Trafficking and ethanol-induced inhibition of AMPA receptors

AMPA receptors are an important class of ionotropic glutamate receptors which participate in fast excitatory synaptic transmission in most brain areas. They have a pivotal role in adjustment of cell membrane excitability as their cell membrane expression levels is altered in brain physiology such as in learning and memory formation. AMPA receptor function and trafficking is regulated by several proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs). NMDA-type glutamate receptors are important target molecules of ethanol. The role of AMPA receptors in the actions of ethanol has not been clarified as thoroughly. Furthermore, the regulation of AMPA receptor synthesis and their possible adaptation in neurons with altered inhibitory mechanisms are poorly understood. In this thesis work AMPA receptor pharmacology, trafficking and synaptic localization was studied using patch-clamp electrophysiology. Both native and recombinant AMPA receptors were studied. Hippocampal slices from transgenic Thy1alfa6 mice with altered inhibition were used to study adaptation of AMPA receptors. Ethanol was found to inhibit AMPA receptor function by increasing desensitization of the receptor, as the steady-state current was inhibited more than the peak current. Ethanol inhibition was reduced when cyclothiazide was used to block desensitization and when non-desensitizing mutant receptors were studied. Ethanol also increased the rate of desensitization, which was increased further by the coexpression of TARP-proteins. We found that the agonist binding capability is important for trafficking AMPA receptors from endoplasmic reticulum to the cell membrane. TARP rescues the surface expression of non-binding AMPA receptor mutants in HEK293 cells, but not in native neurons. Studies with Thy1alfa6 mice revealed that decreased inhibition decrease AMPA receptor mediated excitation keeping the neurotransmission in balance. Thy1alfa6 mice also had lower sensitivity to electroshock convulsions, presumably due to the decreased AMPA receptor function. The results suggest that during alcohol intoxication ethanol may inhibit AMPA receptors by increasing the rate and the extent of desensitization. TARPs appear to enhance ethanol inhibition. TARPs also participate in trafficking of AMPA receptors upon their synthesis in the cell. AMPA receptors mediate also long-term adaptation to altered neuronal excitability, which adds to their well-known role in synaptic plasticity.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Trafficking in human beings and Foucauldian biopower : A case study in the expansion of the human rights phenomenon

Trafficking in human beings has become one of the most talked about criminal concerns of the 21st century. But this is not all that it has become. Trafficking has also been declared as one of the most pressing human rights issues of our time. In this sense, it has become a part of the expansion of the human rights phenomenon. Although it is easy to see that the crime of trafficking violates several of the human rights of its victims, it is still, in its essence, a fairly conventional although particularly heinous and often transnational crime, consisting of acts between private actors, and lacking, therefore, the vertical effect associated traditionally with human rights violations. This thesis asks, then, why, and how, has the anti-trafficking campaign been translated in human rights language. And even more fundamentally: in light of the critical, theoretical studies surrounding the expansion of the human rights phenomenon, especially that of Costas Douzinas, who has declared that we have come to the end of human rights as a consequence of the expansion and bureaucratization of the phenomenon, can human rights actually bring salvation to the victims of trafficking? The thesis demonstrates that the translation process of the anti-trafficking campaign into human rights language has been a complicated process involving various actors, including scholars, feminist NGOs, local activists and global human rights NGOs. It has also been driven by a complicated web of interests, the most prevalent one the sincere will to help the victims having become entangled with other aims, such as political, economical, and structural goals. As a consequence of its fragmented background, the human rights approach to trafficking seeks still its final form, consisting of several different claims. After an assessment of these claims from a legal perspective, this thesis concludes that the approach is most relevant regarding the mistreatment of victims of trafficking in the hands of state authorities. It seems to be quite common that authorities have trouble identifying the victims of trafficking, which means that the rights granted to themin international and national documents are not realized in practice, but victims of trafficking are systematically deported as illegal immigrants. It is argued that in order to understand the measures of the authorities, and to assess the usefulness of human rights, it is necessary to adopt a Foucauldian perspective and to observe the measures as biopolitical defence mechanisms. From a biopolitical perspective, the victims of trafficking can be seen as a threat to the population a threat that must be eliminated either by assimilating them to the main population with the help of disciplinary techniques, or by excluding them completely from the society. This biopolitical aim is accomplished through an impenetrable net of seemingly insignificant practices and discourses that not even the participants are aware of. As a result of these practices and discourses, trafficking victims only very few of fit the myth of the perfect victim, produced by biopolitical discourses become invisible and therefore subject to deportation as (risky) illegal immigrants, turning them into bare life in the Agambenian sense, represented by the homo sacer, who cannot be sacrificed, yet does not enjoy the protection of the society and its laws. It is argued, following Jacques Rancière and Slavoj i ek, that human rights can, through their universality and formal equality, provide bare life the tools to formulate political claims and therefore utilize their politicization through their exclusion to return to the sphere of power and politics. Even though human rights have inevitably become entangled with biopolitical practices, they are still perhaps the most efficient way to challenge biopower. Human rights have not, therefore, become useless for the victims of trafficking, but they must be conceived as a universal tool to formulate political claims and challenge power .In the case of trafficking this means that human rights must be utilized to constantly renegotiate the borders of the problematic concept of victim of trafficking created by international instruments, policies and discourses, including those that are sincerely aimed to provide help for the victims.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

