Immune cells must accurately interpret environmental signals to make robust cell fate decisions and control inflammatory signalling. Many signals (e.g. Tumor Necrosis Factor alpha (TNFα) or interferon gamma (IFNγ)) converge on just a few key signalling systems such as Nuclear Factor kappa B (NF-κB) or Signal Transducers and Activators of Transcription (STAT), which exhibit complex activation dynamics that control patterns of downstream gene expression. Often, seemingly identical cells show heterogeneous or random behaviour to a common stimulus. Therefore, a key question is how can immune cells coordinate inflammatory signalling in the presence of this noise. The NF-kB system dynamics were studied in response to rapidly changing inflammatory signals. It was shown that pulsed TNFa cytokine stimulations induced digital single-cell NF-kB responses, with only a fraction of cells able to respond to repeated pulses. These responses appeared to be reproducible in individual cells, but heterogeneous in the population. Mathematical models of the NF-kB signalling network suggested that single-cell responses were governed through a refractory state potentially encoded via 'extrinsic' noise in the levels of signalling molecules related to the TNFa signal transduction pathway. Such signal processing enabled robust and reproducible single cell responses and maintained acute tissue-level signalling, with fewer cells responding to shorter pulsing intervals. The NF-kB system is involved in effector cytokine propagation in response to pathogen infection. It was shown that in macrophages, the dose of TLR4 stimulation (mimicking the pathogen infection) was encoded in graded (yet heterogeneous) NF-kB dynamics in single cells. This resulted in analogue inflammatory gene expression patterns in the population. However, individual cells substantially differed in their ability to encode TLR4 signal and to regulate TNFa expression, which was explained by extrinsic noise in the NF-kB system. Quantitative mathematical modelling showed that tissue-level environment modulates heterogeneous single-cell TNFa outputs; by effectively removing it from circulation. This may determine the interaction distance between tissue-resident immune cells to enable propagation of cellular inflammation. Heterogeneity of single cell macrophage signalling was also observed in NF-kB and STAT1 system responses to a range of IFN stimulation doses. Although each system showed substantial variability between cells, their responses were surprisingly well correlated in individual cells. It was however apparent (based on gene expression studies) that individual cells may not be able to precisely discriminate different IFNg doses. Overall, this work suggests that heterogeneity in the NF-kB (and other) regulatory networks might be a part of an inherent design motif in the inflammatory response, which enables robust control of the tissue-level inflammatory response by preventing homogeneous and thus potentially harmful activation.

