer cells showed strong durotactic behavior when occupying the softest region of the stiffness gradient with decreasing responsiveness as cells occupied increasingly stiff regions of the gradient. These observations suggest that durotaxis is influenced by the stiffness of a cell&rsquo;s local environment, with soft substrates increasing durotaxis efficiency in the cancer cell lines evaluated. Furthermore, we determined that PI3K inhibition was sufficient to inhibit glioblastoma chemotaxis but not durotaxis, suggesting that alternative signaling is used to respond to durotactic directional cues. Lastly, we evaluated the protrusion and retraction dynamics of cells on a stiffness gradient and have identified a unique mode of discontinuous migration exhibited by these cells. Based on our observations, we developed a durotaxis model to suggest how discontinuously migrating cells can respond to a gradient by moving towards regions of increasing stiffness.</p><p>

<p>Nuclear functions in eukaryotic cells are regulated by the NuA4 histone acetyltransferase complex. This is a highly-conserved protein complex that regulates multiple vital nuclear functions like the cell cycle, DNA repair and transcription. Gene expression is regulated by this complex though epigenetics by adding acetyl groups to lysine residues on histone H4. This affects the expression of genes in the regions of the chromosome where the addition occurred. The planarian flatworm Schmidtea mediterranea is thought to be, effectively, immortal due to its amazing ability to regenerate and maintain pluripotent stem cells throughout its life time. Better understanding of the genes that control differentiation and pluripotency is needed. Humans have gene homologs to the planarian counterparts; therefore, it could be possible to gain knowledge about our own stem cells from these worms. I have identified planarian homologs of 15 proteins in the human NuA4 complex (Ruvbl2, Morf4l2, Mrg15, Epc1, Tip60, Trrap, Gas41, Ruvbl1, Brd8, Yl-1, Baf53a, Dmap1, Ing3, hEaf6-1 and hEaf6-2) and silenced them by RNA interference (RNAi) to examine the role of the complex in stem cell maintenance and regeneration. The RNAi method involves feeding the worms double-stranded RNA with a sequence matching the gene of interest to target the destruction of the mRNA expressed from that gene, thus knocking down its expression. I will observe two groups of RNAi worms; a regenerating group and a homeostasis group. The regenerating will be cut following the knockdown to observe how well they restore their lost tissue. The homeostasis group will be fixed and stained to mark mitotic cells and find out if the stem cells are dividing normally. I will also use in-situ staining to determine where each of these genes are being expressed. I hypothesize that knockdown of these important regulatory complex genes will result in reduced regenerative ability and that the worms? stem cell population will not be properly maintained.

<p> Extracellular adenosine signaling via the adenosine receptor 2B (AR_2B) is a potent profibrotic and proinflammatory molecule that has been shown to contribute to tissue remodeling and excessive matrix protein production under chronic conditions in kidney disease. Investigation of the adenosine pathway on interstitial renal fibroblasts is lacking even though fibroblasts are a major source of ECM production and contribute to the pathogenesis of renal failure in late stage kidney disease. Analysis of mRNA from the interstitial renal fibroblast cell line NRK-49F revealed these cells express AR₁ and AR_2B. Utilizing highly selective small molecule agonists and antagonists specific to each individual adenosine receptor, it was determined by mobilization of intracellular cAMP, both receptors were functional. AR_2B was observed to be the dominant receptor under high &mu;M concentrations of agonism leading to increased pro-inflammatory and profibrotic mediators. Specifically, agonism of AR_2B lead to increased levels of &alpha;SMA and other inflammatory and fibrotic mediators, indicating a transdifferentiation of fibroblasts to a more activated myofibroblasts stage under strong and sustained adenosine signaling. The key fibrotic mediator TGF-&beta;1 was observed to be increased with adenosine signaling while antagonism of AR_2B suppressed TGF-&beta;1 mediated increases in &alpha;SMA and fibronectin, suggesting complementary signaling pathways with significant crosstalk. It was further observed that AR_2B receptor antagonism suppressed TGF-&beta; receptor mediated SMAD binding element associated transcriptional activity the suppression of TGF-&beta; receptor signaling and transcriptional activity was associated with decreased SMAD4, a critical protein necessary for proper formation of the SMAD complex transcription factor. Taken together these data highlight the impact of adenosine signaling on proinflammatory and profibrotic endpoints in renal fibroblasts and describe for the first time direct inhibition of TGF-&beta; receptor signaling by antagonism of the AR_2B receptor.</p><p>

<p> A defining characteristic of alphaherpesviruses is the establishment of lifelong latency in host sensory ganglia with occasional reactivation causing recurrent lytic infections. Much remains unknown regarding the cellular and viral mechanisms involved in HSV exit from latency. We hypothesize that VP16 recruits chromatin-remodeling enzymes to immediate early gene promoters on compact-latent chromatin as a necessary step for reactivation. In order to test this hypothesis, a robust in vitro assay in which HSV latency can be established in neurons was required. In this dissertation, I explored the use of a human sensory neuron cell line as a novel in vitro model of HSV-1 latency and reactivation. HD10.6 cells were derived from embryonic human dorsal root ganglia and immortalized by a tetracycline-regulated v-myc oncogene. HD10.6 cells mature to express a sensory neuron-associated phenotype when treated with doxycycline which suppresses proliferation mediated by the v-myc oncogene. Infection at a low MOI in the presence of acyclovir results in a quiescent infection resembling latency in matured cells. HD10.6 cells provide a novel context in which to study the host and viral mechanisms of HSV-1 latency establishment, maintenance, and reactivation.</p><p>