The development and ultrastructure of intergeneric nuclear transfer embryos using ovine ooplasm.

This thesis encompasses work that aimed to further understand genomic reprogramming, an event crucial in obtaining development in cloned embryos produced by somatic cell nuclear transfer (SCNT). An increasing number of different mammalian species have been cloned using nuclear transfer technology since Dolly the cloned sheep was first successfully produced. However, the biological mechanisms involved in the process of nuclear reprogramming are yet to be fully described. At the centre of this study was an intergeneric SCNT model, which was implemented to determine whether reprogramming factors are conserved across genera. The interaction between donor nucleus and recipient ooplasm was characterised with regard to developmental potential, timing of genome activation, nucleolus formation, and expression of significant proteins. In initial studies, fusion parameters of the intergeneric SCNT procedure were optimised for the ovine cytoplast and porcine donor granulosa cell. Cell fusion and lysis percentages were determined over a range of electrical pulse voltage, duration and repetition. The optimal electrofusion settings were a single DC pulse of 1.5 kV/cm for 20 usec following a 2 sec 400 kHz alignment pulse. In addition, it was demonstrated that ovine oocytes were sensitive to electric stimulation to the extreme that oocyte activation would occur no matter how low the voltage. The practical significance was that it would not be possible to implement a fusion before activation protocol. The ability of the ooplasm of one species to replicate chromosomes and support early embryo cleavage was determined in a preliminary experiment where intergeneric embryos were produced by SCNT using bovine and ovine foetal fibroblasts, and ovine ooplasm. After their construction, the embryos were allowed to develop for 7 days in vitro and the developmental stage determined by Hoechst staining and nuclei counting. In addition, chromosome spreads of the ovine and bovine somatic foetal fibroblast cell lines used in SCNT, as well as the intra- and intergeneric SCNT embryos were prepared to determine whether the ovine ooplasm was replicating the chromosomes according to the karyotype of the donor nucleus. The somatic cells were karyotyped with 54 and 60 chromosomes counted for ovine and bovine cells respectively. Bovine-ovine embryos were characterised as having a bovine karyotype as distinct from an ovine karyotype, due to the presence of only two metacentric chromosomes as compared with six that are found in the latter. These preliminary results indicated that bovine nuclei obtained from foetal fibroblast cells could initiate early pre-implantation embryo development with the support of ovine oocyte cytoplasm. The development of a proportion (33%) of ovine-ovine intrageneric SCNT embryos beyond the 16-cell stage indicated that an extensive characterisation of an intergeneric model could be performed satisfactorily. It was hypothesised that the ovine ooplasm would possess the ability to direct in vitro preimplantation embryo development after nuclear transfer using donor nuclei from a different genus, as has been demonstrated in studies using bovine and rabbit ooplasm. In this study, intergeneric SCNT embryos were constructed by the separate fusion of porcine and bovine cells with ovine cytoplasts (bovine-ovine and porcine-ovine respectively), cultured in vitro and the developmental characteristics compared with ovine-ovine SeNT embryos as well as ovine in vitro produced (IVP) embryos. These four groups of embryos were sampled to determine embryo cell numbers at 24, 36, 48, 72, 96, 120 and 168 h post-activation to compare development over time. Despite cleaving normally and undergoing the first three cleavage divisions at a rate comparable with ovine-ovine SCNT embryos, a major block in development occurred in the intergeneric embryos at the 8-16 cell stage. Consequently, no blastocyst formation was obtained as observed for the IVP and ovine-ovine SCNT controls. These results indicate that unlike the rabbit and bovine ooplasm, the ovine ooplasm is not suitable for intergeneric reprogramming of somatic nuclei from another genus, at least of porcine or bovine origin. To determine the effect of a less differentiated donor nucleus on intergeneric developmental potential, embryonic cell nuclear transfer (ECNT) was conducted in a separate experiment by fusing pluripotent bovine and ovine donor cells (obtained from day-4 preimplantation embryos) to ovine cytoplasts. After 7 days of culture, the cell number of embryos was determined by Hoechst staining and fluorescent observation. Despite observing a single bovine-ovine blastocyst (4.8%), the developmental block remained at the 8-16 cell stage of development. This outcome indicates that a less differentiated nucleus does not increase intergeneric developmental capability. It is well documented that the ooplasm supplies a large amount of mRNA and protein to the newly formed embryo, crucial for normal development leading up to the major activation of the embryonic genome. However, the interaction between the ooplasm as compared with the donor nucleus in SCNT embryos during this developmental period is poorly understood. This intergeneric SCNT model provided an opportunity to determine the role of the ooplasm on nucleolus formation, which is a marker for genome activation. Ultrastructural evidence was obtained that indicates the ovine ooplasm directs the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine SCNT and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-,2-,4-, early 8-, late 8-and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillogranular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcineovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillogranular nucleoli in 16-cell embryos. In parallel, autoradiographic labelling over the nucleoplasm and, in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the 8-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared to comprise of broken down fibrillar material, perhaps formerly of nucleolar origin from the donor cell. These observations indicate that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos. To further characterise nucleolus formation, immunocytochemical localisation by confocal microscopy of nucleolin, fibrillarin and RNA polymerase, three key proteins involved in processing rRNA transcripts, was performed on early 8-, late 8- and 16-cell embryos for ovineovine and porcine-ovine SCNT embryos. Nucleolin was localised throughout the nucleoplasm for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos and, in particular, intensity around the presumptive nucleolar compartments in the later developmental stages. Fibrillarin and RNA polymerase I, on the other hand, were not detected in any ovineovine or porcine-ovine SCNT embryos or ovine IVP controls, although both proteins were detected in control bovine IVP blastocysts. This result indicates that the antifibrillarin and anti-RNA polymerase I were not compatible with the ovine form of these respective proteins. As nucleolin is not present in porcine in vivo embryos before the major activation of the embryonic genome, its presence in porcine-ovine SCNT embryo nucleus indicates that nucleolin is derived from the abundant protein and mRNA stored in the ovine ooplasm. The intergeneric SCNT model established in this thesis demonstrates that the ovine ooplasm lacks the ability to support embryonic development beyond the 16-cell stage. The TEM and autoradiographical studies, in combination with the protein immunocytochemistry study, confirmed that these embryos are unable to undergo the major activation of the embryonic genome, and that the ooplasm influences the initial nucleolar assembly in these embryos.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 200

