Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caractérisation bioinformatique des nouvelles protéines mitochondriales chez les moules d’eau douce (Bivalvia : Unionoida)

Malgré que le contenu des génomes mitochondriaux animaux soit dit bien conservé, des nouveaux gènes mitochondriaux ont été identifiés chez plusieurs espèces, surtout des invertébrés. Par exemple, les bivalves exhibant la double transmission uniparentale de leurs génomes mitochondriaux possèdent des nouveaux gènes spécifiques au sexe (M-ORF dans l’ADN de type M, F-ORF dans l’ADN de type F) qui ont été caractérisés in silico chez trois espèces de l’ordre Mytiloida, une espèce de Veneroida et une espèce de Unionoida par une précédente étude. Même si les séquences varient beaucoup entre ces trois ordres, cette étude à montré que des hélices transmembranaires ainsi que des peptides signaux sont conservés pour toutes les séquences. L’étude a aussi montré que les nouveaux gènes pourraient avoir des rôles dans la signalisation cellulaire, le cycle cellulaire et la réponse immunitaire et qu’ils pourraient être le résultat de l’endogénisation de l’ADN viral. Le projet présenté ici a pour but de mieux caractériser ces nouveaux gènes et leur origine potentielle, en plus d’étudier le H-ORF particulier aux hermaphrodites, en ciblant les espèces des unionidés. Les résultats montrent que les hélices transmembranaires et peptides signaux sont conservés chez les unionidés, les protéines semblent être associées à la membrane et être capables de lier des acides nucléiques et protéines, et les fonctions potentielles sont conservées. Les M-ORFs semblent avoir un rôle dans le transport et des processus cellulaires tels que la signalisation, le cycle cellulaire et la division, et l’organisation du cytosquelette. Les F-ORFs semblent être impliqués dans le trafic et transport cellulaire et la réponse immunitaire. Finalement, les H-ORFs semblent être des glycoprotéines structurales avec des rôles dans la signalisation, le transport et la transcription. Les résultats de ce projet pourraient supporter une origine virale ou mitochondriale pour ces gènes.

Caractérisation bioinformatique des nouvelles protéines mitochondriales chez les moules d’eau douce (Bivalvia : Unionoida).

Malgré que le contenu des génomes mitochondriaux animaux soit dit bien conservé, des nouveaux gènes mitochondriaux ont été identifiés chez plusieurs espèces, surtout des invertébrés. Par exemple, les bivalves exhibant la double transmission uniparentale de leurs génomes mitochondriaux possèdent des nouveaux gènes spécifiques au sexe (M-ORF dans l’ADN de type M, F-ORF dans l’ADN de type F) qui ont été caractérisés in silico chez trois espèces de l’ordre Mytiloida, une espèce de Veneroida et une espèce de Unionoida par une précédente étude. Même si les séquences varient beaucoup entre ces trois ordres, cette étude à montré que des hélices transmembranaires ainsi que des peptides signaux sont conservés pour toutes les séquences. L’étude a aussi montré que les nouveaux gènes pourraient avoir des rôles dans la signalisation cellulaire, le cycle cellulaire et la réponse immunitaire et qu’ils pourraient être le résultat de l’endogénisation de l’ADN viral. Le projet présenté ici a pour but de mieux caractériser ces nouveaux gènes et leur origine potentielle, en plus d’étudier le H-ORF particulier aux hermaphrodites, en ciblant les espèces des unionidés. Les résultats montrent que les hélices transmembranaires et peptides signaux sont conservés chez les unionidés, les protéines semblent être associées à la membrane et être capables de lier des acides nucléiques et protéines, et les fonctions potentielles sont conservées. Les M-ORFs semblent avoir un rôle dans le transport et des processus cellulaires tels que la signalisation, le cycle cellulaire et la division, et l’organisation du cytosquelette. Les F-ORFs semblent être impliqués dans le trafic et transport cellulaire et la réponse immunitaire. Finalement, les H-ORFs semblent être des glycoprotéines structurales avec des rôles dans la signalisation, le transport et la transcription. Les résultats de ce projet pourraient supporter une origine virale ou mitochondriale pour ces gènes.

