Characterization of the TRIM37 gene and mutations underlying mulibrey nanism

Mulibrey nanism is a hereditary developmental disorder, characterized by prenatal onset growth failure without postnatal catch-up growth, distinctive craniofacial features, progressive cardiopathy and failure of sexual maturation. In addition, the patients develop insulin resistance syndrome and type 2 diabetes and they have an increased risk of developing tumors. The TRIM37 gene that underlies mulibrey nanism encodes for a member of the tripartite motif (TRIM) protein family. The physiological function of TRIM37 and the pathogenetic mechanisms leading from TRIM37 dysfunction to the mulibrey nanism phenotype are unknown. However, TRIM37 localizes at least partially to peroxisomes, and possesses ubiquitin E3-ligase activity. Thus, it may mediate ubiquitin dependent protein degradation, suggesting that accumulation of yet unknown substrate proteins may underlie the disease pathogenesis. In this study, the TRIM37 gene was characterized in detail. A transcription initiation window, with several separate transcription start sites, was identified and the putative promoter region immediately upstream from the transcription initiation window was shown to possess basal promoter activity. Further, several alternative splice variants of the gene were identified, including a highly expressed testis specific variant, encoding for an identical protein product with the main transcript. Expression of TRIM37 mRNA was detected in several different tissues, with highest expression seen in testis and in brain, when the expression patterns of the two major transcripts in different human tissues were studied by quantitative real-time PCR. Several mulibrey nanism patients were studied and thirteen novel mutations in TRIM37 were found, including three mutations (p.Gly322Val, p.Cys109Ser, p.Glu271_Ser287), that are likely to express mutant TRIM37 proteins. These mutations were further shown to alter the subcellular localization of the mutant proteins. Most of the mulibrey nanism associated mutations however, lead to premature termination codons and degradation of mRNA. All the TRIM37 mutations identified to date predict loss-of-function alleles, and thus no phenotype-genotype correlation is seen among the patients. In order to understand the pathogenetic mechanisms underlying mulibrey nanism, an animal model for the disorder is needed. For the development of a Trim37 knock-out mouse, the mouse Trim37 gene was characterized. Alternative splice variants, were identified, including a testis specific variant predicting a longer protein product. Further, a strictly tissue and cell-specific pattern of Trim37 expression was observed in developing and adult mouse tissues, when studied by immunohistochemical methods. This distribution of Trim37 expression in mouse tissues is in agreement with the clinical findings in human mulibrey nanism patients. This thesis work gives new tools for the diagnostics of mulibrey nanism as well as for studying the molecular pathogenesis behind this interesting disorder.

A participatory whole farm modelling approach to understand impacts and increase preparedness to climate change in Australia

This study presents the use of a whole farm model in a participatory modelling research approach to examine the sensitivity of four contrasting case study farms to a likely climate change scenario. The newly generated information was used to support discussions with the participating farmers in the search for options to design more profitable and sustainable farming systems in Queensland Australia. The four case studies contrasted in key systems characteristics: opportunism in decision making, i.e. flexible versus rigid crop rotations; function, i.e. production of livestock or crops; and level of intensification, i.e. dryland versus irrigated agriculture. Tested tactical and strategic changes under a baseline and climate change scenario (CCS) involved changes in the allocation of land between cropping and grazing enterprises, alternative allocations of limited irrigation water across cropping enterprises, and different management rules for planting wheat and sorghum in rainfed cropping. The results show that expected impacts from a likely climate change scenario were evident in the following increasing order: the irrigated cropping farm case study, the cropping and grazing farm, the more opportunistic rainfed cropping farm and the least opportunistic rainfed cropping farm. We concluded that in most cases the participating farmers were operating close to the efficiency frontier (i.e. in the relationship between profits and risks). This indicated that options to adapt to climate change might need to evolve from investments in the development of more innovative cropping and grazing systems and/or transformational changes on existing farming systems. We expect that even though assimilating expected changes in climate seems to be rather intangible and premature for these farmers, as innovations are developed, adaptation is likely to follow quickly. The multiple interactions among farm management components in complex and dynamic farm businesses operating in a variable and changing climate, make the use of whole farm participatory modelling approaches valuable tools to quantify benefits and trade-offs from alternative farming systems designs in the search for improved profitability and resilience.