Successful stent graft repair of multiple thoracoabdominal mycotic aortic aneurysms in the presence of Kommerell diverticulum and complicated by aortoesophageal fistula

We describe the management of a woman who presented with synchronous mycotic aortic aneurysms of the aortic arch in the presence of Kommerell diverticulum, the distal thoracic, and the juxtarenal aorta. A staged stent graft repair was undertaken due to rapid expansion of the aneurysms, which involved placement of multiple thoracic quadruple-fenestrated and infrarenal bifurcated stent grafts. Despite complications of an aortoesophageal fistula and transitory spinal cord ischemia, she has been managed successfully and is doing well at 36 months. This case illustrates that stent graft repair of mycotic aneurysms can offer a successful treatment option in selected patients

Assessment of Marine Gill Disease in Farmed Atlantic Salmon (Salmo salar) in Chile Using a Novel Total Gross Gill Scoring System:A Case Study

Gill disorders have become more prevalent and widespread in finfish aquaculture in recent years. Their aetiology is often considered to be multifactorial. Effective diagnosis, control and prevention are hindered by the lack of standardised methodologies to characterise the aetiological agents, which produce an array of clinical and pathological presentations. The aim of this study was to define a novel gross pathological scoring system suitable for field-based macroscopic assessment of complex or multifactorial gill disease in farmed Atlantic salmon, using samples derived from a gill disease outbreak in Chile. Clinical assessment of gross gill morphology was performed, and gill samples were collected for qPCR and histology. A novel total gill scoring system was developed, which assesses gross pathological changes combining both the presumptive or healed amoebic gill disease (AGD) and the presence of other types of gill lesions. This scoring system offers a standardised approach to characterise the severe proliferative pathologies in affected gills. This total gill scoring system can substantially contribute to the development of robust mitigation strategies and could be used as an indicator trait for incorporating resistance to multifactorial gill disease into breeding goals

The use of pedobarographic analysis to evaluate movement patterns in unstable total knee arthroplasty: a proof of concept study

