Thesis (M.S.)--Central Connecticut State University, 2004. / Thesis advisor: Cheryl Watson. " ... in partial fulfillment of the requirements for the degree of Master of Science in Biology." Includes bibliographical references (leaves 43-47). Also available via the World Wide Web.

Thesis--PlanA (M.S.)--University of Wisconsin--Stout, 2003. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 135-149). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Phil.)--University of Hong Kong, 1988.

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 84-91). Also available for download via the World Wide Web; free to University of Oregon users.

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The spray freezing into liquid (SFL) and thin film freezing (TFF) processes were utilized to produce 300 nm protein particles with surface areas on the order of 31 - 73 m²/g and 100% protein activities. Despite a cooling rate of ~10²-10³ K/s in SFL and TFF, the particle sizes and surface areas were similar to those observed in the widely reported process, spray freeze-drying (SFD), where cooling rates reach 10⁶ K/s. In SFL and TFF, the thin liquid channels between the ice domains were sufficiently thin and freezing rates of the thin channels sufficiently fast to achieve the similar particle morphologies. Therefore, the extremely rapid cooling rate in the SFD process was not necessary to form the desired submicron protein particles. In SFL and TFF the surface area/volume ratio of the gas-liquid formed on the liquid protein formulations (46-600 cm⁻¹) was 1-2 orders of magnitude lower than in SFD (6000 cm⁻¹), leading to far less protein adsorption and aggregation. This larger exposure to the gas-liquid interface resulted in lower protein activities in SFD. Although protein stabilities are high in conventional lyophilization, cooling rates are on the order of 1 K/min resulting in large 30 to 100 [mu]m sized particles. Thus, the intermediate cooling rate regime for SFL and TFF, relative to SFD and lyophilization, offers a promising route to form stable submicron protein particles of interest in pulmonary and parenteral delivery applications. The rod-shaped protein particles produced by SFL and TFF are beneficial for forming suspensions stable against settling in hydrofluoroalkanes (HFA) for pressurized metered dose inhaler (pMDI) delivery. The flocculated rods are templated by atomized HFA droplets that evaporate and shrink to form particles with optimal aerodynamic diameters for deep lung delivery. Fine particle fractions of 38-48% were achieved. This novel concept for forming stable suspensions of flocs of rod shaped particles, and templating and shrinking the flocs to produce particles for efficient pMDI deep lung delivery is applicable to a wide variety of drugs. / text

Many neuro-degenerative and metabolic diseases like Parkinson’s and Alzheimer’s are attributed to the effect of mis-folded and aggregated state of proteins in cells. This suggests that the phenomenon of in vivo protein aggregation may be relatively common, perhaps more than currently appreciated. In this study, we aimed to decipher the cause behind an intriguing and potentially related phenomenon observed in yeast cells - a widespread reorganization of hundreds of cytosolic proteins into punctate foci under starvation conditions. The key question that emerges is whether this phenomenon represents organization of proteins into functional assemblies or catastrophic aggregation. This thesis supports the aggregation hypothesis and provides evidence of its role in shaping the dynamics of cellular proteomes. We have been able to demonstrate that the proteins forming foci share a high propensity to aggregate and that these foci may represent sites of homogenous protein aggregation, structures which are typically associated with chaperones. A link between the formation of foci to the yeast aging process has also been established. With evidence correlating protein aggregation propensities to the cellular energy state, we have extended the current "living on the edge" hypothesis (which demonstrates an inverse correlation between protein expression levels and their aggregation propensities). For a specific case of the "purinosome", which is inferred to be a functional enzyme complex responsible for purine biosynthesis, we have shown that the observations may be explained alternatively as a generic protein aggregation phenomenon. This study highlights a systems approach to studying cellular proteins, which can corroborate or provide an alternative explanation to inferences drawn from traditional reductionistic analysis. / text

Apoptosis is a biologically essential phenomenon executed in large part by caspases. Members of the caspase family are activated at different points during apoptosis to proteolyze specific substrates. Given that both excessive and insufficient apoptosis is related to the pathogenesis of various diseases, proper regulation of caspases and apoptosis is necessary for the health of living organisms. Inhibitor of apoptosis (IAP) proteins are endogenous inhibitors of caspases, and since XIAP, the prototypical IAP, binds to and inhibits caspases, all IAPs have been speculated to engage in similar inhibition mechanisms. However, in this dissertation, I demonstrate that cIAP1 binds to the effector caspases-3 and -7, through distinct mechanisms. cIAP1 readily binds to and ubiquitinates, but dos not directly inhibit the activity of fully mature caspase-7. By contrast, cIAP1 does not bind to caspase-3. cIAP1 binding to caspase-7 is mediated primarily by the N-terminus of the large subunit of caspase-7. An AKPD motif located on the N-terminus of caspase-7 is involved in the proteasome-mediated degradation of caspase-7 in cells, thereby decreasing the sensitivity of these cells to apoptosis. Thus, I demonstrate for the first time that cIAP1 is capable of inhibiting caspase-dependent apoptosis through indirect regulation of caspase activity. / text

The c-Jun N-terminal kinases (JNKs) are MAPK family members and are activated by stress, growth factors and cytokines. They are encoded by three separate genes (jnk 1, 2, and 3), spliced alternately creating 10 isoforms. JNK signaling promotes both cell death and cell survival in a stimuli and tissue specic manner and is also implicated in tumorigenesis. Using the Polyoma Virus Middle T Antigen (PyVMT) transgenic mouse model where jnk2 was either expressed or deleted, we found that the PyVMTjnk2-/- tumors expressed higher Epidermal Growth Factor Receptor Substrate 8 (EPS8) mRNA and protein. EPS8 regulates EGFR signaling from Ras to Rac and EGFR tracking via Rab5 and RN-Tre. EPS8 is a prime candidate for connecting the EGFR signaling to actin cytoskeleton remodeling, thus mediating cell migration, a critical step in metastasis. In migration assays, PyVMTjnk2+/+ cells migrated ve fold more than the PyVMTjnk2-/- cells. Re-expression of JNK2[alpha] in the PyVMTjnk2-/- cells rescued this phenotype. Expression of shRNA EPS8 in the PyVMTjnk2-/- cell increased migration in vitro. EPS8 localization at dorsal rues and internalization of EGF-EGFR complexes coincided with JNK2 expression. Expression of shEPS8 in the PyVMTjnk2-/- cells increased EGF internalization suggesting that in absence of JNK2, EPS8 participates in Rab5-RN-Tre complex that inhibits EGFR internalization. Finally, we report that in absence of JNK2, EPS8 protein stability is greatly increased, suggesting that JNK2 is essential for endosomal sorting and degradation of EGFR associated cargo, of which EPS8 is a critical part. In contrast, silencing JNK1 (p46) in 4T1.2 mammary tumor cells, consistently enhanced cell invasion and tumor growth. Tumors derived from orthotopic injection of the 4T1.2shJNK1 expressing cells into the mammary fat pad reached target volume signicantly earlier than non-silencing vector expressing tumors. When injected intravenously, signicantly higher lung metastasis was observed in the 4T1.2shJNK1 group. The more aggressive behavior of 4T1.2shJNK1 tumors was associated with an increase in CCR5 and pAkt as detected by microarray analysis. Taken together, our data suggest that JNK1 suppresses the expression of proteins associated with tumor growth and invasive phenotype, contributing to tumor progression. / text