Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

Study of fracture behaviour of bond coats on nickel superalloy by three-point bending of microbeams

A microbeam testing geometry is designed to study the variation in fracture toughness across a compositionally graded NiAl coating on a superalloy substrate. A bi-material analytical model of fracture is used to evaluate toughness by deconvoluting load-displacement data generated in a three-point bending test. It is shown that the surface layers of a diffusion bond coat can be much more brittle than the interior despite the fact that elastic modulus and hardness do not display significant variations. Such a gradient in toughness allows stable crack propagation in a test that would normally lead to unstable fracture in a homogeneous, brittle material. As the crack approaches the interface, plasticity due to the presence of Ni3Al leads to gross bending and crack bifurcation.

Improving diagnosis of tumor-induced osteomalacia with gallium-68 DOTATATE PET/CT

Context: Tumor-induced osteomalacia (TIO) is a rarely diagnosed disorder presenting with bone pain, fractures, muscle weakness, and moderate-to-severe hypophosphatemia resulting from fibroblast growth factor 23-mediated renal phosphate wasting. Tumors secreting fibroblast growth factor 23 are often small and difficult to find with conventional imaging. Objective: We studied the utility of 68Ga-DOTA-octreotate (DOTATATE) somatostatin receptor positron emission tomography (PET)/computed tomography (CT) imaging in the diagnosis of TIO. Design and Setting: A multicenter case series was conducted at tertiary referral hospitals. Patients and Methods: Six patients with TIO diagnosed between 2003 and 2012 in Australia were referred for DOTATATE PET imaging. We reviewed the clinical history, biochemistry, imaging characteristics, histopathology, and clinical outcome of each patient. Results: Each case demonstrated delayed diagnosis despite severe symptoms. DOTATATE PET/CT imaging demonstrated high uptake and localized the tumor with confidence in each case. After surgical excision, there was resolution of clinical symptoms and serum phosphate, except in one patient who demonstrated residual disease on PET/CT. All tumors demonstrated high somatostatin receptor subtype 2 cell surface receptor expression using immunohistochemistry. Conclusions: In patients with TIO, DOTATATE PET/CT can successfully localize phosphaturic mesenchymal tumors and may be a practical first step in functional imaging for this disorder. Serum phosphate should be measured routinely in patients with unexplained muscle weakness, bone pain, or stress fractures to allow earlier diagnosis of TIO. - See more at: http://press.endocrine.org/doi/abs/10.1210/jc.2012-3642#sthash.eXD0CopL.dpuf

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 x 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 x 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 x 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Status on pre-surgical deformation apparatus for fracture fixation plates

Purpose: This paper reviews the apparatus used for deformation of bone fracture fixation plates during orthopaedic surgeries including surgical irons, pliers and bending press tools. This paper extends the review to various machineries in non-medical industries and adopts their suitability to clinics-related applications and also covers the evolution of orthopaedic bone plates. This review confirms that none of the studied machineries can be implemented for the deformation of bone fracture fixation plates during orthopaedic surgeries. In addition, this paper also presents the novel apparatus that are designed from scratch for this specific purpose. Several conceptual designs have been proposed and evaluated recently. It has been found that Computer Numerical Control (CNC) systems are not the golden solution to this problem and one needs to attempt to design the robotic arm system. A new design of robotic arm that can be used for facilitating orthopaedic surgeries is being completed.

Surfactant-Mediated Synthesis of Functional Metal Oxide Nanostructures Via Microwave Irradiation-Assisted Chemical Synthesis

In the present work we report a rapid microwave irradiation-assisted chemical synthesis technique for the growth of nanoparticles, nanorods, and nanotubes of a variety of metal oxides in the presence of an appropriate surfactant (cationic, anionic, non ionic and polymeric), without the use of any templates. The method is simple, inexpensive, and helps one to prepare nanostructures in quick time, measured in seconds and minutes. This method has been applied successfully to synthesize nanostructures of a variety of binary and ternary metal oxides such as ZnO, CdO, Fe2O3, CuO, Ga2O3, Gd2O3, ZnFe2O4, etc. There is an observed variation in the morphology of the nanostructures with changes in different process parameters, such as microwave power, irradiation time, identity of solvent, type of surfactant, and its concentration.