Quality-driven resource utilization methods for video streaming in wireless communication networks

This research is focused on the optimisation of resource utilisation in wireless mobile networks with the consideration of the users’ experienced quality of video streaming services. The study specifically considers the new generation of mobile communication networks, i.e. 4G-LTE, as the main research context. The background study provides an overview of the main properties of the relevant technologies investigated. These include video streaming protocols and networks, video service quality assessment methods, the infrastructure and related functionalities of LTE, and resource allocation algorithms in mobile communication systems. A mathematical model based on an objective and no-reference quality assessment metric for video streaming, namely Pause Intensity, is developed in this work for the evaluation of the continuity of streaming services. The analytical model is verified by extensive simulation and subjective testing on the joint impairment effects of the pause duration and pause frequency. Various types of the video contents and different levels of the impairments have been used in the process of validation tests. It has been shown that Pause Intensity is closely correlated with the subjective quality measurement in terms of the Mean Opinion Score and this correlation property is content independent. Based on the Pause Intensity metric, an optimised resource allocation approach is proposed for the given user requirements, communication system specifications and network performances. This approach concerns both system efficiency and fairness when establishing appropriate resource allocation algorithms, together with the consideration of the correlation between the required and allocated data rates per user. Pause Intensity plays a key role here, representing the required level of Quality of Experience (QoE) to ensure the best balance between system efficiency and fairness. The 3GPP Long Term Evolution (LTE) system is used as the main application environment where the proposed research framework is examined and the results are compared with existing scheduling methods on the achievable fairness, efficiency and correlation. Adaptive video streaming technologies are also investigated and combined with our initiatives on determining the distribution of QoE performance across the network. The resulting scheduling process is controlled through the prioritization of users by considering their perceived quality for the services received. Meanwhile, a trade-off between fairness and efficiency is maintained through an online adjustment of the scheduler’s parameters. Furthermore, Pause Intensity is applied to act as a regulator to realise the rate adaptation function during the end user’s playback of the adaptive streaming service. The adaptive rates under various channel conditions and the shape of the QoE distribution amongst the users for different scheduling policies have been demonstrated in the context of LTE. Finally, the work for interworking between mobile communication system at the macro-cell level and the different deployments of WiFi technologies throughout the macro-cell is presented. A QoEdriven approach is proposed to analyse the offloading mechanism of the user’s data (e.g. video traffic) while the new rate distribution algorithm reshapes the network capacity across the macrocell. The scheduling policy derived is used to regulate the performance of the resource allocation across the fair-efficient spectrum. The associated offloading mechanism can properly control the number of the users within the coverages of the macro-cell base station and each of the WiFi access points involved. The performance of the non-seamless and user-controlled mobile traffic offloading (through the mobile WiFi devices) has been evaluated and compared with that of the standard operator-controlled WiFi hotspots.

Optimising QoE distribution for video applications through LTE-WiFi interworking

Mobile WiFi devices are becoming increasingly popular in non-seamless and user-controlled mobile traffic offloading alongside the standard WiFi hotspots. Unlike the operator-controlled hotspots, a mobile WiFi device relies on the capacity of the macro-cell for the data rate allocated to it. This type of devices can help offloading data traffic from the macro-cell base station and serve the end users within a closer range, but will change the pattern of resource distributions operated by the base station. We propose a resource allocation scheme that aims to optimize user quality of experience (QoE) when accessing video services in the environment where traffic offloading is taking place through interworking between a mobile communication system and low range wireless LANs. In this scheme, a rate redistribution algorithm is derived to perform scheduling which is controlled by a no-reference quality assessment metric in order to achieve the desired trade-offs between efficiency and fairness. We show the performance of this algorithm in terms of the distribution of the allocated data rates throughout the macro-cell investigated and the service coverage offered by the WiFi access point.

Cellular Automata Models of Traffic Behavior in Presence of Speed Breaking Structures

In this article, we study traffic flow in the presence of speed breaking structures. The speed breakers are typically used to reduce the local speed of vehicles near certain institutions such as schools and hospitals. Through a cellular automata model we study the impact of such structures on global traffic characteristics. The simulation results indicate that the presence of speed breakers could reduce the global flow under moderate global densities. However, under low and high global density traffic regime the presence of speed breakers does not have an impact on the global flow. Further the speed limit enforced by the speed breaker creates a phase distinction. For a given global density and slowdown probability, as the speed limit enforced by the speed breaker increases, the traffic moves from the reduced flow phase to maximum flow phase. This underlines the importance of proper design of these structures to avoid undesired flow restrictions.