CD40 is a TNF-Receptor (TNFR) superfamily member that functions to promote several facets of the humoral immune response--including B cell proliferation, differentiation, antibody isotype switching, and cytokine expression. TNFR superfamily members lack intrinsic kinase activity and must recruit members of the TNFR-associated factor (TRAF) family of adaptor proteins to connect the receptor to intracellular signaling pathways. CD40-mediated JNK and NF-κB activation is critical for an intact humoral immune response; however, the precise mechanisms governing the spatiotemporal activation of these pathways are not completely understood. In this study we report that CD40 ligation results in the dual phosphorylation of TRAF2 on Ser-11 and Ser-55 to control the subcellular localization of key pathway intermediates and temporally regulate downstream JNK and IKK/NF-κB pathway activation. Notably, TBK1- mediated TRAF2 Ser-11 phosphorylation elicits the dissociation of a signaling complex, consisting of TRAF2, cIAP1/2, and IKKγ, from the CD40 receptor to potentiate a secondary phase of JNK and IKK activation. In the absence of this phosphorylation event, these proteins translocate to the insoluble lipid rafts along with the membrane-bound receptor complex, where TRAF2 undergoes Ser-55 phosphorylation-dependent self-ubiquitination and degradation necessary for cessation of JNK activation. Furthermore, TRAF2 Ser-11 phosphorylation inhibits non-canonical NF-κB activation by promoting the lipid raft localization of the CD40 receptor complex. This suggests that TRAF2 dual phosphorylation acts as a molecular switch to control canonical and non-canonical NF-κB activation. CD40 signaling is heavily implicated in a wide array of chronic inflammatory and autoimmune diseases--including Alzheimer's, Grave's disease, and diabetes. As such, characterization of the molecular mechanisms directing CD40 signal transduction will provide a foundation for the further development of targeted immunomodulatory therapeutics. In addition, the NF-κB transcriptional program has well-defined roles in oncogenesis and tumor progression, and many B cell lymphomas exploit the CD40L/CD40 dyad to constitutively activate the NF-κB pathway and potentiate neoplastic growth and survival. Through these analyses, we demonstrate that TRAF2 phosphorylation on Ser-11 and Ser-55 promote cell survival in response to genotoxic and oxidative stress, respectively, by regulating JNK and NF-κB pathway activation and coordinating the subcellular localization and stability of key signaling effectors. Furthermore, we show that inhibition of TRAF2 phosphorylation in B-cell lymphoma cells increases their sensitivity to standard frontline chemotherapeutics, including doxorubicin and vincristine, as well as the novel agents bortezomib and arsenic trioxide. These findings are clinically significant, as TRAF2 is found over-expressed and constitutively phosphorylated in DLBCL cell lines and patient biopsies. In addition, mice bearing tumors that harbor TRAF2 Ser-11 phospho-null mutations are more responsive to treatment with doxorubicin and have significantly prolonged survival compared to wild-type counterparts in a syngeneic model of B-cell lymphoma. The tumor microenvironment is characterized by pro-inflammatory cytokines, hypoxia, low glucose, and free radicals, all of which are known to induce chronic cellular stress and NF-κB activation. Cancer cell adaptation to these stressors has profound consequences for malignant progression and therapeutic response. In this regard, our findings present a unique opportunity where the molecular targeting to TRAF2 phosphorylation could increase the efficacy of current therapies by suppressing basal NF-κB activation, thus synergistically sensitizing NF-κB-driven malignancies to chemotherapeutic-induced cell death.

<p>Denna rapport behandlar en prisjämförelse mellan fyra olika elsystem i fyra valda referensmiljöer. Uppdragsgivaren till detta projekt är företaget Arexor som har utvecklat en ellist som de ska släppa på marknaden under hösten och därför vill ha en prisbild på marknaden idag. De olika systemen är i huvuddel Thorsmans TEK-123, KB-systems DWP, Eubiqs PTS samt det traditionella sättet med kablar och VP-rör infällda i väggen. Även en presentation av projektets uppdragsgivare Arexor och deras ETS finns att läsa om. För att kunna utgå från en realistisk bakgrund är referensmiljöerna valda så att en standard kan sättas för utformningen. Dessa miljöer är ett kontor, ett konferensrum, en datorsal och en lägenhet. Elsystemen har modifierats till viss grad för att de skall kunna jämföras med varandra.</p><p>I detta projekt kom gruppen fram till att den traditionella eldragningen är den mest kostnadseffektiva alternativet när det gäller kontor, konferensrum och lägenhet. När det gäller datorsalen blir KB-system billigast. Dock måste viss hänsyn tas till att jämförelsen enbart bygger på elsystem och har bortsett från att vissa system även kan erbjuda el/tele/datainstallation, så kallad triple-play, i samma system vilket man vanligtvis kombinerar i dessa miljöer. I ett sådant fall kan en ny undersökning ge en annan prisbild.</p> / <p>This rapport treats a price comparison between four different electric systems in four chosen reference environments. The assigner of this project is the company Arexor that has developed an electrical track that they will release on the market during the fall and therefore wanted a price picture of the market today. The different systems are in main Thorsmans TEK-123, KB-systems DWP, Eubiqs PTS and the traditional way with cables and VP-pipes hidden in the wall. Also a presentation of the projects assigner Arexor and their ETS is to read about. To start from a realistic background, the reference environments are chosen so a standard can be maid for the design. These environments are an office, a conference room, a computer room and an apartment. The electrical systems have been modified a bit so that they could be compared with each other.</p><p>In this project the group came to the conclusion that the traditional way is the most cost efficient alternative when it comes to the office, the conference room and the apartment. When it comes to the computer room, the KB-system is the cheapest. However, there must be a consideration to the fact that the comparison only relies on electrical systems and has disregarded from the fact that some systems also can provide for electricity/tele/computer installation, so called triple-play, in the same system, wich is common to combine in these environments. If that is the case, a new investigation could give another price picture.</p>

