In vitro systems to study the relationship between apoptosis in multicellular organisms and yeast : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Turitea, Palmerston North

Apoptosis is a distinct form of cell death that is characterised by specific morphological and biochemical markers, such as chromatin condensation and internucleosomal DNA cleavage. This type of cell death is evolutionarily conserved in higher eukaryotes. Homologues of the main apoptosis regulators, such as the Bcl-2 family of proteins and caspases, have been found in multicellular organisms. However, homologues of these proteins have not been found in the unicellular organism Saccharomyces cerevisiae, although in certain circumstances S. cerevisiae will exhibit features of apoptosis. In this project, we developed in vitro systems to explore the relationship between mammalian apoptosis and any similar mechanism that may be present in yeast. Components derived from yeast and mammalian cells were incubated together in vitro and assessed for the activation of apoptosis. Rat cytochrome c activates apoptosis in mammalian cell-free extracts (human neuroblastoma SY5Y cells). Internucleosomal DNA cleavage was observed in S. cerevisiae spheroplasts when they were incubated in mammalian cell-free extracts activated by rat cytochrome c. Although yeast cytochrome c is similar to rat cytochrome c, it failed to induce apoptosis in mammalian cell-free extracts. Yeast cytosol caused internucleosomal DNA cleavage in PCl2 nuclei. This cleavage was enhanced by rat cytochrome c and was mostly inhibited by the caspase inhibitor DEVD-CHO, but only in the presence of rat cytochrome c. Yeast cytosol did not cause chromatin condensation in PCl2 nuclei or cleavage of Parp (a downstream caspase substrate). Yeast cytosol was therefore unable to induce apoptosis in PCl2 nuclei. Mitochondria play a central role in most forms of mammalian apoptosis. We developed a cell-free system in which we could examine the role of mitochondria in apoptosis. We attempted to activate apoptosis in SY5Y cytosol by the addition of mitochondria subjected to rupture-inducing treatment, with limited success. However, we found that mitochondria purified from healthy SY5Y cells protected PCl2 nuclei from undergoing apoptosis in vitro

parkin counteracts symptoms in a Drosophila model of Parkinson's disease

BACKGROUND: Parkinson's disease, a prevalent neurodegenerative disease, is characterized by the reduction of dopaminergic neurons resulting in the loss of motor control, resting tremor, the formation of neuronal inclusions and ultimately premature death. Two inherited forms of PD have been linked to mutations in the α-synuclein and parkin genes. The parkin protein functions as an ubiquitin ligase targeting specific proteins for degradation. Expression of human α-synuclein in Drosophila neurons recapitulates the loss of motor control, the development of neuronal inclusions, degeneration of dopaminergic neurons and the ommatidial array to provide an excellent genetic model of PD. RESULTS: To investigate the role of parkin, we have generated transgenic Drosophila that conditionally express parkin under the control of the yeast UAS enhancer. While expression of parkin has little consequence, co-expression of parkin with α-synuclein in the dopaminergic neurons suppresses the α-synuclein-induced premature loss of climbing ability. In addition directed expression of parkin in the eye counteracts the α-synuclein-induced degeneration of the ommatidial array. These results show that parkin suppresses the PD-like symptoms observed in the α-synuclein-dependent Drosophila model of PD. CONCLUSION: The highly conserved parkin E3 ubiquitin ligase can suppress the damaging effects of human α-synuclein. These results are consistent with a role for parkin in targeting α-synuclein to the proteasome. If this relationship is conserved in humans, this suggests that up-regulation of parkin should suppress α-synucleinopathic PD. The development of therapies that regulate parkin activity may be crucial in the treatment of PD

Hologenome analysis of two marine sponges with different microbiomes

Background: Sponges (Porifera) harbor distinct microbial consortia within their mesohyl interior. We herein analysed the hologenomes of Stylissa carteri and Xestospongia testudinaria, which notably differ in their microbiome content. Results: Our analysis revealed that S. carteri has an expanded repertoire of immunological domains, specifically Scavenger Receptor Cysteine-Rich (SRCR) like domains, compared to X. testudinaria. On the microbial side, metatranscriptome analyses revealed an overrepresentation of potential symbiosis-related domains in X. testudinaria. Conclusions: Our findings provide genomic insights into the molecular mechanisms underlying host-symbiont coevolution and may serve as a roadmap for future hologenome analyses

