The Chediak-Higashi Syndrome is a disorder affecting lysosome biogenesis. At the cellular level, the Chediak-Higashi syndrome is characterized by the presence of grossly enlarged lysosomes in every tissue. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. The Chediak-Higashi syndrome is caused by the loss of a BEACH protein of unknown function named Lyst. In this work, I have studied the function of the Dictyostelium LvsB protein, the ortholog of mammalian Lyst and a protein that is also important for lysosomal function. Using a knock-in approach we tagged LvsB with GFP and expressed it from its single chromosomal locus. GFP-LvsB was observed on endocytic and phagocytic compartments. Specific analysis of the endocytic compartments labeled by LvsB showed that they represented late lysosomes and postlysosomes. The analysis of LvsB-null cells revealed that loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. This work demonstrated that the abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of lysosomes with postlysosomal compartments. Furthermore, this work provided the first evidence that LvsB is a functional antagonist of the GTPase Rab14 in vesicle fusion events. In particular, we demonstrated that reduction of Rab14 activity suppressed the LvsB-null phenotype by reducing the enlarged post-lysosomes and the enhanced rate of heterotypic fusion. In contrast, expression of an active form of Rab14 enhanced the LvsB-null phenotype by causing an even more severe enlargement of endosome size. The results provided by this work support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early with late compartments and antagonize Rab GTPases in membrane fusion. The LvsB localization studies and the functional assessment of the LvsB-null phenotype helped make unique contributions to the understanding of the molecular function of Lyst proteins.

Fat facets in Mouse (FAM) or mUSP9x is a deubiquitylating enzyme of the USP class. Knockdown of FAM protein levels in mouse pre-implantation embryos by antisense oligonucleotides is known to prevent embryos from progressing to the blastocyst stage indicating an important role for FAM in early mammalian development. In mammals, the Fam gene is located on the X-chromosome. In mice, the Y homologue, Dffry or usp9y, is expressed exclusively in the testes and maps to the Sxrb deletion (Brown et al., 1998). Sxrb is associated with an early post-natal blockage of spermatogonial proliferation and differentiation leading to absence of germ cells (Bishop et al., 1988; Mardon et al., 1989). The human Y homologue of Fam is closely associated with oligozoospermia (Sargent et al., 1999; Sun et al., 1999) and the human X homologue has been linked to the failure of oocytes to pass through the first meitoc prophase in Turner syndrome (Cockwell et al., 1991; Speed, 1986) Despite these associations, the substrates and precise role of Fam and its homologues in these processes have not yet been defined. Due to the complex nature of Fam expression and the lack of data tying FAM to specific cellular functions, much attention has been paid in identifying interacting partners and cellular targets of FAM activity to aid in the definition of its role in the cell and development. Three common molecular biology techniques were applied here in an attempt to further characterise known interactions of FAM, including interactions with the cell adhesion molecule β-catenin and the protein trafficking pathway proteins epsin-1 and itch. The aim of these investigations was to generate FAM mutants that could abolish individual interactions, enabling investigation of individual interactions in cellular function and development. These experiments failed to identify the amino acids of FAM that were critical for its interactions with β-catenin, epsin-1, or itch. Experiments aimed at characterising a novel ubiquitin-like domain located in the N-terminal half of the FAM protein, did however identify novel interactions of FAM with the three Golgi associated adaptor proteins GGA1, GGA2, and GGA3. Further investigations prompted by this interaction, examined the role of FAM in the trafficking of proteins from the Golgi apparatus. Cellular FAM protein levels were altered either by exogenous expression of FAM protein or knockdown of endogenous FAM using FAM specific shRNA triggers. The cellular protein levels and extent of post-translational modification of eleven lysosomal proteins were monitored in each case. It was found that increased FAM protein levels resulted in decreased cellular protein levels of five of the eleven lysosomal proteins studied. In contrast, a reduction in FAM protein levels was found to result in an increase in the cellular protein levels of eight of the eleven lysosomal proteins. This study provides the first evidence of a deubiquitylating enzyme that is able to interact with the GGA proteins. It is also the first to describe a deubiquitylating enzyme that can affect the biosynthesis of lysosomal proteins and provides valuable new insight into the cellular function of FAM/USP9X. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2008

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 9, 2009). Includes bibliographical references.

