Izmir subterranean clover (Trifolium subterraneum L. var. subterraneum)

Izmir is a hardseeded, early flowering, subterranean clover of var. subterraneum (Katz. et Morley) Zohary and Heller collected from Turkey and developed by the collaborating organisations of the National Annual Pasture Legume Improvement Program. It is a more hardseeded replacement for Nungarin and best suited to well-drained, moderately acidic soils in areas with a growing season of less than 4.5 months. Izmir seed production and regeneration densities in 3-year pasture phases were similar to Nungarin in 21 trials across southern Australia, but markedly greater in years following a crop or no seed set. Over all measurements, Izmir produced 10% more winter herbage and 7% more spring herbage than Nungarin. Its greater hardseededness and good seed production, makes it better suited to cropping rotations than Nungarin. Softening of Izmir hard seeds occurs later in the summer–autumn period than Nungarin, giving it slightly greater protection from seed losses following false breaks to the season. Izmir is recommended for sowing in Western Australia, New South Wales, Victoria, South Australia and Queensland. Izmir has been granted Plant Breeders Rights in Australia.

Dry sliding wear behaviour of magnesium alloy based hybrid composites in the longitudinal direction

In the present investigation, the wear behaviour of a creep-resistant AE42 magnesium alloy and its composites reinforced with Saffil short fibres and SiC particles in various combinations is examined in the longitudinal direction i.e., the plane containing random fibre orientation is perpendicular to the steel counter-face. Wear tests are conducted on a pin-on-disc set-up under dry sliding condition having a constant sliding velocity of 0.837 m/s for a constant sliding distance of 2.5 km in the load range of 10-40 N. It is observed that the wear rate increases with increase in load for the alloy and the composites, as expected. Wear rate of the composites is lower than the alloy and the hybrid composites exhibit a lower wear rate than the Saffil short fibres reinforced composite at all the loads. Therefore, the partial replacement of Saffil short fibres by an equal volume fraction of SiC particles not only reduces the cost but also improves the wear resistance of the composite. Microstructural investigation of the surface and subsurface of the worn pin and wear debris is carried out to explain the observed results and to understand the wear mechanisms. It is concluded that the presence of SiC particles in the hybrid composites improves the wear resistance because these particles remain intact and retain their load bearing capacity even at the highest load employed, they promote the formation of iron-rich transfer layer and they also delay the fracture of Saffil short fibres to higher loads. Under the experimental conditions used in the present investigation, the dominant wear mechanism is found to be abrasion for the AE42 alloy and its composites. It is accompanied by severe plastic deformation of surface layers in case of alloy and by the fracture of Saffil short fibres as well as the formation of iron-rich transfer layer in case of composites.

Introduction

[Excerpt] There is perhaps no more visible segment of the American economy than the arts and entertainment sector. When the Writers guild engaged its members in a strike against the Alliance of Motion Picture and Television Producers in 1988, the popular culture of the vast majority of the American public was deeply affected. New television shows were delayed and the networks scrambled to find replacement programming. Virtually everyone was aware of the labor-management conflict, though probably not of its cause, and conscious of its impact on their lives. It could be argued that strikes in any of a half-dozen industries over the course of that year had less impact on the average American life, even though many times the number of workers were effected.

Approaches to selecting native plant replacements for fleshy-fruited invasive species

Invasive plants are a serious threat to biodiversity. Yet, in some cases, they may play an important ecological role in heavily modified landscapes, such as where fleshy-fruited invasive plants support populations of native frugivores. How can such conservation conflicts be managed? We advocate an approach in which native fleshy-fruited plants are ranked on their ability to provide the fruit food resources for native frugivores currently being provided by invasive plants. If these native taxa are preferentially used, where ecologically appropriate, in plantings for restoration and in park and garden settings, they could help support native frugivore populations in the event of extensive invasive plant control. We develop and critically examine six approaches to selecting candidate native plant taxa: a multivariate approach based on the frugivore assemblage, a scoring model, and several multivariate approaches (including trait combinations having the greatest correlation with the diet of the native frugivore assemblage) based on the functional traits of fruit morphology, phenology, conspicuousness, and accessibility. To illustrate these approaches, we use a case study with Bitou bush (Chrysanthemoides monilifera subsp. rotundata) (Asteraceae), an Australian Weed of National Significance. The model using a dissimilarity value generated from all available traits identified a set of species used by the frugivores of C. monilifera more than null models. A replacement approach using species ranked by either all traits available or the frugivore community appears best suited to guide selection of plants in restoration practice.

