The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

Quantum well infrared photodetectors (QWIP) utilize quantum wells of large bandgap materials to detect infrared radiation. When compared to conventional low bandgap LWIR photodetectors, the QWIP technology offers largest format thermal imagers with much better uniformity. The theoretical part of this study includes the development of a QWIP ensemble Monte-Carlo simulator. Capture paths of electrons to quantum wells are simulated in detail. For standard AlGaAs/GaAs QWIPs, at medium and high E-fields L valley quantum well (QW) is a trap for electrons which causes higher capture probability when compared with InP/InGaAs and GaAs/InGaAs QWIPs. The results suggest that high photoconductive gain observed in InP/InGaAs and GaAs/InGaAs QWIPs is not due to good transport properties of binary barrier material but due to higher &amp / #61511 / -L valley energy separation. The experimental part of the study includes the fabrication and characterization of InP/InGaAs and InP/InGaAsP QWIPs and 640x512 FPAs with the main objective of investigating the feasibility of these material systems for QWIPs. The InP/InGaAs and InP/InGaAsP QWIP detectors showed specific detectivity values above 1010 cm.Hz1/2/W (70K, f/2, background limited). The devices offer higher allowable system noise floor when compared with the standard AlGaAs/GaAs QWIP technology. It is also experimentally shown that for strategic applications LWIR InP based QWIPs have advantages over the standard QWIP technology. The InP/InGaAs 640x512 QWIP FPA reached 36 mK average NETD value at 70 K with f/1.5 optics and 10 ms integration time. The InP/InGaAsP QWIP on the other hand yielded 38 mK NETD histogram peak at 70 K with f/1.5 optics and 5 ms integration time on 320x256 window of the 640x512 FPA.

Spiking representing a periodic melting and solidification in the depth of fusion zone during high-intensity beam welding is experimentally and theorectically investigated in this work . A spike is a sudden increase in penetration beyond what might be called the average penetration line. Many spikes have voids in their lower portions because molten metal does not fuse to the sides of the hole, producing a condition similar to a cold shut in a casting. These defects seriously reduce the strength of the joint. Due to the significant role of specular reflection on absorption, an investigation of the beam characteristics, especially the focal location, on spiking is important. Furthermore, as the cavity base oscillates upward and downward relatively from the focal location, a central region subject to direct irradiation changes instantaneously from maximum to zero and vice versa. This leads to several hundred time difference in energy absorption and strongly periodic melting at the cavity base. Physical phenomenon of spiking is obtained by comparing between the measured and predicted data based on scale anlaysis of transport process near the cavity base and energy absorption as a function of focal location.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 83-95).

Thesis (Ph. D.)--School of Electrical and Computer Engineering, Georgia Institute of Technology, 2004. Directed by D. Scott Wills. / Vita. Includes bibliographical references (leaves 105-111).