The influence of HIV infection to the periodontium; a clinical, microbiological, and enzymological study

The main targets of human immunodeficiency virus (HIV) are CD4 receptors of CD4+ lymphocytes and many other cells such as monocytes/macrophages, megakaryocytes, peripheral blood dendritic cells, follicular dendritic cells (DC), epidermal Langerhans cells, and astrocytes. Infection and killing of CD4+ lymphocytes or false reaction of the body to HIV infection and the spontaneous apoptosis of CD4+ lymphocytes decrease CD4+ lymphocyte counts leading to immunosuppression, further disease progression, and appearance of opportunistic infections and malignancies. Oral manifestations are considered to be among the first signs of HIV infection. Enhanced degradation of extracellular matrix and basement membrane components in oral diseases including periodontitis is caused by Zn-dependent enzymes called matrix metalloproteinases (MMPs). The levels and degrees of activation of MMP-1, -2, -3, -7, -8, -9, -25, -26, tissue inhibitors of MMPs (TIMP)-1 and -2, and myeloperoxidase (MPO) and collagenolytic/gelatinolytic activities, and also Ig A, -G, and -M, total protein, and albumin levels in a two-year follow-up were studied from salivary samples. The expression of MMP-7, -8, -9, -25, and -26 immunoreactivities in gingival tissue specimens were studied. Healthy HIV-negative subjects served as controls. All studied clinical periodontal parameters and microbiological evaluation of the periodontopathogens showed that periodontal health of the HIV-positive patients was moderately decreased in comparison to the healthy controls. The levels of Candida in the periodontal pockets and salivary MPO increased with the severity of HIV infection. Immunoreactivities and levels of MMPs and TIMPs, and MMP activities (collagenase, gelatinase) were enhanced in the HIV-positive patient salivary samples relative to the healthy controls regardless of the phase of HIV infection. However, these parameters did not reflect periodontal status in a similar way as in the generally healthy periodontitis patients. Salivary total protein, albumin, IgA, -G, and -M levels were significantly higher in all phases of HIV infection compared to the controls, and salivary total protein, IgG and IgM levels remained higher after two years follow-up, partly correlating with the disease progression and which may reflect the leakage of serum components into the mouth and thus a decreased mucosal barrier. Salivary analyses of MMPs and TIMPs with immunohistochemical analyses showed that HIV infection could predispose to periodontal destruction when compared with healthy controls or the body s defence reactions associated with HIV infection may have been reflected or mediated by MMPs.

Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.