Selecting replacements for invasive plants to support frugivores in highly modified sites: a case study focusing on Lantana camara

Invasive plants are regarded as a major threat to biodiversity worldwide. Yet, in some cases, invasive plants now perform important ecological functions. For example, fleshy-fruited invasive plants provide food that supports indigenous frugivore populations. How can the disparate goals of conservation versus invasive weed control be managed? We suggest using the fruit characteristics of the invasive plant to select replacement indigenous plants that are functionally similar from the perspective of frugivores. These could provide replacement food resources at sites where plants with these characteristics are part of the goal plant community and where such plants would not otherwise regenerate. Replacement plants could also redirect seed dispersal processes to favour indigenous, rather than invasive, plant species. We investigated the utility of this approach by ranking all indigenous fleshy-fruited plant species from a region using a simple model that scored species based upon measures of fruit phenology, morphology, conspicuousness and accessibility relative to a target invasive species, Lantana (Lantana camara). The model successfully produced high scores for indigenous plant species that were used by more of the frugivores of Lantana than a random selection of plants, suggesting that this approach warrants further investigation.

The development of a component to improve the loading safety of bone-anchored prostheses

Use of socket prostheses Currently, for individuals with limb loss, the conventional method of attaching a prosthetic limb relies on a socket that fits over the residual limb. However, there are a number of issues concerning the use of a socket (e.g., blisters, irritation, and discomfort) that result in dissatisfaction with socket prostheses, and these lead ultimately a significant decrease in quality of life. Bone-anchored prosthesis Alternatively, the concept of attaching artificial limbs directly to the skeletal system has been developed (bone anchored prostheses), as it alleviates many of the issues surrounding the conventional socket interface.Bone anchored prostheses rely on two critical components: the implant, and the percutaneous abutment or adapter, which forms the connection for the external prosthetic system (Figure 1). To date, an implant that screws into the long bone of the residual limb has been the most common intervention. However, more recently, press-fit implants have been introduced and their use is increasing. Several other devices are currently at various stages of development, particularly in Europe and the United States. Benefits of bone-anchored prostheses Several key studies have demonstrated that bone-anchored prostheses have major clinical benefits when compared to socket prostheses (e.g., quality of life, prosthetic use, body image, hip range of motion, sitting comfort, ease of donning and doffing, osseoperception (proprioception), walking ability) and acceptable safety, in terms of implant stability and infection. Additionally, this method of attachment allows amputees to participate in a wide range of daily activities for a substantially longer duration. Overall, the system has demonstrated a significant enhancement to quality of life. Challenges of direct skeletal attachment However, due to the direct skeletal attachment, serious injury and damage can occur through excessive loading events such as during a fall (e.g., component damage, peri-prosthetic fracture, hip dislocation, and femoral head fracture). These incidents are costly (e.g., replacement of components) and could require further surgical interventions. Currently, these risks are limiting the acceptance of bone-anchored technology and the substantial improvement to quality of life that this treatment offers. An in-depth investigation into these risks highlighted a clear need to re-design and improve the componentry in the system (Figure 2), to improve the overall safety during excessive loading events. Aim and purposes The ultimate aim of this doctoral research is to improve the loading safety of bone-anchored prostheses, to reduce the incidence of injury and damage through the design of load restricting components, enabling individuals fitted with the system to partake in everyday activities, with increased security and self-assurance. The safety component will be designed to release or ‘fail’ external to the limb, in a way that protects the internal bone-implant interface, thus removing the need for restorative surgery and potential damage to the bone. This requires detailed knowledge of the loads typically experienced by the limb and an understanding of potential overload situations that might occur. Hence, a comprehensive review of the loading literature surrounding bone anchored prostheses will be conducted as part of this project, with the potential for additional experimental studies of the loads during normal activities to fill in gaps in the literature. This information will be pivotal in determining the specifications for the properties of the safety component, and the bone-implant system. The project will follow the Stanford Biodesign process for the development of the safety component.

Pozzolanic properties of rice husk ash, burnt clay and red mud

In this paper materials like rice husk ash, burnt clay and red mud are examined for their pozzolanic properties. Rice husk ash, obtained from various sources, is analysed by X-ray diffraction. Compressive strength properties of lime-pozzolana mortars with rice husk ash, burnt clay and red mud as pozzolana are studied. Influence of grinding of rice husk ash and intergrinding with lime are also investigated. Combination pozzolana with partial replacement of burnt clay and red mud by rice husk ash are examined for their pozzolanic properties. Long term strength behaviour of lime-pozzolana mortars is investigated to understand the durability of lime-pozzolana cements.