Invasion and metastasis are the major causes of treatment failure and high mortality rate in hepatocellular carcinoma (HCC) patients. Cell motility is crucial to tumor invasion and metastasis, requiring the ability of tumor cells to interact with extracellular matrix, which is regulated by integrins and integrin-associated molecules at the focal adhesions. Recent studies have demonstrated the role of β1 integrin (CD29) overexpression in HCC and its correlation with cancer cell invasiveness and metastastic potential, as well as its protective role against cancer cells against chemotherapeutic drug-induced apoptosis, yet the mechanism is not fully known. Focal adhesion proteins serve as binding platforms for additional cytoskeletal and signaling molecules in the CD29 signaling pathway. Recently, Actopaxin has been demonstrated to form complex with numerous molecules at the focal adhesions, including ILK, which interacts with the cytoplasmic tail of CD29. Through these interactions, Actopaxin has been shown to regulate different cellular events, including cell survival, spreading and cell migration. In this study, the role of Actopaxin in HCC was investigated. In particular, its role in the regulation of tumor invasion and metastasis of HCC cells was demonstrated. This study showed that Actopaxin expression was overexpressed in HCC specimens when compared with the adjacent non-tumorous liver, and that its overexpression positively correlated with tumor size, stage and metastasis in HCC specimens. Actopaxin expression was also correlated with the metastatic potential in HCC cell-lines. Functional studies established that overexpression of Actopaxin conferred invasive phenotypes in primary, non-metastatic HCC cells, whereas down-regulation of Actopaxin could revert the invasive phenotypes and metastatic potential of metastatic HCC cells in vitro and in vivo. Suppression of Actopaxin expression was associated with reduced expression of ILK, PINCH, Paxillin and cdc42, whereas expressions of E-cadherin, β-catenin and GSK3β were induced, indicative of a less invasive and invasive phenotype. Conversely, overexpression of Actopaxin in primary, non-metastasis HCC cells accordingly up-regulated the expression of ILK, PINCH, Paxillin and cdc42, and down-regulation of of E-cadherin, β-catenin and GSK3β, suggestive of an enhanced invasive phenotype. The expression of Actopaxin was found to be correlated with CD29 level, indicating that Actopaxin is a CD29-associated protein and involved in CD29-regulated signaling. Finally, Actopaxin down-regulation enhanced chemosensitivity of of HCC cells towards chemotherapeutic treatment. Treatment with Oxaliplatin was enhanced in Actopaxin-deficient HCC cells, which showed a stronger inhibitory effect on cell proliferation and cell cycle progression, accompanied with induction on apoptosis. The enhanced chemosensitivity effect was a collective result of suppression of Survivin protein, β-catenin and mTOR pathways; and up-regulation of p53. To conclude, this study demonstrated for the first time that Actopaxin is involved in HCC invasion, metastasis and chemosensitization, providing the basis to further investigate the potential role of this protein or its downstream effectors as a therapeutic target for inhibiting the development of metastasis and enhancing chemotherapy efficacy to combat HCC, and perhaps other invasive cancers. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

H κινάση εστιών προσκόλλησης (FAK) συμμετέχει στη μεταγωγή μηνυμάτων κατά την κυτταροφαγία. Στα αιμοκύτταρα των εντόμων υπάρχει σε διαφορετικές ποσότητες κατά την ανάπτυξη. Ενεργοποιείται με φωσφορυλίωση στην Tyr-397. Kατά την κυτταροφαγία του βακτηρίου Ε. coli ενεργοποιείται άμεσα. Ανάμεσα στα μόρια που σχηματίζουν σύμπλοκο με την FAK είναι και οι πρωτείνες pinch, Src και οι ΜΑΡΚ. Κάποια από τα παραπάνω σχηματίζουν σύμπλοκο κατά την κυτταροφαγία ενώ για άλλα το σύμπλοκο προυπάρχει. Ο κυτταροσκελετός ακτίνης και τουμπουλίνης χρειάζονται για την κυτταροφαγία, όπως χρειάζεται και η έκκριση. Τέλος διαπιστώθηκε πως κατά την κυτταροφαγία του βακτηρίου Ε. coli και του πεπτιδίου RGD, συμμετέχουν οι πρωτείνες Src, Ras, Rho και JNK. / Focal adhesion kinase (FAK) participates in signal transduction at phagocytosis. Insect hemocytes have FAK in different mounts during development. FAK becomes activated after phosphorylation at Tyr-397. During phagocytosis of E. coli, FAK becomes immediately activated. Pinch, Src and MAPK are some of the molecules which are in complex with FAK. Some of these molecules are in complex with FAK during phagocytosis, but some others there are in complex at any time. Phagocytosis, in order to happen, needs actin and tubulin cytoskeleton. Secretion is also needed for this purpose. Finally, we found that the proteins Src, Rho, Ras and JKN participate in phagocytosis of the RGD peptide and in phagocytosis of the microbe Escherichia coli.