The Functions and Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) regulates the synthesis of polyamines which are involved in many cellular functions e.g. proliferation and differentiation. Due to its critical role, ODC is a tightly regulated enzyme by antizymes and antizyme inhibitors. If the regulation fails, the activity of ODC increases and may lead to malignant transformation of a cell. Increased ODC activity is found in many common cancers, including colon, prostate, and breast cancer. In a transformed cell, dynamics of the actin cytoskeleton is disturbed. A small G-protein, RhoA regulates organization of the cytoskeleton, and its overactivity increases malignant potential of the cell. The present results indicate that covalent attachment of polyamines by transglutaminase is a physiological means of regulating the activity of RhoA. The translocation of RhoA to the plasma membrane, where it exerts its activity is dependent on the presence of catalytically active ODC. As the overactivity of ODC and RhoA are implicated in cell transformation, the results provide a mechanistic explanation of the interrelationship between the polyamine metabolism and the reorganization of the actin cytoskeleton occurring in cancer cells. ODC and polyamines have also an important role in the function of central nervous system. They participate in the regulation of brain morphogenesis in embryos. In adult nervous tissue, polyamines regulate K+ and glutamate channels. K+ inward rectifying channels control membrane potentials and NMDA-type glutamate receptors (NMDAR) regulate synaptic plasticity. High ODC activity and polyamine levels are considered important in the development of ischemic brain damage and they are implicated in the pathogenesis of Alzheimer s disease (AD). A homolog of ODC was cloned from a human brain cDNA library, and several alternatively spliced variants were detected in human brain and testis. The novel protein was nevertheless devoid of ODC catalytic activity. It was subsequently found to be a novel inductor of ODC activity and polyamine synthesis, called antizyme inhibitor 2 (AZIN2). The accumulation of AZIN2 in vesicle-like formations along the axons and beneath the plasma membrane of neurons as well as in steroid hormone producing Leydig cells and luteal cells of the gonads implies that AZIN2 plays a role in secretion and vesicle trafficking. An accumulation of AZIN2 was detected also in specimens of AD brains. This increased expression of AZIN2 was specific for AD and was not found in brains with other neurodegenerative diseases including CADASIL or dementia with Lewy bodies.

Studies on immune activation in rheumatoid arthritis and reactive arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, progressive joint destruction, and disability. Reactive arthritis (ReA) is a sterile joint inflammation following a distant mucosal infection. The clinical course of these diseases is variable and cannot be predicted with reasonable accuracy by clinical and laboratory markers. The predictive value of circulating soluble interleukin-2 receptor (sIL-2R), a marker of lymphocyte activation, measured by Immulite® automated immunoassay analyzer, was evaluated in two cohorts of RA patients. In 175 patients with active early RA randomized to treatment with either on disease-modifying antirheumatic drug (DMARD) or a combination of 3 DMARDs and prednisolone, low baseline sIL-2R level predicted remission after 6 months in patients treated with a single DMARD. In 24 patients with active RA refractory to DMARDs, low baseline sIL-2R level predicted rapid clinical response to treatment with infliximab, an anti-tumour necrosis factor antibody. Furthermore, in a cohort of 26 patients with acute ReA, high baseline sIL-2R level predicted remission after 6 months. Levels of circulating soluble E-selectin (sE-selectin), a marker of endothelial activation, were measured annually by enzyme-linked immunosorbent assay (ELISA) in a cohort of 85 patients with early RA. During a five-year follow-up, sE-selectin levels were associated with activity and outcome of RA. The levels of neutrophil and monocyte CD11b/CD18 expression measured by flow cytometry, and circulating levels of sE-selectin measured by ELISA, and procalcitonin by immunoluminometric assay, were compared in 28 patients with acute ReA and 16 patients with early RA. The levels of the markers were comparable in ReA, RA, and healthy control subjects. In conlusion, sIL-2R may provide a new predictive marker in early RA treated with a single DMARD and refractory RA treated with infliximab. In addition, sIL-2R level predicts remission in acute ReA.

Molecular and Cellular Mechanisms Behind Juvenile Neuronal Ceroid Lipofuscinosis (JNCL, Batten disease)