Thesis (Ph.D.) - University of St Andrews, February 2010.

No description available.

Two-photon fluorescence microscopy (2PFM) has become a powerful technique for bioimaging in non-invasive cancer diagnosis and also investigating the mechanization and original of a variety of diseases by tracking various biological processes. Because the fluorescence emission by two photon absorbing (2PA) is directly proportional to the square of the intensity of excitation light, this intrinsic property of 2PA provides 2PFM great advantages over traditional one-photon fluorescence microscopy (1PFM), including high 3D spatial localization, less photodamage and interference from biological tissue because of using longer wavelength excitation (700-1300 nm). However, most 2PA probes are hydrophobic and their photostabilities are questionable, severely limiting their biological and medical applications. In addition, probes with significant specificity for certain organelles for tracking cellular processes or metal ions for monitoring neural transmission are somewhat rare. Moreover, it is also very significant to deliver the probes to specific disease sites for early cancer diagnosis. In order to increase the water solubility of probes, polyethylene glycol (PEG) was introduced to a fluorene-based 2PA probe LT1 for lysosomal 2PFM cell imaging. The 2PFM bioimaging application of the novel two-photon absorbing fluorene derivative LT1, selective for the lysosomes of HCT 116 cancer cells is described in Chapter II. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for 2PFM lysosomal imaging. After the investigation of the cytotoxicity of this new probe, colocalization studies of the probe with commercial lysosomal probe Lysotracker Red in HCT 116 cells were conducted. A high colocalization coefficient (0.96) was achieved and demonstrated the specific localization of the probe in lysosomes. A figure of merit, F[subscript M], was introduced by which all fluorescent probes for 2PFM can be compared. LT1 was demonstrated to have a number of properties that far exceed those of commercial lysotracker probes, including much higher 2PA cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. Consequently, 2PFM was used to demonstrate lysosomal tracking with LT1. In addition to lysosomes, it is also very significant to investigate the physiological roles of free metal ions in biological processes, especially Zn²⁺, because Zn²⁺ normally serves either as the catalytic elements in enzymatic activity centers or as structural elements in enzymes and transcription factors. However, biocompatible and effective Zn²⁺ probes for 2PFM bioimaging are infrequent. In Chapter III, 2PFM bioimaging with a hydrophilic 2PA Zn²⁺ sensing fluorescent probe, bis(1,2,3-triazolyl)fluorene derivative, is described. 2PFM bioimaging of the probe in living HeLa cancer cells was demonstrated. The results revealed a significant fluorescence increase upon introduction of Zn²⁺ into the cancer cells, and a reversible Zn²⁺ binding to the probe was also demonstrated, providing a robust probe for two-photon fluorescence zinc ion sensing. Early cancer diagnosis is another critical application for 2PFM, but there are still huge challenges for this new technique in clinical areas. Most 2PA probes with large two-photon absorbing cross sections and fluorescence quantum efficiency are synthetically more accessible in hydrophobic forms. In order to increase the efficiency of the probes and minimize the effect of the probe on the human body, delivery of the probe specifically to cancer sites is desired. The synthesis and characterization of narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, are reported in Chapter IV. The surface of the SiNPs was functionalized with folic acid to specifically deliver the probe to folate receptor (FR) over-expressing HeLa cells, making these folate 2PA dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing HeLa cells demonstrated specific cellular uptake of the functionalized nanoparticles. However, when the concentration of the dye in SiNPs increased for higher signal output, the fluorescence quantum efficiency of a probe normally decreases because of self-quenching. In Chapter V, a near-infrared (NIR) emitting probe is reported to overcome this limitation through both aggregate-enhanced fluorescence emission and aggregate enhanced two-photon absorption. The dye was encapsulated in SiNPs and the surface of the nanoparticles was functionalized with PEG followed by a folic acid derivative to specifically target folate receptors. NIR emission is important for deep tissue imaging. In vitro studies using HeLa cells that upregulate folate receptors indicated specific cellular uptake of the folic acid functionalized SiNP nanoprobe. Meanwhile, the probe was also investigated for live animal imaging by employing mice bearing HeLa tumors for in vivo studies. Ex vivo 2PFM tumor imaging was then conducted to achieve high quality 3D thick tissue tumor images.