Modelling heterogeneity of concrete using 2D lattice network for concrete fracture and comparison with AE study

In this paper, numerical modelling of fracture in concrete using two-dimensional lattice model is presented and also a few issues related to lattice modelling technique applicable to concrete fracture are reviewed. A comparison is made with acoustic emission (AE) events with the number of fractured elements. To implement the heterogeneity of the plain concrete, two methods namely, by generating grain structure of the concrete using Fuller's distribution and the concrete material properties are randomly distributed following Gaussian distribution are used. In the first method, the modelling of the concrete at meso level is carried out following the existing methods available in literature. The shape of the aggregates present in the concrete are assumed as perfect spheres and shape of the same in two-dimensional lattice network is circular. A three-point bend (TPB) specimen is tested in the experiment under crack mouth opening displacement (CMOD) control at a rate of 0.0004 mm/sec and the fracture process in the same TPB specimen is modelled using regular triangular 2D lattice network. Load versus crack mouth opening isplacement (CMOD) plots thus obtained by using both the methods are compared with experimental results. It was observed that the number of fractured elements increases near the peak load and beyond the peak load. That is once the crack starts to propagate. AE hits also increase rapidly beyond the peak load. It is compulsory here to mention that although the lattice modelling of concrete fracture used in this present study is very similar to those already available in literature, the present work brings out certain finer details which are not available explicitly in the earlier works.

Outcome of singleton pregnancy after assisted reproductive treatment

Singleton pregnancies achieved by means of assisted reproductive treatment (ART) are associated with increased obstetric and neonatal risks in comparison with spontaneously conceived singleton pregnancies. The impact of infertility- and treatment-related factors on these risks is not properly understood. In addition, the psychological effects of infertility and its treatment on the experience of pregnancy have scarcely been studied. Thus, the aim of the present study was to evaluate the importance of infertility- and treatment-related factors on prediction of pregnancy outcome, obstetric and neonatal risks, fear-of-childbirth and pregnancy-related anxiety. The subjects consisted of infertile women who achieved a singleton pregnancy by means of in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). The control groups comprised spontaneously conceiving women with singleton gestations. Early pregnancy outcome was assessed by means of assay of serum human chorionic gonadoptrophin (hCG) in single samples. Other outcome data were collected from patient records, national Health Registers and via prospective questionnaire surveys. Viable pregnancies were associated with significantly higher serum hCG levels 12 days after embryo transfer than non-viable pregnancies. Among singleton pregnancies, aetiological subgroup, treatment type or the number of transferred embryos did not impair the predictive value of single hCG assessment. According to the register-based data, age-, parity- and socioeconomic status- adjusted risks of gestational hypertension, preterm contractions and placenta praevia were more frequent in the ART pregnancies than in the control pregnancies. Significantly higher rates of induction of delivery and Caesarean section occurred in the ART group than in the control group. The risks of preterm birth and low birth weight (LBW) were increased after ART pregnancy. Duration or aetiology of infertility, treatment type (fresh or frozen IVF or ICSI) or rank of treatment did not contribute to the risks of preterm birth or LBW. In addition, the risks of preterm birth and LBW remained elevated in spite of of the number of transferred embryos. Although mean duration of pregnancy was shorter and mean birth weight lower in the ART pregnancies than in the control pregnancies, these differences were hardly of clinical significance. Fear-of-childbirth and pregnancy-related anxiety were equally common to women conceiving by means of ART, or spontaneously. Partnership of five to ten years appeared to be protective as regards severe fear-of-childbirth, whereas long preceding infertility (≥ seven years) had the opposite effect. In conclusion, an early hCG assessment maintained its good predictive value regardless of infertility- or patient-related factors. Further, we did not recognise any infertility- or patient-related factors that would expose infertile women to increased obstetric or neonatal risks. However, a long period of infertility was associated with severe fear-of-childbirth.

