Molecular Study of Intracellular Changes as Response of Microorganisms to Environment

Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.In nature yeast cells live in a rapidly changing environment. In order to survive constant fluctuations in their external surroundings cells quickly and effectively adapt their internal systems to meet the challenges of each new environment. One aspect of this cellular adaptation is the reorganization of genomic expression to the program required for growth in each environment. This genomic reorganization results in altered cell’s metabolome and physiology. The molecular responses elicited by the cells dictate whether the organism adapts, survives or, if injured beyond repair, undergoes death. In this work, genomic, proteomic and metabolic profiles of some carotenogenic yeasts grown in optimal and stress conditions were compared. To increase the yield of carotenoid pigments, several types of environmental changes as well as nutrition stress were applied. Yeasts were cultivated under osmotic, oxidative and metal stress, on different carbon sources or on various waste materials (whey, potato extract). Some chemical and UV mutants were also prepared and tested. Changes on yeast metabolome level were studied by RP-HPLC/MS technique used to carotenoid analysis. Another possibility to study stress response is preparation of transformed cells with deletion of definite genes. For setup, the method was first optimized for fission yeasts Schizosaccharomyces pombe. Technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. As transformation vector plasmid pCloneKanmx confering resistance to geneticine was used. The vector and constructs were digested by relevant restriction endonucleases, ligated, amplified in E.coli and transformed into the yeast Schizosaccharomyces pombe sp286. Positive transformants were selected according to resistance to geneticine and checking PCR products. By analysis of transformants proteins required for meiotic chromosome segregation were identified.

Studies of genetic mosaicism in rare diseases

Mosaicism in human genetics refers to an individual harboring two or more genetic compositions, all derived from the same fertilized egg. Common signs of genetic mosaicism are asymmetric growth, skin aberrations or vascular malformations. Each clinical picture is in itself rare, but together mosaic disorders form a growing group of identifiable characteristic abnormalities. Interestingly, several pharmacological treatment possibilities for these conditions have evolved in the last couple of years. In study I, we found mosaic hotspot PIK3CA variants in two patients with ectopic muscles and muscular overgrowth, by performing whole genome sequencing and digital PCR. This adds information about timing of PIK3CA mutagenesis during embryogenesis in correlation to phenotype and confirms the diagnostic entity PIK3CA-related muscular overgrowth with ectopic muscles. In study II, we describe a genetic mechanism in DICER1-related overgrowth. We show that a constitutional DICER1 variant encoding the RNase IIIa domain causes a severe subtype of DICER1 syndrome with intellectual disability, macrocephaly, extensive bilateral lung cysts, early onset of Wilms tumor, and well-differentiated fetal lung adenocarcinoma. This phenotype is similar to, but distinct from, the phenotype reported in two patients with GLOW syndrome caused by mosaic hotspot variants encoding the RNase IIIb domain. In study III, we add knowledge of genotype-phenotype correlations in male focal dermal hypoplasia patients by describing a previously unknown disease-causing variant in a male patient, and by highlighting that focal dermal hypoplasia can be suspected in patients with characteristic limb malformations, such as ectrodactyly, or ocular manifestations, even in the absence of typical skin findings. In study IV, we used droplet digital PCR to analyze blood- and sperm-derived DNA from 87 parents to children with intellectual disability syndromes caused by de novo variants. We found germline mosaicism in two fathers and showed that analysis of blood alone may underestimate germline mosaicism. Taken together, these studies have improved our understanding of methodological approaches in mosaicism diagnostics. In addition, these studies contribute to our understanding of the phenotypic and/or genetic spectrum of PIK3CA-related overgrowth, DICER1-related overgrowth, focal dermal hypoplasia and germline mosaicism in rare diseases

Influence of fetal tissue transplant on the morphology of the neuromuscular junctions of tibialis anterior and medial gastrocnemius following spinal transection in the rat

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Perinatal Outcomes in fetuses with early-onset fetal growth restriction at Chris Hani Baragwanath Academic Hospital (CHBAH) and accuracy of Doppler findings in predicting outcomes

