Quality of service and resource management in IP and wireless networks

A common theme in the publications included in this thesis is the quality of service and resource management in IP and wireless networks. This thesis presents novel algorithms and implementations for admission control in IP and IEEE 802.16e networks, active queue management in EGPRS, WCDMA, and IEEE 802.16e networks, and scheduling in IEEE 802.16e networks. The performance of different algorithms and mechanisms is compared with the prior art through extensive ns-2 simulations. We show that similar active queue management mechanisms, such as TTLRED, can be successfully used to reduce the downlink delay (and in some cases even improve the TCP goodput) in different bottlenecks of IP, EGPRS, WCDMA, and IEEE 802.16e access networks. Moreover, almost identical connection admission control algorithms can be applied both in IP access networks and at IEEE 802.16e base stations. In the former case, one just has to first gather the link load information from the IP routers. We also note that DiffServ can be used to avoid costly overprovisioning of the backhaul in IEEE 802.16e networks. We present a simple mapping between IEEE 802.16e data delivery services and DiffServ traffic classes, and we propose that IEEE 802.16e base stations should take the backhaul traffic load into account in their admission control decisions. Moreover, different IEEE 802.16e base station scheduling algorithms and uplink channel access mechanisms are studied. In the former study, we show that proportional fair scheduling offers superior spectral efficiency when compared to deficit round-robin, though in some cases at the cost of increased delay. Additionally, we introduce a variant of deficit round-robin (WDRR), where the quantum value depends on the modulation and coding scheme. We also show that there are several ways to implement ertPS in an efficient manner, so that during the silence periods of a VoIP call no uplink slots are granted. The problem here, however, is how to implement the resumption after the silence period while introducing as little delay as possible

Profilin 1 negatively regulates osteoclast migration in postnatal skeletal growth, remodeling, and homeostasis in mice

Profilin 1 (Pfn1), a regulator of actin polymerization, controls cell movement in a context‐dependent manner. Pfn1 supports the locomotion of most adherent cells by assisting actin‐filament elongation, as has been shown in skeletal progenitor cells in our previous study. However, because Pfn1 has also been known to inhibit migration of certain cells, including T cells, by suppressing branched‐end elongation of actin filaments, we hypothesized that its roles in osteoclasts may be different from that of osteoblasts. By investigating the osteoclasts in culture, we first verified that Pfn1‐knockdown (KD) enhances bone resorption in preosteoclastic RAW264.7 cells, despite having a comparable number and size of osteoclasts. Pfn1‐KD in bone marrow cells showed similar results. Mechanistically, Pfn1‐KD osteoclasts appeared more mobile than in controls. In vivo, the osteoclast‐specific conditional Pfn1‐deficient mice (Pfn1‐cKO) by CathepsinK‐Cre driver demonstrated postnatal skeletal phenotype, including dwarfism, craniofacial deformities, and long‐bone metaphyseal osteolytic expansion, by 8 weeks of age. Metaphyseal and diaphyseal femurs were drastically expanded with suppressed trabecular bone mass as indicated by μCT analysis. Histologically, TRAP‐positive osteoclasts were increased at endosteal metaphysis to diaphysis of Pfn1‐cKO mice. The enhanced movement of Pfn1‐cKO osteoclasts in culture was associated with a slight increase in cell size and podosome belt length, as well as an increase in bone‐resorbing activity. Our study, for the first time, demonstrated that Pfn1 has critical roles in inhibiting osteoclast motility and bone resorption, thereby contributing to essential roles in postnatal skeletal homeostasis. Our study also provides novel insight into understanding skeletal deformities in human disorders

Connexin-mimetic peptide Gap 27 decreases osteoclastic activity

BACKGROUND: Bone remodelling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. We have shown previously that osteoclasts contain gap-junctional protein connexin-43 and that a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor. METHODS: A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption. We utilised the well-characterised pit-formation assay to study the effects of the specific gap-junctional inhibitor to the survival and activity of osteoclasts. RESULTS: Gap 27 caused a remarked decrease in the number of both TRAP-positive mononuclear and multinucleated rat osteoclasts cultured on bovine bone slices. The decrease in the cell survival seemed to be restricted to TRAP-positive cells, whereas the other cells of the culture model seemed unaffected. The activity of the remaining osteoclasts was found to be diminished by measuring the percentage of osteoclasts with actin rings of all TRAP-positive cells. In addition, the resorbed area in the treated cultures was greatly diminished. CONCLUSIONS: On the basis of these results we conclude that gap-junctional communication is essential for the action of bone resorbing osteoclasts and for proper remodelling for bone

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’

Murtumapotilaiden osteoporoosin lääkehoidossa on isoja alueellisia eroja

Lähtökohdat : Lääkekattavuudesta osteoporoottisten murtumien sekundaaripreventiossa Suomessa ei ole tietoa. Tässä tutkimuksessa tarkastellaan pienienergiaisen murtuman jälkeen osteoporoosi-lääkemääräyksen saaneiden potilaiden osuutta ja lääkemääräyskattavuuden alueellisia eroja. Menetelmät : Tutkimus on retrospektiivinen rekisteritutkimus, joka toteutettiin hoitoilmoitusten ja Kelan reseptitietojen pohjalta. Tulokset : Potilaista 9 %:lle määrättiin osteoporoosilääkitys 6 kuukauden sisällä murtumasta. Lääkemääräyskattavuus suureni 12 %:iin 12 kuukauden sisällä murtumasta. Lonkka- (11 %) ja nikamamurtumien (18 %) kohdalla lääkemääräyksen saaneiden osuus oli hiukan keskiarvoa suurempi. Lonkkamurtuman jälkeinen lääkemääräyskattavuus oli eri sairaanhoitopiireissä 3–32 %. Päätelmät : Kansainvälisiin tuloksiin verrattuna osteoporoosilääkemääräyksen saaneiden murtumapotilaiden osuutta voi pitää pienenä. Lääkemääräyskattavuudessa oli eroja sairaanhoitopiirien välillä, joskaan tulokset eivät ota huomioon tsoledronihappohoitoja. Osteoporoosin sekundaariprevention tehostamiselle ja lääkehoidon yhtenäistämiselle on tarvetta.Peer reviewe

Roles of hyaluronan in bone resorption

BACKGROUND: Hyaluronan, an unsulfated glycosaminoglycan, while being closely linked to osteoclast function several years ago, has received little attention lately. Given recent new knowledge of hyaluronan's possible cell binding abilities, it is important to re-examine the role of this polysaccharide in bone homeostasis. DISCUSSION: Previously published data demonstrating a linkage between induction of hyaluronan synthesis and osteoclast-mediated bone resorption are reviewed. Suggestions are made involving the cell binding ability of hyaluronan and its potential to mediate osteoclast binding to bone surfaces and its potential to serve as a diffusion barrier and participate in the sealing zone required for osteoclast-mediated bone resorption. SUMMARY: This brief article summarizes previous studies linking HA to bone resorption and suggests roles for hyaluronan in the process of bone resorption

Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems

BACKGROUND: Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. RESULTS: We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. CONCLUSION: Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of α(v)β(3 )integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection

The Tyrosine Kinase Pyk-2/Raftk Regulates Natural Killer (Nk) Cell Cytotoxic Response, and Is Translocated and Activated upon Specific Target Cell Recognition and Killing

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK–target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK–target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response