The effects of low-intensity ultrasound in bioabsorbable self-reinforced poly-L-lactide -fixed cancellous bone fracture

The purpose of the present study was to investigate the effects of low-intensity ultrasound on bioabsorbable self-reinforced poly-L-lactide (SR-PLLA) screws and on fracture healing after SR-PLLA device fixation in experimental and clinical cancellous bone fracture. In the first experimental study, the assessment of the mechanical strengths of the SR-PLLA screws was performed after 12 weeks of daily 20-minute ultrasound exposure in vitro. In the second experimental study, 32 male Wistar rats with an experimental distal femur osteotomy fixed with an SR-PLLA rod were exposed for daily low-intensity ultrasound treatment for 21 days. The effects on the healing bone were assessed. The clinical studies consist of three prospective, randomized, and placebo-controlled series of dislocated lateral malleolar fractures fixed with one SR-PLLA screw. The total number of the patients in these series was 52. Half of the patients were provided randomly with a sham ultrasound device. The patients underwent ultrasound therapy 20 minutes daily for six weeks. Radiological bone healing was assessed both by radiographs at two, six, nine, and 12 weeks and by multidetector computed tomography (MDCT) scans at two weeks, nine weeks, and 18 months. Bone mineral density was assessed by dual-energy X-ray absorptiometry (DXA). The clinical outcome was assessed by both Olerud-Molander scoring and clinical examination of the ankle. Low-intensity ultrasound had no effects on the mechanical properties and degradation behaviour of the SR-PLLA screws in vitro. There were no obvious signs of low-intensity ultrasound-induced enhancement in the bone healing in SR-PLLA-rod-fixed metaphyseal distal femur osteotomy in rats. The biocompatibility of low-intensity ultrasound treatment and SR-PLLA was found to be good. In the clinical series low-intensity ultrasound was observed to have no obvious effects on the bone mineral density of the fractured lateral malleolus. There were no obvious differences in the radiological bone healing times of the SR-PLLA-screw-fixed lateral malleolar fractures after low-intensity ultrasound treatment. Low-intensity ultrasound did not have any effects on radiological bone morphology, bone mineral density or clinical outcome 18 months after the injury. There were no obvious findings in the present study to support the hypothesis that low-intensity pulsed ultrasound enhances bone healing in SR-PLLA-rod-fixed experimental metaphyseal distal femur osteotomy in rats or in clinical SR-PLLA-screw-fixed lateral malleolar fractures. It is important to limit the conclusions of the present set of studies only to lateral malleolar fractures fixed with an SR-PLLA screw.

Verification of the applicability of lattice model to concrete fracture by AE study

Notched three-point bend specimens (TPB) were tested under crack mouth opening displacement (CMOD) control at a rate of 0.0004 mm/s and the entire fracture process was simulated using a regular triangular two-dimensional lattice network only over the expected fracture proces zone width. The rest of the beam specimen was discretised by a coarse triangular finite element mesh. The discrete grain structure of the concrete was generated assuming the grains to be spherical. The load versus CMOD plots thus simulated agreed reasonably well with the experimental results. Moreover, acoustic emission (AE) hits were recorded during the test and compared with the number of fractured lattice elements. It was found that the cumulative AE hits correlated well with the cumulative fractured lattice elements at all load levels thus providing a useful means for predicting when the micro-cracks form during the fracturing process, both in the pre-peak and in the post-peak regimes.