The analysis of parkin in Drosophila melanogaster

Parkinson's disease (PD) is a highly prevalent neurodegenerative disease characterized by loss of motor control and resting tremor. Mutations in a number of genes, including α-synuclein and parkin, have been associated with inherited forms of PD and many of these genes are linked to the ubiquitin/proteasome degradation system (UPS). Studies of the effects of mutations in these genes suggest that impairment of the UPS is central to PD. Expression of wild-type or PD-associated forms of human α-synuclein in Drosophila melanogaster neurons recapitulates some of the symptoms of PD such as the loss of motor control, development of neuronal inclusions, and degeneration of dopaminergic neurons. Parkin, an E3 ubiquitin protein ligase, may be involved in targeting α-synuclein for degradation. To analyse this interaction I generated transgenic flies expressing parkin under the control of the yeast enhancer upstream activating sequence (UAS). The α-synuclein and parkin transgenes were expressed in combination to examine their interaction in vivo. I showed that expression of parkin prevents the toxic effects of both mutant and wild-type human α-synuclein. Although the yeast protein Gal4 is a key component of the UAS/Gal4 ectopic expression system in Drosophila melanogaster, I showed that this protein can be toxic. Transgenic flies that express high levels of Gal4 in the developing eye show elevated apoptosis in the eye imaginal disc, which leads to a disorganised ommatidial array in the adult. Suppression of apoptosis by expression of the caspase inhibitor p35 prevents this. High levels of Gal4 expression in dopaminergic neurons produce larvae that have excessive apoptosis in the brain and reduced longevity in adult flies. I showed that parkin can suppress apoptosis and development defects in the eye. The ability of parkin to counter the toxicity of exogenous and endogenous proteins may provide great insight into our understanding of toxic protein-induced diseases

Hologenome analysis of two marine sponges with different microbiomes

Background:Sponges (Porifera) harbor distinct microbial consortia within their mesohyl interior. We herein analysed the hologenomes of Stylissa carteri and Xestospongia testudinaria, which notably differ in their microbiome content.Results:Our analysis revealed that S. carteri has an expanded repertoire of immunological domains, specifically Scavenger Receptor Cysteine-Rich (SRCR)-like domains, compared to X. testudinaria. On the microbial side, metatranscriptome analyses revealed an overrepresentation of potential symbiosis-related domains in X. testudinaria.Conclusions:Our findings provide genomic insights into the molecular mechanisms underlying host-symbiont coevolution and may serve as a roadmap for future hologenome analyses.publishe

Employment Relationship in Terms of the Optimization of Wage Costs

The Bachelor’s thesis deals with the issues of labour-law relations, above all their establishment. The aim of the paper is to propose a solution to the problem concerning lack of applicants for the work positions offered, or rather optimization of the number of employees working for an employer in relation to the optimization of labour costs. Based on the results of a conducted analysis and survey, the employer is recommended taking measures which would optimize the number of employees as well as the labour costs

Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Is a Fully Penetrant, Lethal Arrhythmic Disorder Caused by a Missense Mutation in the TMEM43 Gene

Autosomal-dominant arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) causes sudden cardiac death and is characterized by clinical and genetic heterogeneity. Fifteen unrelated ARVC families with a disease-associated haplotype on chromosome 3p (ARVD5) were ascertained from a genetically isolated population. Identification of key recombination events reduced the disease region to a 2.36 Mb interval containing 20 annotated genes. Bidirectional resequencing showed one rare variant in transmembrane protein 43 (TMEM43 1073C→T, S358L), was carried on all recombinant ARVD5 ancestral haplotypes from affected subjects and not found in population controls. The mutation occurs in a highly conserved transmembrane domain of TMEM43 and is predicted to be deleterious. Clinical outcomes in 257 affected and 151 unaffected subjects were compared, and penetrance was determined. We concluded that ARVC at locus ARVD5 is a lethal, fully penetrant, sex-influenced morbid disorder. Median life expectancy was 41 years in affected males compared to 71 years in affected females (relative risk 6.8, 95% CI 1.3–10.9). Heart failure was a late manifestation in survivors. Although little is known about the function of the TMEM43 gene, it contains a response element for PPARγ (an adipogenic transcription factor), which may explain the fibrofatty replacement of the myocardium, a characteristic pathological finding in ARVC