Schwann cells (SCs) are crucial for peripheral nerve development and regeneration; however, the intrinsic regulatory mechanisms governing post-injury responses are poorly understood. Activation and deacetylation of nuclear factor-κB (NF- κB) in SCs have been implicated as prerequisites for peripheral nerve myelination. Using GFAP-IκBα-dn mice, in which NF- κB transcriptional activation is inhibited in SCs, we found no discernable differences in the quantity or structure of myelinated axons in adult facial nerves. Following crush injury, axonal regeneration was impaired at 31 days and greatly improved at 65 days in GFAP-IκBα-dn mutants. Compact re-myelination and sensory fiber organization were significantly compromised at 31 days and restored by 65 days. Together, these data indicate that NF- κB activation in SCs is dispensable for peripheral nerve myelination in adults, but required for early re-myelination and axonal regeneration. SC myelination during development and following injury in adult mice may hinge on different transcriptional cascades; these findings may offer new therapeutic avenues for PNS and CNS regeneration.

Le facteur de transcription NF-κB joue un rôle majeur dans lorchestration de nombreux processus biologiques, tels que les réponses immunitaires innée et adaptative, la division cellulaire, lapoptose et le développement. Le NF-κB est activé en réponse à un grand nombre de stimuli, comme les cytokines pro-inflammatoires, les agents viraux et bactériens et la stimulation antigénique des cellules du système immunitaire. Il peut être aussi activé dans des conditions de stress oxydant, par exemple après une exposition à des concentrations physiologiques (de lordre du µM) de peroxyde dhydrogène (H2O2). Au début de ce travail, le mécanisme conduisant à lactivation du NF-κB par le stress oxydant était mal connu et sujet à maintes controverses, ce qui nous a poussé à investiguer cette voie plus avant. Dans une première partie, nous nous sommes attachés à étudier le mécanisme dactivation du NF-κB dans des lymphocytes T soumis à un stress oxydant. Les cellules du système immunitaire sont en effet très sensibles à lenvironnement redox, et sont fréquemment en contact avec des espèces réactives de loxygène libérées par les cellules phagocytaires (monocytes/macrophages et neutrophiles) lors dune réponse inflammatoire. Comme le NF-κB est une protéine cruciale pour le développement et lhoméostasie des lymphocytes T, létude de sa modulation lors dun stress oxydant savère particulièrement importante. Nous avons pu mettre en évidence une activation importante du facteur de transcription lors dun traitement oxydant, ainsi quune dégradation presque compète de linhibiteur IκBα. Létude approfondie du mécanisme menant à cette dégradation a mis au jour un mécanisme dactivation tout à fait inédit, impliquant lactivation du complexe IKK via lintervention de lInositol Phosphatase SHIP-1. Dans cette première partie, nous avons également mis en évidence le rôle crucial de la protéine SHIP-1 dans la protection des lymphocytes T contre lapoptose induite par le stress oxydant, ce qui en fait une protéine clé dans la lhoméostasie des lymphocytes T. Dans un second temps, nous nous sommes intéressés au mécanisme influençant la fixation du NF-κB à lADN. Nous avons pu démontrer le rôle important de la protéine IKKα comme déterminant la fixation du NF-κB aux promoteurs de certains gènes, mettant ainsi au jour un mode daction inconnu pour cette protéine. Ce rôle est dautant plus intéressant quil est spécifique, ce qui pourrait déboucher sur des applications thérapeutiques intéressantes.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