Compiler-assisted instruction decoder energy optimization for clustered VLIW architectures

Traditionally, an instruction decoder is designed as a monolithic structure that inhibit the leakage energy optimization. In this paper, we consider a split instruction decoder that enable the leakage energy optimization. We also propose a compiler scheduling algorithm that exploits instruction slack to increase the simultaneous active and idle duration in instruction decoder. The proposed compiler-assisted scheme obtains a further 14.5% reduction of energy consumption of instruction decoder over a hardware-only scheme for a VLIW architecture. The benefits are 17.3% and 18.7% in the context of a 2-clustered and a 4-clustered VLIW architecture respectively.

Highly biocompatible and water-dispersible, amine functionalized magnetite nanoparticles, prepared by a low temperature, air-assisted polyol process: a new platform for bio-separation and diagnostics

A low temperature polyol process, based on glycolaldehyde mediated partial reduction of FeCl3 center dot 6H(2)O at 120 degrees C in the presence of sodium acetate as an alkali source and 2,2'-(ethylenedioxy)-bis-(ethylamine) as an electrostatic stabilizer has been used for the gram-scale preparation of biocompatible, water-dispersible, amine functionalized magnetite nanoparticles (MNPs) with an average diameter of 6 +/- 0.75 nm. With a reasonably high magnetization (37.8 e.m.u.) and amine groups on the outer surface of the nanoparticles, we demonstrated the magnetic separation and concentration implications of these ultrasmall particles in immunoassay. MRI studies indicated that these nanoparticles had the desired relaxivity for T-2 contrast enhancement in vivo. In vitro biocompatibility, cell uptake and MR imaging studies established that these nanoparticles were safe in clinical dosages and by virtue of their ultrasmall sizes and positively charged surfaces could be easily internalized by cancer cells. All these positive attributes make these functional nanoparticles a promising platform for further in vitro and in vivo evaluations.

Transition to parenthood after assisted reproductive treatment: Follow-up study of singleton pregnancies

Infertility treatments are relatively easily available in most Western countries today, but the psychological consequences of these high-tech treatments have scarcely been addressed. The purpose of this controlled longitudinal study was to explore the early environment of the infant born by assisted reproductive treatment (ART). We focused on the parents mental well-being, marital relations and experience of parenting. In addition to this, we assessed parent child interaction and parents mental representations of their child after long-standing infertility and several unsuccessful ART attempts. The subjects were infertile couples who achieved a singleton pregnancy by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The control group comprised of spontaneously conceiving couples with singleton pregnancies. ART women showed fewer depressive symptoms than controls during pregnancy and after delivery, but the difference vanished by the end of the child s first year. ART men consistently had lower levels of anxiety symptoms, sleeping difficulties, and social dysfunction than control men. Control women experienced a decrease in dyadic consensus during the child s first year, which did not happen among ART women. After the child was born, ART men reported a higher level of sexual affection compared with control men. Psychic symptoms and stressful life events were differently related to marital relations in ART and control groups. The parenting experiences of ART mothers were in general at a higher level, compared with controls, and they changed in a positive direction during the child s first year. Fathering experiences were at the same level in both groups, and they changed positively in both groups by the end of the child s first year. The parenting experiences of ART mothers and fathers were more resilient to certain child-related stressors than those of control group. Both mothers and fathers with long-term infertility showed more sensitive behaviour with their child in toddler-age than in infancy. Correspondingly, children s cooperation increased. Mothers often mentioned a fear of miscarriage and difficulty in creating representations of the child during pregnancy. Descriptions of the infants were mainly rich, vivid and loaded with positive features. In conclusion, ART parents in general seem to adapt well to the transition to parenthood. Former infertility and ART do not seem to constitute a risk for parents mental health, marital relations or experience of parenting. Even longstanding infertility with several unsuccessful treatment attempts did not create a risk as regards parenting behaviour or parents mental representations of their child. In this group, however, women were found to have fear for losing the child and difficulty in creating representations of the child during pregnancy, which in some cases may indicate need for psychosocial support. Even though our results are encouraging, infertility and infertility treatments are generally considered as a stressful experience. It is a challenge for health authorities to recognize those couples who need professional help to overcome the distressing experiences of infertility and ART.