No description available.

Lysosomal storage diseases (LSDs) are metabolic diseases which occur as a result of a deficiency of one of the essential lysosomal enzymes, called glycohydrolases. A mutation in the gene encoding one of these enzymes leads to an accumulation of unwanted substrates, resulting in a variety of clinical manifestations. A common symptom found in LSDs is skeletal muscle dysfunction, which includes muscle weakness, atrophy and loss of muscle mass. The genes for lysosomal hydrolases are well characterized; however, much less is known about how mutations in these genes affect the cell and lead to the muscle dysfunction observed. One pathway of interest is autophagy; it has been shown to be essential for maintenance of skeletal muscles. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of Sialidosis, Sandhoff Disease, and GM1-Gangliosidosis in order to assess autophagy and its impact on myogenic differentiation in skeletal muscles. Our results demonstrated that autophagy is induced upstream (ULK1 phosphorylation) but is inhibited at the autophagosome to lysosome fusion (p62 upregulation) in LSDs. We also found that myoblast fusion and myogenic differentiation are impaired. We conclude that blocking autophagy impairs myogenic differentiation, which potentiates the muscle dysfunction observed in LSDs. This work highlights autophagy as a new pathway of interest and possible therapeutic target to alleviate muscle dysfunction in LSDs, and other similar neurodegenerative diseases. / Thesis / Master of Science (MSc) / Lysosomal storage diseases (LSDs) occur because of a deficiency of lysosomal glycohydrolases. A common symptom found in LSDs is skeletal muscle dysfunction. Little is known about how a deficiency of these enzymes leads to the clinical manifestations observed. However, one pathway of interest is autophagy. This study sought to investigate the impact of LSDs on autophagy and how this may potentiate muscle dysfunction. We utilized in-vivo and in-vitro models of LSDs to assess autophagy and its impact on myogenic differentiation in skeletal muscles. We demonstrated that autophagy is induced and blocked, and that myoblast fusion and myogenic differentiation is impaired. We concluded that the induction and block of autophagy impairs myogenic differentiation, which potentiates muscle dysfunction.

The cysteine proteinases cathepsins B, L, and S are lysosomal enzymes responsible for the degradation of endocytosed proteins. Their presence in human cell monocytic lines THP1 and U937 was detected by the use of the membrane-permeable, irreversible, active-site directed inhibitor Fmoc-(¹²⁵I)Tyr- Ala-CHN₂ followed by immunoprecipitation of the enzymes, SDSPAGE, and autoradiography. All three enzymes were detected in THP1 cells; only after differentiation of U937 cells to macrophage-like cells were the enzymes detectable. Both cell lines show multiple forms of cathepsin S, at 35 kDa, 28 kDa, and 26 kDa, suggesting the presence of an active pro-form of cathepsin S as well as the processing of cathepsin S into single- and two-chain forms. This is the first evidence for an active pro-form of a cysteine proteinase and for the processing of cathepsin S to a two-chain enzyme form. Multiple forms of cathepsin L were analyzed by isoelectric focusing followed by denaturing polyacrylamide gel electrophoresis. The multiple forms are not due to the presence of carbohydrate chains on the protein. The inhibitor Fmoc-Tyr-Ala-CHN₂ synthesized and its inhibitory properties against cathepsins B, L, and S were determined. Both in vitro and in vivo studies show that this inhibitor is an effective reagent for studying lysosomal cysteine proteinases. In order to be useful in the study of the delivery of lysosomal enzymes to vesicles containing recently internalized compounds, the deblocked peptidyl diazomethane inhibitor NH₂-Tyr-Ala-CHN₂ was cross-linked to bovine serum albumin (BSA) using the heterobifunctional crosslinking agent sulfo-SANPAH. This non-reducible cross-linked complex was used to characterize the inhibitory properties of the protein-inhibitor complex against cathepsins B, L, S and papain in vitro and in vivo. / Ph. D.