Glucocorticoids are exploited for the treatment of hematological malignancies due to their ability to cause apoptosis in lymphoid cells. Innate and acquired resistance, however, limits their efficacy in the clinic. The mechanisms contributing to resistance are poorly understood. A better understanding of the critical events during glucocorticoid-induced apoptosis are needed in order to develop novel agents that will exploit these critical targets and improve the response to glucocorticoid-based therapies. Previously, using WEHI7.2 murine thymic lymphoma cells, our laboratory demonstrated that the levels of reactive oxygen species (ROS) increase during glucocorticoid-induced apoptosis signaling. WEHI7.2 cell variants with increased catalase exhibit increased resistance to glucocorticoids, suggesting that oxidative stress plays a role in glucocorticoid-induced apoptosis and that increasing the intracellular production of ROS may be a potential strategy for sensitizing lymphoma cells to glucocorticoid treatment. The following studies demonstrate that an increase in H₂O₂ is essential for lymphoma cells to undergo apoptosis and that the ability to remove cellular H₂O₂ protects the cells from glucocorticoid-mediated cell death. The redox-cycling agent, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin, increased glucocorticoid-induced oxidative stress in WEHI7.2 cells and sensitized the cells to glucocorticoid treatment. MnTE-2-PyP⁵⁺ glutathionylated NF-κB and inhibited its activity. Collectively, these findings suggest that manipulating the redox environment with MnTE-2-PyP⁵⁺ is a promising approach for lymphoma therapy.

Denna rapport behandlar en prisjämförelse mellan fyra olika elsystem i fyra valda referensmiljöer. Uppdragsgivaren till detta projekt är företaget Arexor som har utvecklat en ellist som de ska släppa på marknaden under hösten och därför vill ha en prisbild på marknaden idag. De olika systemen är i huvuddel Thorsmans TEK-123, KB-systems DWP, Eubiqs PTS samt det traditionella sättet med kablar och VP-rör infällda i väggen. Även en presentation av projektets uppdragsgivare Arexor och deras ETS finns att läsa om. För att kunna utgå från en realistisk bakgrund är referensmiljöerna valda så att en standard kan sättas för utformningen. Dessa miljöer är ett kontor, ett konferensrum, en datorsal och en lägenhet. Elsystemen har modifierats till viss grad för att de skall kunna jämföras med varandra. I detta projekt kom gruppen fram till att den traditionella eldragningen är den mest kostnadseffektiva alternativet när det gäller kontor, konferensrum och lägenhet. När det gäller datorsalen blir KB-system billigast. Dock måste viss hänsyn tas till att jämförelsen enbart bygger på elsystem och har bortsett från att vissa system även kan erbjuda el/tele/datainstallation, så kallad triple-play, i samma system vilket man vanligtvis kombinerar i dessa miljöer. I ett sådant fall kan en ny undersökning ge en annan prisbild. / This rapport treats a price comparison between four different electric systems in four chosen reference environments. The assigner of this project is the company Arexor that has developed an electrical track that they will release on the market during the fall and therefore wanted a price picture of the market today. The different systems are in main Thorsmans TEK-123, KB-systems DWP, Eubiqs PTS and the traditional way with cables and VP-pipes hidden in the wall. Also a presentation of the projects assigner Arexor and their ETS is to read about. To start from a realistic background, the reference environments are chosen so a standard can be maid for the design. These environments are an office, a conference room, a computer room and an apartment. The electrical systems have been modified a bit so that they could be compared with each other. In this project the group came to the conclusion that the traditional way is the most cost efficient alternative when it comes to the office, the conference room and the apartment. When it comes to the computer room, the KB-system is the cheapest. However, there must be a consideration to the fact that the comparison only relies on electrical systems and has disregarded from the fact that some systems also can provide for electricity/tele/computer installation, so called triple-play, in the same system, wich is common to combine in these environments. If that is the case, a new investigation could give another price picture.

Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.

KB-1 is an anaerobic Dehalococcoides-containing microbial culture used industrially to bioremediate sites impacted with chlorinated solvents. The culture is typically grown at pH 7. However, lower pH is often encountered and therefore the effect of pH was investigated. Both sudden and stepwise decreases in pH from 7 to 6 and 5.5 were investigated over a period of 450 days. An electron balance was also calculated to look at the flow of electrons for dechlorination. More than 95% of the reducing equivalents went towards methanogenesis and acetogenesis. Select microorganisms were compared by quantitative Polymerase Chain Reaction. It was found that lower rates of dechlorination correspond to low Dehalococcoides numbers and that different methanogens were enriched on different electron donors.