Clathrin-mediated endocytosis is the process whereby specific cargoes are internalized into coated vesicles from the plasma membrane. Numerous clathrin adaptors facilitate this process by linking the coat protein clathrin to the plasma membrane by associating with PI(4,5)P2 and binding to membrane-bound cargo. Here, I investigated the role of two clathrin adaptors, APβ1/2 and Hip1r, in clathrin-mediated endocytosis. I found that Dictyostelium APβ1/2 functions in both the AP1 and AP2 complexes, unlike vertebrates, which have distinct β subunits for each AP complex. I found that APβ1/2 function is required for several clathrin-dependent processes, including cytokinesis, development and osmoregulation. I also uncovered a role for APβ1/2 in the stability other subunits of the AP1 and AP2 complexes. Finally, phenotypic comparisons of APβ1/2 mutant cells with cells missing subunits that are specific to the AP1 or AP2 complex allowed me to distinguish between endocytic defects and endosomal trafficking defects in clathrin mutants. My investigation of Hip1r centered on the known requirement for Hip1r in actin dynamics during endocytosis and a possible role for Hip1r phosphorylation in regulating actin. To determine how phosphorylation contributes to Hip1r function, I identified a specific serine residue that serves as a Hip1r phosphorylation site. I also identified a novel role for the kinase PKB in Hip1r phosphorylation. I determined that phosphorylation is not required for Hip1r localization to the plasma membrane. Similar to Hip1r, PKB is required for proper actin dynamics during endocytosis. My results support a model in which epsin recruits Hip1r to the plasma membrane during formation of clathrin-coated vesicles. Here, Hip1r functions as both a clathrin adaptor and a negative regulator of actin polymerization. I propose that phosphorylation of Hip1r by PKB triggers a reduction in the affinity of Hip1r for clathrin, which may stimulate actin polymerization and tethering of clathrin-coated vesicles with the actin cytoskeleton. / text

No description available.

The mammalian immune system is equipped to both eliminate pathogenic microorganisms and tumors, while remaining in homeostasis with commensal species at mucosal surfaces and tolerant towards self. Suppressor regulatory T cells (Tregs) are a major sentinel of this immunological tolerance. Induced Tregs (iTregs) arise in the periphery following the integration of cues from the metabolites, cytokines, etc. which make up its milieu. Dysregulation of iTreg development, function or homing underlies the etiology of many autoimmune diseases and immunopathologies. The amelioration or prevention of multiple murine disease models by boosting Treg cell numbers foreshadows clinical efficacy of iTreg therapy, but an incomplete understanding of Treg development has thus far prevented successful translation. Therefore, we considered the basic biology of T cell fate decision making from two unique, but integrated angles. First, we show that the stimulation of PPARγ in human T cells upregulates RDH10, a molecule which catalyzes the rate limiting step in the oxidation of retinol to transcriptionally active all-trans retinoic acid (ATRA), a positive regulator of iTreg development. This functionally intact pathway endows T cells the ability to autonomously sense and respond to retinoid signals present during Treg development and at tissue sites. Next, we asked questions about how T cells sense nutrient and oxygen availability as they differentiate. Tregs lacking the serine/threonine kinase PINK1 have limited activation-induced phosphorylation of Akt and oxidative phosphorylation rates, and reduced suppressor function. Notably, the uncoupling of iTreg function from normal FoxP3 expression reinforces the recent hypothesis that the PI3K/Akt/mTORC1 axis and metabolic checkpoints are decisive players in the acquisition of suppressor activity. Ultimately, the studies described herein converge on Akt and metabolism, and contribute to our understanding of how T cells integrate diverse signals present during fate determinism, provoking future Treg based therapeutics.

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.

Zugl.: Berlin, Freie Univ., Diss., 2005

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 150-182).

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Tübingen, Univ., Diss., 2004.

University, Diss., 2004--Tübingen.

Vít Neznal’s PhD thesis begins by defining theatre as a specific sphere of media. According to the author, such notion is made possible by following up to the tradition of Czech theatre theory (immediacy as a specific feature of the medium, a synthetic basis, a non-substantial conception of a work, acting as a decisive art, a total three-dimensional feedback-based communication with cardinal impact on the specific nature of stage acting, and an active position of the spectator). On this basis, Neznal proceeds to explain why there is no justifiable reason for theatre theory to abolish the dichotomy between art and reality. His premise is that while theatre is rooted in life, it is at the same time automatically and irreversibly taken out of it as a medium. As a result, there is, from the point of view of theatre theory, a difference between an intensified “life” entering the stage and becoming the basis of expression (intensification of “life” is the basis of means of expression and is therefore made intentional) and between its integration with other pillars forming the constitutive basis of a theatre act as such on the actual level of media (the artificial pervaded by “life”).