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of the Master of Medicine in Obstetrics and Gynaecology. MMed (O&G) Johannesburg, December 2016.Background: Intrauterine growth restriction (IUGR) is a pregnancy-related complication that is diagnosed more frequently due to improved antenatal surveillance with greater access to ultrasound equipment and training in ultrasound and it affects 3-5% of all pregnancies. The addition of Doppler surveillance in high risk pregnancies has been found to decrease morbidity and mortality by 29%. Methods: This was a prospective cross-sectional descriptive study that took place at Chris Hani Baragwaneth Academic Hospital (CHBAH), a tertiary hospital in Soweto, Johannesburg, which conducts an average of 23,000 deliveries a year. Study participants were collected over a 15 month period and early-onset IUGR was defined as an abdominal circumference of less than the 5th percentile for a given gestation and diagnosed between 26+0 and 32+0 weeks of gestation. Multivessel Doppler surveillance was performed to monitor these fetuses and optimally time delivery. Pregnancy and neonatal outcomes were followed up. Results: A total of 60 women were included in the study. Detailed data on outcomes was available for 47 study participants. Nineteen babies (40.6%) were discharged home after an average hospital stay of 62.6 days and 52.6% of these babies fulfilled the criteria for major morbidity. Twenty eight babies (59.4%) were mortalities, with 9 intrauterine fetal deaths, 2 medical terminations of pregnancy for maternal indications, 3 fresh still births, 9 early neonatal deaths, 3 late neonatal deaths and 2 infant deaths. The average birth weight of babies that survived was 969g and the gestation was 29 weeks and 2 days, while in the group that demised, the average birth weight was 775.8g and the mean gestation was 28weeks and 4 days. All study participants had abnormalities on Doppler studies, with 38 of the 48 study participants having abnormalities in more than one vessel. Abnormalities in the Ductus Venosus, as well as a low birth weight, were shown to be independent risk factors for perinatal mortality. Conclusion Babies born with IUGR at CHBAH tend to have severe, early onset IUGR at the time of diagnosis, with many compounding factors affecting their outcomes, the most important of which is their birth weight and the presence of an abnormal Ductus Venosus Doppler.LG201

Identification and Validation of Regulatory Genetic Variation in Yeasts

Genomic variation impacts on the molecular network that performs all cellular functions. As these functions are largely carried out by proteins, it is crucial to understand how genomic variation contributes to differences in protein abundances and activities. We investigated the effects of genetic variation on the molecular network in two independent studies in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae through the mapping of quantitative trait loci (QTL). To be able to observe the molecular network in more states, we exposed fission yeast samples to oxidative stress. The effects of most QTL differed between these two experimental conditions. We identified QTL-hotspots that affected the expression of large numbers of stress response genes. For one of these hotspots we identified and validated pka1 as the causal gene: a missense mutation in pka1 caused a reduction in RAS signaling, which mediates part of the stress response in fission yeast. In the second study, we integrated quantitative phosphorylation traits for hundreds of proteins with matched transcriptomic and proteomic data. We found the phosphoproteome to be controlled by genetic variation and identified numerous associations between genetic variants and phosphorylation traits. Hotspots for other molecular layers and local variation both impacted on the phosphorylation of proteins. The additional information of phsophorylation was further demonstrated by the analysis of affected signaling pathways. The comprehensive proteomic data in both projects allowed us to investigate the effects of changes in transcript levels on protein levels. We found the relationship between transcript and protein levels to be complex and variable across functional groups of genes. This work contributes to the understanding of molecular networks by identifying the effects of genetic variation on the stress response, proteome, and phosphoproteome in the yeast models, studied here. We demonstrate how perturbations of this network can be tracked through multiple molecular layers

Deeper insights into SRB-driven biocorrosion mechanisms

Dissertação para obtenção do Grau de Doutor em Química SustentávelMicrobially Influenced Corrosion (MIC) is recognized as an important category of corrosion and represents one of the most challenging to study and prevent. In biocorrosion, Sulphate Reducing Bacteria (SRB) are the best suited organisms because its ability to produce biogenic hydrogen sulphide. Still little is known about the mechanisms related to corrosion involving those microorganisms. In that regard, this thesis describes the analysis of the interactions, and its consequences, between Desulfovibrio desulfuricans ATCC 27774 and metal surfaces using electrochemistry, of surface analysis and biochemical techniques. The Open Circuit Potential (OCP) showed that the equilibrium of sulphide in the solution/gas phases is a key factor on determining the evolution of the corrosion process. Also it have been demonstrated that, although the trend in the behaviours remains, long measurements periods are necessary to avoid overestimations when dealing with biocorrosion. Our results also indicate that the nitrate injection strategy in oil fields has to be considered carefully as it can increase the uniform corrosion and the SRB population growth rate. High-throughput analysis techniques were used to characterize the surface of metal plates after incubation with SRB with different respiratory substrate. The precipitation of inorganic salts as calcium and phosphates, has been detected in the samples incubated in sulphate and could be related to iron dissolution areas in the metal surface. Also micropitting underneath the bacteria cells could be observed when the bacteria were incubated. In nitrate medium, a high concentration of precipitated chloride ions was observed on the surface which could be responsible for the enhanced corrosions rates observed. The Extracellular Polymeric Substance chemical composition corroborated the previous results regarding the iron uptake and the influence of phosphates and other functional groups in the corrosion process. The protein profile characterization has demonstrated heterogeneity of the expressed proteins depending on the respiratory substrate.Project BIOCOR ITN, "People" Programme, under grant agreement n° 23857