Performance of cellular CDMA with voice/data traffic with an SIR based admission control

We analyze the performance of an SIR based admission control strategy in cellular CDMA systems with both voice and data traffic. Most studies In the current literature to estimate CDMA system capacity with both voice and data traf-Bc do not take signal-tlFlnterference ratio (SIR) based admission control into account In this paper, we present an analytical approach to evaluate the outage probability for voice trafllc, the average system throughput and the mean delay for data traffic for a volce/data CDMA system which employs an SIR based admission controL We show that for a dataaniy system, an improvement of about 25% In both the Erlang capacity as well as the mean delay performance is achieved with an SIR based admission control as compared to code availability based admission control. For a mixed voice/data srtem with 10 Erlangs of voice traffic, the Lmprovement in the mean delay performance for data Is about 40%.Ah, for a mean delay of 50 ms with 10 Erlangs voice traffic, the data Erlang capacity improves by about 9%.

Cellular automaton model for molecular traffic jams

We consider the time evolution of an exactly solvable cellular automaton with random initial conditions both in the large-scale hydrodynamic limit and on the microscopic level. This model is a version of the totally asymmetric simple exclusion process with sublattice parallel update and thus may serve as a model for studying traffic jams in systems of self-driven particles. We study the emergence of shocks from the microscopic dynamics of the model. In particular, we introduce shock measures whose time evolution we can compute explicitly, both in the thermodynamic limit and for open boundaries where a boundary-induced phase transition driven by the motion of a shock occurs. The motion of the shock, which results from the collective dynamics of the exclusion particles, is a random walk with an internal degree of freedom that determines the jump direction. This type of hopping dynamics is reminiscent of some transport phenomena in biological systems.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals

Novel Radio Link Buffer Management Schemes for End-User Multi-class Traffic in High Speed Packet Access Networks

The requirement to provide multimedia services with QoS support in mobile networks has led to standardization and deployment of high speed data access technologies such as the High Speed Downlink Packet Access (HSDPA) system. HSDPA improves downlink packet data and multimedia services support in WCDMA-based cellular networks. As is the trend in emerging wireless access technologies, HSDPA supports end-user multi-class sessions comprising parallel flows with diverse Quality of Service (QoS) requirements, such as real-time (RT) voice or video streaming concurrent with non real-time (NRT) data service being transmitted to the same user, with differentiated queuing at the radio link interface. Hence, in this paper we present and evaluate novel radio link buffer management schemes for QoS control of multimedia traffic comprising concurrent RT and NRT flows in the same HSDPA end-user session. The new buffer management schemes—Enhanced Time Space Priority (E-TSP) and Dynamic Time Space Priority (D-TSP)—are designed to improve radio link and network resource utilization as well as optimize end-to-end QoS performance of both RT and NRT flows in the end-user session. Both schemes are based on a Time-Space Priority (TSP) queuing system, which provides joint delay and loss differentiation between the flows by queuing (partially) loss tolerant RT flow packets for higher transmission priority but with restricted access to the buffer space, whilst allowing unlimited access to the buffer space for delay-tolerant NRT flow but with queuing for lower transmission priority. Experiments by means of extensive system-level HSDPA simulations demonstrates that with the proposed TSP-based radio link buffer management schemes, significant end-to-end QoS performance gains accrue to end-user traffic with simultaneous RT and NRT flows, in addition to improved resource utilization in the radio access network.

End-to-End QoS Enhancement ofHSDPA End-User Multi-flow Traffic Using RAN Buffer Management

High speed downlink packet access (HSDPA) was introduced to UMTS radio access segment to provide higher capacity for new packet switched services. As a result, packet switched sessions with multiple diverse traffic flows such as concurrent voice and data, or video and data being transmitted to the same user are a likely commonplace cellular packet data scenario. In HSDPA, radio access network (RAN) buffer management schemes are essential to support the end-to-end QoS of such sessions. Hence in this paper we present the end-to-end performance study of a proposed RAN buffer management scheme for multi-flow sessions via dynamic system-level HSDPA simulations. The scheme is an enhancement of a time-space priority (TSP) queuing strategy applied to the node B MAC-hs buffer allocated to an end user with concurrent real-time (RT) and non-real-time (NRT) flows during a multi-flow session. The experimental multi- flow scenario is a packet voice call with concurrent TCP-based file download to the same user. Results show that with the proposed enhancements to the TSP-based RAN buffer management, end-to-end QoS performance gains accrue to the NRT flow without compromising RT flow QoS of the same end user session

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.