Physical activity in mid-age and older women: Lessons from the Australian longitudinal study on women’s health

The Australian Longitudinal Study on Women’s Health (ALSWH) commenced in Australia in 1996 when researchers recruited approximately 40,000 women in three birth cohorts: 1973–1978, 1946–1951, and 1921–1926. Since then participants have completed surveys on a wide range of health issues, at approximately three-year intervals. This overview describes changes in physical activity (PA) over time in the mid-age and older ALSWH cohorts, and summarizes the findings of studies published to date on the determinants of PA, and its associated health outcomes in Australian women. The ALSWH data show a significant increase in PA during mid-age, and a rapid decline in activity levels when women are in their 80s. The study has demonstrated the importance of life stages and key life events as determinants of activity, the additional benefits of vigorous activity for mid-age women, and the health benefits of ‘only walking’ for older women. ALSWH researchers have also drawn attention to the benefits of activity in terms of a wide range of physical and mental health outcomes, as well as overall vitality and well-being. The data indicate that maintaining a high level of PA throughout mid and older age will not only reduce the risk of premature death, but also significantly extend the number of years of healthy life.

Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post-flowering stress. Overseas reports of M.phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperaturexsoil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M.phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M.phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.

Twitter (probably) isn't dying, but is it becoming less sociable?

Twitter’s demise has been announced so many times over its lifetime that it’s hard to keep track of all the premature eulogies (and this one from a year ago is actually pretty insightful), but there seems to be a new intensity in the circulation of decline narratives at the moment. A couple of weeks ago there was quite a bit of heat on umair haque’s Medium story, in which he proclaimed..

Scoping study to inform the development of the new national nutrition policy for Australia

This comprehensive report (748 pages) scopes a new nutrition policy for Australia (RFT 028/1213) Australian Department of Health and Ageing. Optimum nutrition is fundamental to good health. It is essential for the normal growth and physical and cognitive development of infants and children, contributes significantly to quality of life, wellbeing and workforce productivity, enhances resistance to infection and reduces the risk of obesity, chronic disease and premature death (AIHW 2012; NHMRC 2013). This scoping study reviews the current literature to identify: - key population health issues related to diet and nutrition within the Australian population; and - where gaps in current policy are evident. The scoping study reviews the literature on past and present national and international nutrition policies, strategies (policy actions), interventions and evaluations. The study analyses the evidence from the literature review and formulates recommendations regarding the key elements of effective nutrition policies including scope, guiding principles, format, key inclusions (content), development processes, governance, implementation, timelines, monitoring and surveillance, evaluation and review to guide the development of a new comprehensive National Nutrition Policy for Australia.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

Lipid-Containing Icosahedral dsDNA Bacteriophages: Entry, Exit and Structure

In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.

The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection

Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

Neuromagnetic studies on cortical somatosensory functions in infants and children : Normal development and effect of early brain lesions

