No description available.

No description available.

The lamina is a cage-like structure, composed of lamins, found underneath the inner nuclear membrane (INM). In mammals, Lamins are type V intermediate filaments. Three genes encode seven different lamin proteins. These genes are LMNA, LMNB1 and LMNB2. Lamins are classified into A- and B-types. A-type lamins, Lamin A, Lamin C, Lamin A?10 and Lamin C2, are mainly expressed in differentiated tissues. All encodes by the LMNA gene and products of alternative splicing. Recent studies have shown the expression of Lamin A/C in colon crypt epithelia cells. This expression was greater in differentiated epithelial and stem cells than in the proliferation zone. Since 1999, mutations in LMNA have been shown to cause several different inherited diseases in muscle, fat, bone, skin and nerve tissues. LAP2? is one of the strong binding partners to Lamin A/C. LAP2? has a specific function as a non-membrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with Lamin A/C. To understand more about Lamin A/C and LAP2? in colon epithelial development,a Yeast 2-hybrid screen was used to look for novel protein interactions to Lamin A/C and/or LAP2a. It was found Cytochrome c oxidase subunit II (Cox2) is a putative binding partner to Lamin A/C and LAP2 ?. Cox2 is encoded by the mitochondrial genome and imported into complex IV (COX) of the mitochondrial respiratory chain (MRC). The majority of mitochondrial proteins are encoded by nuclear DNA. However, 13 essential subunits of the MRC are encoded by mtDNA. The MRC is composed of five multi-subunit complexes (I-V). MRC is implicated in ATP generation and is involved in apoptosis in response to different stimuli. Deficiency in COX in colon cancer cells results in resistance to apoptosis and increases in reactive oxygen species (ROS). Biochemical assays like Coimmunopreciptation (IP) and western blot (WB) and Immuno-gold labeling (TEM) were used. IP confirmed the putative interaction of Cox2 to Lamin A/C and LAP2?. WB showed the expression of lamin A/C and LAP2? in the mitochondrial and nuclear fractions but not in cytosol. TEM is alternative method showed the distribution of lamin A/C and LAP2? in the nucleus and mitochondria.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved eukaryotic cellular response to starvation, resulting in the formation of double-membrane vesicles known as autophagosomes. These sequester cytoplasmic contents to the lysosome, where they are degraded to generate amino acids which can be used for protein synthesis during periods of nutrient deprivation. Membrane targeting of LC3, the major structural protein of the autophagosome, is essential for autophagy. LC3 is processed from its cytosolic form (LC3-I) to its lipidated form (LC3-II) in a series of ubiquitin-like conjugation events. Of particular importance is Atg16-like-1 (Atg16L1), which through its interactions with Atg5-Atg12, specifies the site of LC3 incorporation into the expanding autophagosomal membrane. In this study tools have been generated for the study and characterisation of Atg16L1 domains in cell culture. Atg16L1 -/- MEFs have also been generated and used for reconstitution experiments, which confirm that the coiled-coil domain of Atg16L1 is sufficient to restore autophagy in response to starvation and to a surrogate pathogen. A novel interaction between Atg16L1 and the serine/threonine kinase MEKK4 has been characterised in this study. Existing research has established an interaction between the pathogen recognition receptors NOD1/2 and Atg16L1. The NOD receptors are activated by bacterial peptidoglycan (PG), which has been shown to drive autophagy. Interestingly, MEKK4 inhibits NOD2 signalling by binding the downstream adaptor protein RIP2, leading to the hypothesis that MEKK4 may play a role in autophagy. The interaction was verified by co-immunoprecipitation, with domain analysis indicating that MEKK4 binds to the linker region of Atg16L1. The kinase activity of MEKK4 was also required for the interaction. The complex between Atg16L1 and MEKK4 persisted during starvation induced autophagy. Lipidation of LC3 in response to starvation required expression of MEKK4, with a redistribution of LC3 to large perinuclear aggregrates that co-localised with p62 and ubiquitin in siRNA silenced cells and CRISPR/Cas MEKK4 knockout cells. The aggregates were also able to recruit LC3 I, indicating a role of aggresome-like induced structures (ALIS). This thesis documents an interaction between Atg16L1 and MEKK4 that is vital for LC3 processing. The potential role for MEKK4 in regulating autophagy through Atg16L1 is discussed and experiments proposed to further explore this interaction in the context of autophagy.

No description available.

No description available.

Endothelial cells line all of our blood vessels. They monitor and respond to signals generated during injury, infection and disease by releasing a wide range of molecules that regulate blood flow, coagulation, inflammatory responses and vessel growth. Protein mediators are released by exocytosis of intracellular organelles, and a major trigger for this type of secretion is an increase in intracellular free calcium ion concentration ([Ca2+]i). Mitochondria are thought to influence Ca2+ homeostasis through local Ca2+ buffering. Due to a lack of sensitive and time-resolved assays for endothelial exocytosis little is known about the precise relationship between Ca2+-signalling and exocytosis, and the influence of Ca2+ buffering by mitochondria. Using fluorescence and biochemical techniques I have investigated the relationship between secretagogue-evoked Ca2+-signalling and the influence of mitochondrial function on the exocytosis of two distinct organelle populations in cultures of Human Umbilical Vein Endothelial Cells (HUVEC); 1) the Weibel-Palade body (WPB) the main storage organelle for pro-coaguland and inflammatory mediators, and 2) the non-WPB, a non-stored and morphologically distinct organelle that can contain a range of inflammatory and anti-coagulant molecules. These two distinct organelle populations were labeled for fluorescence microscopy by targeted expression of chimeras of green (EGFP) or red (mRFP) fluorescent proteins in living HUVEC. Exocytosis was evoked by both physiological and pharmacological secretogogues that increase [Ca2+]i. The times of exocytosis of individual organelles were monitored by flashes of light from granule EGFP, produced by pH changes within the organelle upon fusion. In the same experiments, [Ca2+]i and intra-mitochondrial Ca2+ concentration ([Ca2+]m) were monitored using fluorescent Ca2+-indicators. The data obtained has defined more precisely the relationship between agonist-evoked changes in [Ca2+]i and secretory vesicle exocytosis in HUVEC. These studies will contribute to a better understanding of the processes that regulate secretion of biomolecules from the endothelium.

No description available.

No description available.

No description available.