Desensitisation is defined as a failure of a signaling pathway to respond to chronic or repeated stimulation. The β-adrenergic receptor signaling pathway of the healthy adult heart is known to desensitise, and then regain the sensitivity to stimulation if given enough time to rest between stimulations (resensitisation). The fetal heart does not desensitise, and in animal models of heart failure, a permanent desensitisation have been observed. No isolated element of the signaling pathway have yet been proven to be the sole modulator of the desensitisation behavior. Therefore a mathematical model of the signaling pathway has been constructed, minimized against theoretical desensitisation data and tested for resensitisation. The minimal models and the original model were capable of describing the theoretical de- and resensitisation of the pathway, and only one receptor type with three states was required in the minimal models, but one feedback from the kinases either to phosphorylation of the receptor or to breakdown of cAMP. The original model was also capable of describing experimental data of contraction force from chicken cardiac tissue. The cardiac tissue displays the peak behavior of the desensitisation when stimulated with ISO for ten minutes, and resensitises in less than 5 minutes.

Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects. There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies. In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear. We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions. We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells. Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.

The signal transducer and activator of transcription-3 (Stat3) is a latent cytoplasmic protein that is activated through phosphorylation of tyrosine-705 by a number of receptor and non-receptor tyrosine kinases. This leads to Stat3 dimerization by reciprocal SH2-ptyr interactions, followed by translocation to the nucleus to initiate transcription of genes involved in cell growth, survival, and differentiation. Many of these signaling molecules known to activate Stat3 concentrate in specialized plasma membrane microdomains called caveolae, and are sequestered in an inactive state to the caveolin scaffolding domain (CSD) of the main caveolae resident protein, caveolin-1 (cav1). Since many of these signaling molecules are known, potent Stat3 stimulators, we set out to examine the effect of cav1 upon Stat3 activity. To this effect, cav1 was downregulated using a cholesterol chelator (methylcyclodextrin), or an antisense approach. Since we previously found that cell density can dramatically activate Stat3, all experiments were conducted at several densities. The results show that cav1 downregulation causes an increase in Stat3-tyr705 phosphorylation at all densities examined. We next examined the effect of cav1 upregulation upon Stat3 activity by transfecting an EGFP-cav1 construct. The results revealed that cav1 overexpression using this construct reduces Stat3 activity and induces apoptosis, which can be overcome by expression of a constitutively active form of Stat3. Finally, by expressing a Stat3 shRNA with an adenovirus vector, we demonstrated that Stat3 downregulation leads to an increase in cav1 levels. These results reveal the presence of a potent, negative regulatory relationship between cav1 and Stat3 phosphorylation. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2008-11-09 20:53:54.787

Bacterial cytoskeleton is an exciting and relatively new field of research. Recent findings have proven that microbes are well-organized and neatly structured organisms. In this study we have shown that intermediate filament-like proteins with a characteristic rod domain architecture of coiled coil segments separated by non-coiled coil linkers, are widely spread among bacteria. We identified and characterized an intermediate filament-like protein (named FilP after filamentous protein) in Streptomyces coelicolor. It shares the characteristic biochemical property of eukaryotic intermediate filaments of formation of spontaneous filaments in vitro without requiring any energy or co-factor. We have provided here a preliminary model of its assembly in vitro. FilP also forms in vivo filaments in S. coelicolor hyphae, which are strongest at the sub-apical location of growing vegetative hyphae. We have proposed that FilP cytoskeletal network provides rigidity to the hyphae, especially at the growing tips, by interacting with an essential coiled coil protein DivIVA and possibly other partner elements, yet to be found. S. coelicolor is a well-studied model organism with a complicated life cycle. It germinates from a spore and spreads by forming branched vegetative hyphae. Lack of nutrients in the environment initiates formation of aerial hyphae in the air, perpendicular to the vegetative ones. The aerial hyphae differentiate into spore chains and eventually grey-pigmented dispersed individual spores are released. The signals involved in sporulation including cell division and chromosome segregation are not clear yet. We characterized here a novel locus consisting of two genes: a small putative membrane protein with no defined function, named SmeA and a member of the SpoIIIE/FtsK family, called SffA. The expression of this locus appears to be dependent on whiA and whiG-whiH-whiI pathways. This finding is intriguing as it can provide insight to the relationship between two apparently unrelated pathways, both leading to the same function of septation and maturation during sporulation.

Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.

As global meat consumption is rising, the negative impact the animal husbandry sector has on the environment will increase. Greenhouse gas emissions have increased by 40 % during the last 200 years, and the animal husbandry sector is today responsible for 18 % of the total greenhouse gas emissions from food production. More environmentally friendly protein sources, such as soy and pea, must therefore be developed. Pisum sativum can (unlike the most popular meat alternative – soy) be grown all over Europe and might thus be a good alternative that allows for locally sourced alternatives to meat protein. Identifying genes with important agricultural properties might aid the development of pea cultivars with a more reliable protein content. One such gene was hypothesised to be the OMT gene, which is strongly expressed during the embryonic development of P. sativum and seems involved in functions such as seed storage and protein synthesis. Thirty-one accessions of P. sativum were tested to see if different improvement types differed from each other regarding protein content and seed weight, but no such differences were found. DNA was extracted from all accessions, sequenced, and successful sequences were tested to determine if variation in the gene correlated with protein content. Two haplotypes were identified, but there was no correlation between them and protein content found. Based on the results of this study, there is little evidence that the OMT gene correlates with protein content in the studied accessions.

Trichome formation in Arabidopsis lyrata is a naturally occurring trait with phenotypic polymorphisms within wild populations. In Swedish accessions of A. lyrata, three genetic polymorphisms situated in the coding region of GL1, an important transcription factor in trichome production, have been identified, and these are candidates for being the cause of a glabrous phenotype. In this study a complementation test has been performed to clarify which mutation/mutations that are detrimental for trichome formation. A set of constructs has been transformed into A. thaliana, a close relative to A. lyrata, and subsequent generations of plants were examined for phenotype, genotype and gene expression. A SNP (Single Nucleotide Polymorphism) in the R3 MYB domain of GL1, resulting in a change of an alanine to aspartic acid, was identified as the critical polymorphism. The other two mutations, two indels, were harmless to protein function. The inserted constructs were under control of the native GL1 promoter. Plants that, because of the SNP, lacked trichome production, became totally glabrous.

Hydrogen peroxide (H2O2), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H2O2 is produced as a defense mechanism against pathogens, as being released by activated phagocytes. In recent years, H2O2 has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H2O2 is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H2O2 production. The aim of this study was to investigate H2O2 production induced by the proteins gp120 and Trx80. In order to detect H2O2 production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H2O2 released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H2O2 production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.

L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation. L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother. To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing. Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G>C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.

Peripheral neuropathy includes a wide range of diseases affecting millions around the world, and many of these diseases have unknown etiology. Peripheral neuropathy in diabetes represents a large proportion of peripheral neuropathies. Nerve damage can also be caused by trauma. Peripheral neuropathies are a significant clinical problem and efficient treatments are largely lacking. In the case of a transected nerve, different methods have been used to repair or reconstruct the nerve, including the use of nerve conduits, but functional recovery is usually poor. Autophagy, a cellular mechanism that recycles damaged proteins, is impaired in the brain in many neurodegenerative diseases affecting animals and humans. No research, however, has investigated the presence of autophagy in the human peripheral nervous system. In this study, I present the first structural evidence of autophagy in human peripheral nerves. I also show that the density of autophagy structures is higher in peripheral nerves of patients with chronic idiopathic axonal polyneuropathy (CIAP) and inflammatory neuropathy than in controls. The density of these structures increases with the severity of the neuropathy. In animal model, using Goto-Kakizaki (GK) rats with diabetes resembling human type 2 diabetes, activation of autophagy by local administration of rapamycin incorporated in collagen conduits that were used for reconnection of the transected sciatic nerve led to an increase in autophagy proteins LC3 and a decrease in p62 suggesting that the autophagic flux was activated. In addition, immunoreactivity of neurofilaments, which are parts of the cytoskeleton of axons, was increased indicating increased axonal regeneration. I also show that many proteins involved in axonal regeneration and cell survival were up-regulated by rapamycin in the injured sciatic nerve of GK rats four weeks after injury. Taken together, these findings provide new knowledge about the involvement of autophagy in neuropathy and after peripheral nerve injury and reconstruction using collagen conduits.