Until recently, objective investigation of the functional development of the human brain in vivo was challenged by the lack of noninvasive research methods. Consequently, fairly little is known about cortical processing of sensory information even in healthy infants and children. Furthermore, mechanisms by which early brain insults affect brain development and function are poorly understood. In this thesis, we used magnetoencephalography (MEG) to investigate development of cortical somatosensory functions in healthy infants, very premature infants at risk for neurological disorders, and adolescents with hemiplegic cerebral palsy (CP). In newborns, stimulation of the hand activated both the contralateral primary (SIc) and secondary somatosensory cortices (SIIc). The activation patterns differed from those of adults, however. Some of the earliest SIc responses, constantly present in adults, were completely lacking in newborns and the effect of sleep stage on SIIc responses differed. These discrepancies between newborns and adults reflect the still developmental stage of the newborns’ somatosensory system. Its further maturation was demonstrated by a systematic transformation of the SIc response pattern with age. The main early adult­like components were present by age two. In very preterm infants, at term age, the SIc and SIIc were activated at similar latencies as in healthy fullterm newborns, but the SIc activity was weaker in the preterm group. The SIIc response was absent in four out of the six infants with brain lesions of the underlying hemisphere. Determining the prognostic value of this finding remains a subject for future studies, however. In the CP adolescents with pure subcortical lesions, contrasting their unilateral symptoms, the SIc responses of both hemispheres differed from those of controls: For example the distance between SIc representation areas for digits II and V was shorter bilaterally. In four of the five CP patients with cortico­subcortical brain lesions, no normal early SIc responses were evoked by stimulation of the palsied hand. The varying differences in neuronal functions, underlying the common clinical symptoms, call for investigation of more precisely designed rehabilitation strategies resting on knowledge about individual functional alterations in the sensorimotor networks.

Juvenile neuronal ceroid lipofuscinosis; psychiatric symptoms, GAD-autoantibodies, and response to medication

Juvenile neuronal ceroid lipofuscinosis (JNCL) is one of the most common neurodegenerative diseases in childhood. Its clinical onset, with visual failure as the first sign, is between the ages of 4 to 8 years. During the disease progress, epilepsy, motor symptoms, cognitive decline, and psychiatric symptoms become apparent. It leads to premature death between ages 15 and 30. Treatment consists of symptomatic drug administration and various forms of rehabilitation, but to date, no curative treatment exists. To gain a more comprehensive picture of psychiatric problems, symptoms were evaluated by the Child Behavior Checklist, the Teacher Report Form, and the Children s Depression Inventory. The JNCL patients had a great number of severe psychiatric symptoms, with wide inter-individual variability. The most common symptoms were social, thought, attention, and sleep problems, somatic complaints, and aggressive behaviour. Patients with psychotropic treatment had more problems than did those without psychotropic treatment, and female patients had more problems than did males. Between 10 and 20% of the patients reported depressive symptoms. In a 5-year follow-up, [123I]β-CIT SPECT and MRI revealed a tendency of decreasing serotonin transporter (SERT) availability and progressive brain atrophy. The correlation between changes in midbrain SERT and total brain volume was positive; no correlation appeared between SERT or brain atrophy and depressive symptoms. Thus, it seems likely that the low SERT availability is associated with progressive brain atrophy; it may also predispose towards depression, however. An open survey of psychotropic drugs and their efficacy was performed on JNCL patients in Finland. The most commonly used psychotropic drugs were the antidepressant citalopram and the antipsychotic risperidone. Their efficacy was good or satisfactory in the majority of cases and they seemed well tolerated. Quetiapine had a marked effect on one patient with a history of severe psychotic symptoms. Glutamate decarboxylase 65 autoantibodies (GAD65ab), found in JNCL patients, indicate that an immunomediated reaction against GAD or GABAergic neurons may play a part in the underlying pathogenetic mechanism. GAD65ab s also appeared in the serum of all eight JNCL patients included and intermittent corticosteroid therapy was initiated in all cases. After one year, the GAD65ab s had disappeared in the two oldest patients, who experienced an improvement in motor symptoms and alertness associated with their prednisolone therapy. Two younger patients experienced a significant IQ increase, but no change in GADab s. A randomized study with longer follow-up time is needed, however, to clarify the effect of prednisolone on disease progression.