Neurodegenerative disorders are chronic, progressive, and often fatal disorders of the nervous system caused by dysfunction, and ultimately, death of neuronal cells. The underlying mechanisms of neurodegeneration are poorly understood, and monogenic disorders can be utilised as disease models to elucidate the pathogenesis. Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease) is a recessively inherited lysosomal storage disorder with progressive neurodegeneration and accumulation of autofluorescent storage material in most tissues. It is caused by mutations in the CLN3 gene; however, the exact function of the corresponding CLN3 protein, as well as the molecular mechanisms of JNCL pathogenesis have remained elusive. JNCL disease exclusively affects the central nervous system leaving other organs unaffected, and therefore it is of a particular importance to conduct studies in brain tissue and neuronal cells. The aim of this thesis project was to elucidate the molecular and cell biological mechanisms underlying JNCL. This was the first study to describe the endogenous Cln3 protein, and it was shown that Cln3 localised to neuronal cells in the mouse brain. At a subcellular level, endogenous Cln3 was localised to the presynaptic terminals and to the synaptosome compartment, but not to the synaptic vesicles. Studies with the CLN3-deficient cells demonstrated an impaired endocytic membrane trafficking, and established an interconnection between CLN3, microtubulus-binding Hook1 and Rab proteins. This novel data was not only important in characterising the roles of CLN3 in cells, but also provided significant information delineating the versatile role of the Rab proteins. To identify affected cellular pathways in JNCL, global gene expression profiling of the knock-out mouse Cln3-/- neurons was performed and systematically analysed; this revealed a slight dysfunction of the mitochondria, cytoskeletal abnormality in the microtubule plus-end, and an impaired recovery from depolarizing stimulus when specific N-type Ca2+ channels were inhibited, thus leading to a prolonged time of higher intracellular calcium. All these defective pathways are interrelated, and may together be sufficient to initiate the neurodegenerative process. Results of this thesis also suggest that in neuronal cells, CLN3 most likely functions at endocytic vesicles at the presynaptic terminal, potentially involved in the regulation of the calcium-mediated synaptic transmission.

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.

Genetics of T cell co-stimulatory receptors -CD28, CTLA4, ICOS and PDCD1 in immunity and transplantation

Co-stimulatory signals are essential for the activation of naïve T cells and productive immune response. Naïve T cells receive first, antigen-specific signal through T cell receptor. Co-stimulatory receptors provide the second signal which can be either activating or inhibitory. The balance between signals determines the outcome of an immune response. CD28 is crucial for T cell activation; whereas cytotoxic T lymphocyte associated antigen 4 (CTLA4) mediates critical inhibitory signal. Inducible co-stimulator (ICOS) augments cytokine expression and plays role in immunoglobulin class switching. Programmed cell death 1 (PDCD1) acts as negative regulator of T cell proliferation and cytokine responses. The co-stimulatory receptor pathways are potentially involved in self-tolerance and thus, they provide a promising therapeutic strategy for autoimmune diseases and transplantation. The genes encoding CD28, CTLA4 and ICOS are located adjacently in the chromosome region 2q33. The PDCD1 gene maps further, to the region 2q37. CTLA4 and PDCD1 are associated with the risk of a few autoimmune diseases. There is strong linkage disequilibrium (LD) on the 2q33 region; the whole gene of CD28 exists in its own LD block but CTLA4 and the 5' part of ICOS are within a same LD block. The 3' part of ICOS and PDCD1 are in their own separate LD blocks. Extended haplotypes covering the 2q33 region can be identified. This study focuses on immune related conditions like coeliac disease (CD) which is a chronic inflammatory disease with autoimmune features. Immunoglobulin A deficiency (IgAD) belongs to the group of primary antibody deficiencies characterised by reduced levels of immunoglobulins. IgAD co-occurs often with coeliac disease. Renal transplantation is needed in the end stage kidney diseases. Transplantation causes strong immune response which is tried to suppress with drugs. All these conditions are multifactorial with complex genetic background and multiple environmental factors affecting the outcome. We have screened ICOS for polymorphisms by sequencing the exon regions. We detected 11 new variants and determined their frequencies in Finnish population. We have measured linkage disequilibrium on the 2q33 region in Finnish as well as other European populations and observed conserved haplotypes. We analysed genetic association and linkage of the co-stimulatory receptor gene region aiming to study if it is a common risk locus for immune diseases. The 2q33 region was replicated to be linked to coeliac disease in Finnish population and CTLA4-ICOS haplotypes were found to be associated with CD and IgAD being the first non-HLA risk locus common for CD and immunodeficiencies. We also showed association between ICOS and the outcome of kidney transplantation. Our results suggest new evidence for CTLA4-ICOS gene region to be involved in susceptibility of coeliac disease. The earlier published contradictory association results can be explained by involvement of both CTLA4 and ICOS in disease susceptibility. The pattern of variants acting together rather than a single polymorphism may confer the disease risk. These genes may predispose also to immunodeficiencies as well as decreased graft survival and delayed graft function. Consequently, the present study indicates that like the well established HLA locus, the co-stimulatory receptor genes predispose to variety of immune disorders.

Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.