Chronic thromboembolic pulmonary vascular disease: physiological concepts and genetic predisposition

Chronic thromboembolic pulmonary hypertension (CTEPH) is an uncommon sequela of acute pulmonary embolism and, untreated, leads to right ventricular (RV) failure and death. Despite its growing recognition, methods for the detection of early RV insufficiency and prediction of clinical deterioration, important to optimum preservation of RV function, are currently suboptimal. Furthermore, underlying genetic predisposition to CTEPH, unexplained by defective fibrinolysis, remains largely unexplored. The RV’s physiological response to chronic thromboembolic obstruction is arguably best described by RV pressure volume loops which, historically, are best obtained using the conductance catheter. Although invasive, conductance has an indisputable advantage over current imaging modalities; catheters measure dynamic ventricular pressure and volume throughout the cardiac cycle. Using this technique, abnormal RV pressure volume loops are demonstrated in response to chronic thrombotic obstruction, independent of resting haemodynamic criteria diagnostic of CTEPH. Pressure volume differences and accrual of an exercise gas exchange deficit further suggest early ‘subclinical’ RV adaptation. The genetic architecture of CTEPH is also explored using high-throughput sequencing of unrelated patients. This shows that rare DNA variants in CTEPH that are predicted to harbour deleterious effects are not over-represented in fibrinolytic pathways. Finally, prognostication in CTEPH is evaluated using a clinical deterioration model which is shown to be predicted by patient-reported outcomes at diagnosis. In conclusion, RV and pulmonary circulatory function in chronic thromboembolic pulmonary vascular disease are inadequately characterised by existing routine methods. Links between the observed physiological deficits, risk of clinical deterioration and abnormal genetic architecture warrant further evaluation in this rare disease.Open Acces

Determinants of Archetype BK Polyomavirus Replication.

BK polyomavirus (BKPyV) is a widespread, small double-stranded DNA virus that is an emerging pathogen in immunocompromised individuals. Following an initial asymptomatic infection, it establishes persistence in the urinary tract of its human host. BKPyV can reactivate and cause diseases such as polyomavirus-associated nephropathy and hemorrhagic cystitis under conditions of immune suppression in renal and bone marrow transplant recipients, respectively. The focus of this dissertation is to better understand viral mechanisms of regulation that control replication, specifically how the virus is able to establish and maintain a state of persistence following initial infection. Therefore we set out to characterize the transmissible form of BKPyV, archetype virus, which is thought to establish persistence in the host. This virus is defined as having a linear O-P-Q-R-S arrangement of sequence blocks in the non-coding control region (NCCR), of the genomic DNA, as opposed to rearranged variants which have deletions and duplications in their blocks. Rearranged variants can arise during reactivation and are found almost exclusively in patients with BKPyV-associated diseases. Our laboratory developed a natural cell culture model of rearranged variant replication in primary renal proximal tubule epithelial (RPTE) cells. However, archetype virus does not produce progeny in RPTE cells. We determined that of the three major genetic regions that make up the BKPyV genome, the NCCR is responsible for the replication phenotype that differentiates archetype and rearranged virus in RPTE cells. However, we can artificially induce archetype virus propagation in 293TT cells, which overexpress large T antigen, a protein that is required for DNA replication. Furthermore, archetype virus uniquely displays differential promoter activity, driving high levels of a regulatory miRNA early, prior to DNA replication. This miRNA targets early mRNA, thereby limiting virus replication in RPTE cells. This means of repressing viral replication likely contributes to the mechanism of archetype virus persistence in the host. Since there is currently no model of persistence for any human polyomavirus, we have begun to directly examine archetype BKPyV persistence in a cell culture model of urothelial cells. Overall, our results advance our current understanding of archetype polyomavirus replication and persistence.PhDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97966/1/broekenm_1.pd