Juvenile rheumatoid knee : Management of knee growth disturbances in children with juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is associated with growth disturbances, especially leg length discrepancy (LLD) and knee valgus deformity (KVD). Studies have demonstrated growth plate stimulation with chronic arthritis. In the context of surgical treatment of LLD or KVD of a growing knee, the less invasive procedures, which allow immediate mobilisation, are preferred. Establishment of the skeletal age and the correction potential in the knees of rheumatic children is difficult due to rheumatic changes. In this present work, an analysis of the efficacy, safety and long-term results of temporary epiphyseal arrests performed in Rheumatism Foundation Hospital (Heinola, Finland). The distribution of diagnoses among children (n=71) with JIA and LLD (68 knees) was consistent with the normal oligoarthritis-predominated population of children with JIA. A higher male:female ratio (1:1.7 vs. 1:2.4 in population-based studies (PBS)) and earlier mean onset age (4 vs. 7 years in PBSs) were, however, distinct features in the study population. In most cases the correction was reliable and temporary arrest produced a mean correction of 1mm per month. The time of arrest required, however, varied significantly, probably due to the effect of underlying diseases and medication, and the age of the child. All complications encountered (10%) were minor. The correction achieved persisted in long-term follow-up. KVD (n=112, 177 knees) was associated with a high proportion of polyarthritic disease subtype (45% vs. 12-31% in PBSs), and the male:female distribution was grossly female-dominated (1:4.9 vs. 1:2.4 in PBSs). The early mean onset age (3 vs. 7 years in PBSs) was also notable in this cohort. Successful correction was achieved in 2/3 cases and the mean angular correction was 0.7 degrees per month. The required time of arrest, however, varied considerably. In 13% of knees the paucity of follow-up visits resulted in over-correction to varus. The complication rate (3%) in the knees operated for KVD was considerably lower compared to ten per cent in the management of LLD. Most of the complications related to epiphyseal stapling were reversible. However, the risk of premature closure of growth plates does exist. The number of over-corrections was notably high, with 13% knees turning to varus. The correction achieved persisted in long-term follow-up.

IL-10 in Human Newborns : Ontogeny of IL-10 secretion and relation to the secretion of IFN-g, immunoglobulin M, G, and A, and mononuclear cell composition in newborns

The purpose of this work was to elucidate the ontogeny of interleukin-10 (IL-10) secretion from newborn mononuclear cells (MCs), and to examine its relation to the secretion of interferon-g (IFN-g) and immunoglobulins (Igs). The initial hypothesis was that the decreased immunoglobulin (Ig) synthesis of newborn babies was the result of immature cytokine synthesis regulation, which would lead to excessive IL-10 production, leading in turn to suppressed IFN-g secretion. Altogether 57 full-term newborns and 34 adult volunteers were enrolled. Additionally, surface marker compositions of 29 premature babies were included. Enzyme-linked immunoassays were used to determine the amount of secreted IL-10, IFN-g, and Igs, and the surface marker composition of MC were analyzed with a FACScan flow cytometer. The three most important findings were: 1. Cord blood MC, including CD5+ B cells, are able to secrete IL-10. However, when compared with adults, the secretion of IL-10 was decreased. This indicates that reasons other than excessive IL-10 secretion are responsible of reduced IFN-g secretion in newborns. 2. As illustrated by the IL-10 and IFN-g secretion pattern, newborn cytokine profile was skewed towards the Th2 type. However, approximately 25% of newborns had an adult like cytokine profile with both good IL10 and IFN-g secretion, demonstrating that fullterm newborns are not an immunologically homogenous group at the time of birth. 3. There were significant differences in the surface marker composition of MCs between individual neonates. While gestational age correlated with the proportion of some MC types, it is evident that there are many other maternal and fetal factors that influence the maturity and nature of lymphocyte subpopulations in individual neonates. In conclusion, the reduced ability of neonates to secrete Ig and IFN-g is not a consequence of high IL-10 secretion. However, individual newborns differ significantly in their ability to secrete cytokines as well as Igs.

Evaluation of material crack using acoustic emission technique

Cracks in civil structures can result in premature failure due to material degradation and can result in both financial loss and environmental consequences. This thesis reports an effective technique using Acoustic Emission (AE) technique to assess the severity of the crack propagation in steel structures. The outcome of this work confirms that combination of AE parametric analysis and signal processing techniques can be used to evaluate crack propagation under different loading configurations. The technique has potential application to assess and monitor the condition of civil structures.