BackgroundDefinition and clinical diagnosis of instability in TKA is challenging. Sensitive and objective biomechanical tools to aid diagnosis are currently lacking. This proof-of-concept study evaluates the use of pressure mat analyses to identify abnormal biomechanical loading patterns associated with TKA instability within an outpatient clinical setting.MethodsTwenty participants were examined: 10 patients with suspected unilateral TKA instability and 10 healthy controls. Participants underwent bilateral stance and gait tests measuring time and limb loading pressure parameters. Gait was divided into three phases: heel strike, mid-foot and toe off. Pressure recordings are expressed relative to bodyweight. Between-limb loading discrepancies were calculated in TKA patients and controls, and these differences were then compared between groups. Statistical significance was accepted at p < 0.05.ResultsTKA patients consistently offloaded pressure away from the operated limb, whereas healthy controls exhibited more even limb loading throughout bilateral stance ( p < 0.05). TKA patients exhibited greater discrepancy in overall step contact time between limbs (−0.09 s ± 0.16 s; p = 0.016) compared to controls (0.06 s ± 0.08 s; p = 0.04). Post-hoc tests showed significant between-group differences during midfoot (−0.04 s ± 0.07 s; p = 0.03) and toe-off (0.05 s ± 0.14 s; p = 0.013). Between-group differences in limb loading discrepancy were evident at heel strike (−9.24% ± 2.11%; p = 0.0166) and toe-off (−10.34% ± 5.51%; p = 0.0496).DiscussionPedobarographic measurements demonstrated differences in mechanical loading patterns in patients with TKA instability compared to healthy controls during functional tasks and warrants further investigation. This may prove to be a useful clinical diagnostic tool in identifying patients that would benefit from revision surgery or physical therapy

Patterns of Hamstring Muscle Tears in the General Population: A Systematic Review

BackgroundHamstring tears are well recognised in the sporting population. Little is known about these injuries in the general population.PurposeEvaluating the rates, patterns and risk factors of non-sporting hamstring tears, compared to sporting related hamstring tears.Data SourcesMEDLINE, EMBASE, CINAHL, and the Cochrane Central Register of Controlled Trials (1989–2015).Study SelectionStudies reporting patients with a grade 2 or 3 hamstring muscle tear, identified clinically, confirmed by MRI imaging or direct visualisation during surgical exploration.Data Synthesis144 sets of linked data were extracted for analysis. Most injuries were in males (81.3%), where mean age at injury was lower (30.2, 95% CI 29.1–31.3) than in females (35.4, 95% CI 32.4–38.4) p = 0.06. Key differences were found in the proportion of non-sporting injuries in patients under and over the age 40 (p = 0.001). The proportion of non-sporting injuries was significantly higher in females compared to males (25.9% female non-sporting injuries, versus 8.5% male; p = 0.02). Avulsions were more frequently reported in non-sporting activities (70.5%). The proportion of such injuries was notably higher in females, though this failed to meet significance (p = 0.124). Grouped by age category a bimodal distribution was noted, with the proportion of avulsions greater in younger (age 40) (p = 0.008). 86.8% of patients returned to pre-injury activity levels with a similar frequency across all study variables; age, activity (sporting vs non-sporting) and injury type (avulsion vs tear).ConclusionThis review highlights a proportion of adults suffering grade 2 or 3 hamstring injuries from activities other than the classic sports trauma. The majority of these non-sporting injuries were avulsion injuries that clustered in older female and skeletally immature patients suggesting a potential link to bone mineral density

Limb lengthening and peripheral nerve function—factors associated with deterioration of conduction

Background and purpose Limb lengthening is performed for a diverse range of orthopedic problems. A high rate of complications has been reported in these patients, which include motor and sensory loss as a result of nerve damage. We investigated the effect of limb lengthening on peripheral nerve function.Patients and methods 36 patients underwent electrophysiological testing at 3 points: (1) preoperatively, (2) after application of external fixator/corticotomy but before lengthening, and (3) after lengthening. The limb-length discrepancy was due to a congenital etiology (n = 19), a growth disturbance (n = 9), or a traumatic etiology (n = 8).Results 2 of the traumatic etiology patients had significant changes evident on electrophysiological testing preoperatively. They both deteriorated further with lengthening. 7 of the 21 patients studied showed deterioration in nerve function after lengthening, but not postoperatively, indicating that this was due to the lengthening process and not to the surgical procedure. All of these patients had a congenital etiology for their leg-length discrepancy.Interpretation As detailed electrophysiological tests were carried out before surgery, after surgery but before lengthening, and finally after completion of lengthening, it was possible to distinguish between the effects of the operation and the effects of lengthening on nerve function. The results indicate that the etiology, site (femur or tibia), and nerve (common peroneal or tibial) had a bearing on the risk of nerve injury and that these factors had a far greater effect than the total amount of lengthening