Kinetics Of The Aminolysis Reactions Of Chlorocyclotriphosphazenes-Changeover From A Sn2(P) To A Sn1(P) Mechanism

A change-over from SN2(P) to SN1(P) mechanism is established for the chlorine replacement reactions of halogenocyclophosphazenes; this mechanistic change-over helps in rationalising the diverse findings reported for this class of reactions.

ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

Restorative treatment practices and dentist-related factors

This study aimed at elucidating real-life aspects of restorative treatment practices. In addition, dentists' views and perceptions of and variation in restorative treatment practices with respect to dentist-related factors were evaluated. Reasons for placement and replacement of restoration, material selection, posterior restoration longevity, and the use of local anesthesia were assessed on two cross-sectional data sets. Data from the Helsinki Public Dental Service (PDS) included details on 3057 restorations performed by dentists (n=134) during routine clinical work in 2001. The other PDS data from Vantaa were based on 205 patient records of young adults containing information on 1969 restorations investigated retrospectively from 1994-1996 backwards; 51 dentists performed the restorations. In addition, dentists’ self-reported use of local anesthesia and estimates of restoration longevity were investigated by means of a nationwide questionnaire sent to 592 general dental practitioners selected by systematic sampling from the membership list of the Finnish Dental Association in 2004. All data sets included some background information on dentists such as gender, year of birth or graduation, and working sector. In PDS in 2001, primary caries was the reason for placement of restoration more often among patients aged under 19 years than among older patients (p<0.001). Among patients over 36 years of age, replacements represented the majority. Regarding dentist-related factors, replacements of restorations were made by younger dentists more frequently than by older dentists (p<0.001). In PDS in 1994-1996, the replacement rate of posterior restorations was greater among female dentists than among male dentists (p=0.01), especially for amalgams (p=0.008). The mean age of replaced posterior restoration among young adults was 8.9 (SD 5.2) years for amalgam and 2.4 (SD 1.4) years for tooth-colored restorations, the actual replacement rate for all existing posterior restorations being 7% in PDS in 1994-1996. Of all restorative materials used, a clear majority (69%) were composites in PDS in 2001. Local anesthesia was used in 48% of cases and more frequently for older patients (55%) than for patients aged under 13 years (35%) (p<0.001). Younger dentists more often used local anesthesia for primary restoration than did the older dentists (p<0.001), especially for primary teeth (p=0.005). Working sector had an impact on dentists’ self-reported use of local anesthesia and estimates of restoration longevity; public sector dentists reported using local anesthesia more frequently than private sector dentists for Class II (p=0.04) and for Class III restorations (p=0.01). Private sector dentists gave longer estimates of posterior composite longevity than public sector dentists (p=0.001). In conclusion, restorative treatment practices seem to vary according to patient age and also dentist-related factors. Replacements of restorations are common for adults. For children, clear underuse of local anesthesia prevails.

Utilisation of local ingredients in commercial pig feeds

The long-term competitiveness of the both the Vietnamese feed and pig production industries are constrained and under pressure whilst the industry is dependent on the use of imported feed ingredients in diets for animal production. These cost pressures are a result of import taxes, transport costs, currency fluctuations and feed supply limitations. By undertaking studies on available resources which are currently under-utilised and with potential as local feeds, we can prove their suitability for use as feedstuffs in pig diets and as replacements for imported feed ingredients. In undertaking this process we can lower feeding costs for pig production in Vietnam by the use of local feeds which are cheaper, generate new industries in Vietnam harvesting or processing these feeds and increase the incomes of Vietnamese workers who are involved in producing these by-products. Our project has shown that rubber seed, when processed correctly to lower the hydrogen cyanide content, is a safe and suitable protein meal feedstuff for use in pig diets with the potential to replace significant quantities of imported soybean and fishmeal in Vietnamese pig diets as long as diets are balanced for any amino acid shortfalls. Our peanut studies have shown that use of binders can help alleviate pig production problems with aflatoxin content in peanut meals. Further work is needed to characterise the fate of the bound aflatoxin to see if there is any meat residue risk. Cassava residue is a resultant by-product from starch extraction in both large and small cassava processing factories. Sub-samples from these two mill types were collected and evaluated for residue HCN. Analyses has shown that the processing and sun drying results in a product with relatively consistent low HCN content. Chemical analyses also reveal that significant residual starch also remains in this by-product. Digestibility studies and pig performance feeding studies have shown that cassava residue can be included in diets at 30% with no adverse effect, although the higher fibre content of this product means that strategically, cassava residue is more suitably used in finisher and sow diets. Research has examined the digestible energy content of a number of sunflower meal types available in Australia and identified major differences in their energy value based on processing, additionally, amino acid analysis has shown a significantly lower lysine content than previous reported. We also examined the digestible energy content of a number of Australian stylo forage legume harvest batches and identified the differences in their energy value based on age/harvest time of the forage legume. Analysis results of various stylo cuts showed that the early cut stylo has a higher starch content and lower fibre fraction content than observed in late and recut stylo which were allowed to grow longer. As a result the faecal digestible energy content was higher for the early cut stylo than for the subsequent cut stylo material which had been allowed to become woody. The results have shown that feeding of stylo meal does provide some nutritive value to the pig with increased energy and nitrogen supply, with a portion of the nitrogen presented which the pig is able to retain. Based on nutrient and fibre content stylo could have a useful role in sow feeding and satiety under non-stall housing situations. With increasing Vietnamese investment in rubber production seen with larger areas under plantations the amounts of rubber seed available for animal feeding will grow significantly over the next 15 years and the importance of the by-product ie rubber seed meal as a protein source in diets for Vietnamese pigs.

Routine Measurement of Health-Related Quality of Life in Assessing Cost-Effectiveness in Secondary Health Care

Aims: The aims of this study were 1) to identify and describe health economic studies that have used quality-adjusted life years (QALYs) based on actual measurements of patients' health-related quality of life (HRQoL); 2) to test the feasibility of routine collection of health-related quality of life (HRQoL) data as an indicator of effectiveness of secondary health care; and 3) to establish and compare the cost-utility of three large-volume surgical procedures in a real-world setting in the Helsinki University Central Hospital, a large referral hospital providing secondary and tertiary health-care services for a population of approximately 1.4 million. Patients and methods: So as to identify studies that have used QALYs as an outcome measure, a systematic search of the literature was performed using the Medline, Embase, CINAHL, SCI and Cochrane Library electronic databases. Initial screening of the identified articles involved two reviewers independently reading the abstracts; the full-text articles were also evaluated independently by two reviewers, with a third reviewer used in cases where the two reviewers could not agree a consensus on which articles should be included. The feasibility of routinely evaluating the cost-effectiveness of secondary health care was tested by setting up a system for collecting HRQoL data on approximately 4 900 patients' HRQoL before and after operative treatments performed in the hospital. The HRQoL data used as an indicator of treatment effectiveness was combined with diagnostic and financial indicators routinely collected in the hospital. To compare the cost-effectiveness of three surgical interventions, 712 patients admitted for routine operative treatment completed the 15D HRQoL questionnaire before and also 3-12 months after the operation. QALYs were calculated using the obtained utility data and expected remaining life years of the patients. Direct hospital costs were obtained from the clinical patient administration database of the hospital and a cost-utility analysis was performed from the perspective of the provider of secondary health care services. Main results: The systematic review (Study I) showed that although QALYs gained are considered an important measure of the effectiveness of health care, the number of studies in which QALYs are based on actual measurements of patients' HRQoL is still fairly limited. Of the reviewed full-text articles, only 70 reported QALYs based on actual before after measurements using a valid HRQoL instrument. Collection of simple cost-effectiveness data in secondary health care is feasible and could easily be expanded and performed on a routine basis (Study II). It allows meaningful comparisons between various treatments and provides a means for allocating limited health care resources. The cost per QALY gained was 2 770 for cervical operations and 1 740 for lumbar operations. In cases where surgery was delayed the cost per QALY was doubled (Study III). The cost per QALY ranges between subgroups in cataract surgery (Study IV). The cost per QALY gained was 5 130 for patients having both eyes operated on and 8 210 for patients with only one eye operated on during the 6-month follow-up. In patients whose first eye had been operated on previous to the study period, the mean HRQoL deteriorated after surgery, thus precluding the establishment of the cost per QALY. In arthroplasty patients (Study V) the mean cost per QALY gained in a one-year period was 6 710 for primary hip replacement, 52 270 for revision hip replacement, and 14 000 for primary knee replacement. Conclusions: Although the importance of cost-utility analyses has during recent years been stressed, there are only a limited number of studies in which the evaluation is based on patients own assessment of the treatment effectiveness. Most of the cost-effectiveness and cost-utility analyses are based on modeling that employs expert opinion regarding the outcome of treatment, not on patient-derived assessments. Routine collection of effectiveness information from patients entering treatment in secondary health care turned out to be easy enough and did not, for instance, require additional personnel on the wards in which the study was executed. The mean patient response rate was more than 70 %, suggesting that patients were happy to participate and appreciated the fact that the hospital showed an interest in their well-being even after the actual treatment episode had ended. Spinal surgery leads to a statistically significant and clinically important improvement in HRQoL. The cost per QALY gained was reasonable, at less than half of that observed for instance for hip replacement surgery. However, prolonged waiting for an operation approximately doubled the cost per QALY gained from the surgical intervention. The mean utility gain following routine cataract surgery in a real world setting was relatively small and confined mostly to patients who had had both eyes operated on. The cost of cataract surgery per QALY gained was higher than previously reported and was associated with considerable degree of uncertainty. Hip and knee replacement both improve HRQoL. The cost per QALY gained from knee replacement is two-fold compared to hip replacement. Cost-utility results from the three studied specialties showed that there is great variation in the cost-utility of surgical interventions performed in a real-world setting even when only common, widely accepted interventions are considered. However, the cost per QALY of all the studied interventions, except for revision hip arthroplasty, was well below 50 000, this figure being sometimes cited in the literature as a threshold level for the cost-effectiveness of an intervention. Based on the present study it may be concluded that routine evaluation of the cost-utility of secondary health care is feasible and produces information essential for a rational and balanced allocation of scarce health care resources.

Alternative fruit fly management for market access for apples

Difficulty with control of Queensland fruit fly with four cultivars of apples on the Granite Belt, Qld. Warnings that the insecticides dimethoate & fenthion might be removed from the market for apples, had been current for several years. Dimethoate was widely used as a post harvest control measure as well as an in-field treatment. Fenthion also had and still has in-field usage. The project attempted to find a replacement for these control measures.

Inpatient hospital care and its costs among type 1 diabetic patients in Finland : a nationwide longitudinal study

Background. In Finland, the incidence of type 1 diabetes mellitus (T1DM) is the highest in the world, and it continues to increase steadily. No effective preventative interventions exist either for individuals at high risk or for the population as a whole. In addition to problems with daily lifelong insulin replacement therapy, T1DM patients with long-lasting disease suffer from various diabetes related complications. The complications can lead to severe impairments and reductions in functional capacity and quality of life and in the worst case they can be fatal. Longitudinal studies on the costs of T1DM are extremely rare, especially in Finland. Typically, in these studies, distinctions between the various types of diabetes have not been made, and costs have not been calculated separately for the sexes. Aims. The aim of this study was to describe inpatient hospital care and costs of inpatient care in a cohort of 5,166 T1DM patients by sex during 1973-1998 in Finland. Inpatient care and costs of care due to T1DM without complications, due to T1DM with complications and due to other causes were calculated separately. Material and Methods. The study population consisted of all Finnish T1DM patients diagnosed before the age of 18 years between January 1st in 1965 and December 31st in 1979 and derived from the Finnish population based T1DM register (N=5,120 in 1979 and N=4,701 in 1997). Data on hospitalisations were obtained from the Finnish Hospital Discharge Register. Results. In the early stages of T1DM, the majority of the use of inpatient care was due to the treatment of T1DM without complications. There were enormous increases in the use of inpatient care for certain complications when T1DM lasted longer (from 9.5 years to 16.5 years). For women, the yearly number of bed-days for renal complications increased 4.8-fold, for peripheral vascular disease 4.3-fold and for ophthalmic complications 2.5-fold. For men, the corresponding increases were as follows: 5-fold, 6.9-fold and 2.5-fold. The yearly bed-days for glaucoma increased 8-fold, nephropathy 7-fold and microangiopathy 6-fold in the total population. During these 7 years, the yearly numbers of bed-days for T1DM without complications dropped dramatically. The length of stay in inpatient care decreased notably, but hospital visits became more frequent when the length of duration of T1DM increased from 9.5 years to 16.5 years. The costs of treatments due to complications increased when T1DM lasted longer. Costs due to inpatient care of complications in the cohort 2.5-folded as duration of T1DM increased from 9.5 years to 16.5 years, while the total costs of inpatient care in the cohort dropped by 22% due to an 80% decrease in the costs of care of T1DM without complications. Treating complications of female patients was more expensive than treating complications of men when T1DM had lasted 9.5 years; the mean annual costs for inpatient care of a female diabetic (any cause) were 1,642 , and the yearly costs of care of complications were 237 . The corresponding yearly mean costs for a male patient were 1,198 and 167 . Treating complications of female patients was more expensive than that of male patients also when the duration of diabetes was 16.5 years, although the difference in average annual costs between sexes was somewhat smaller. Conclusions. In the early phases of T1DM, the treatment of T1DM without complications causes a considerable amount of hospital bed-days. The use of inpatient care due to complications of T1DM strongly increases with ageing of patients. The economic burden of inpatient care of T1DM is substantial.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.