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Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.

Please read the abstract in the section 00front of this document / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

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Der G Protein-gekoppelte Rezeptor 30 (GPR30) wurde vornehmlich im Kontext von schnellen Östrogeneffekten auf zelluläre Signaltransduktionskaskaden untersucht und stellt möglicherweise einen neuen Östrogenrezeptor dar. Die physiologische Funktion von GPR30 in vivo konnte jedoch bisher nicht ermittelt werden. Daher wurde in dieser Arbeit ein Gpr30-defizientes Mausmodell charakterisiert, bei dem ein Teil der kodierenden Sequenz durch einen LacZ-Reporter ersetzt wurde (Gpr30-lacZ). Die Integration des Konstruktes in den Gpr30-Locus wurde mittels Southern blotting und Real-time PCR verifiziert. Gpr30-positive Zelltypen wurden durch Kolokalisation von LacZ mit zelltyp-spezifischen Markerproteinen identifiziert. Weitere Versuche dienten der Aufklärung des Phänotyps von Gpr30-lacZ Mäusen. Zur Identifizierung von Proteinen des GPR30-Signalkomplexes wurden Yeast-Two-Hybrid Analysen mit der N- bzw. C-terminalen Domäne des Rezeptors durchgeführt. Die wesentlichen LacZ-positiven Zellpopulationen waren (i) Endothelzellen in kleinen arteriellen Gefäßen, (ii) glatte Muskelzellen, Perizyten und neuronale Subpopulationen im Gehirn, (iii) Hauptzellen in der Magenschleimhaut, (iv) Zellpopulationen in der Adenohypophyse und dem Hypophysenzwischenlappen sowie (vi) chromaffine Zellen im Nebennierenmark. Während der Phenotypisierung des Mausmodells wurde eine Reduktion der CD62L+ T-Zellen von ca. 50% im peripheren Blut festgestellt. Mittels Yeast Two-Hybrid Analyse wurden Pals1-associated tight junction protein (PATJ) und FUN14 domain-containing 2 (FUNDC2) als mögliche Interaktionspartner identifiziert. Zusammenfassend wurde in dieser Arbeit eine zelluläre Basis für die Funktion von Gpr30 in vivo ermittelt. Der Phänotyp in Gpr30-lacZ Mäusen ist wahrscheinlich durch eine verringerte Produktion von naiven T-Zellen im Thymus bedingt. PATJ bindet die C-terminalen Aminosäuren von GPR30 mit einer PDZ-Domäne und könnte ein Gerüst-protein des GPR30-Signal¬komplexes darstellen. / The orphan G protein coupled receptor 30 (GPR30) was predominantly analyzed in the context of membrane-initiated estrogen signaling suggesting that GPR30 represents a novel estrogen receptor. However, the physiological function of GPR30 in vivo remained unknown. To unravel the physiological role of murine Gpr30 in vivo, a Gpr30-deficient mouse model was analyzed that harbors a LacZ reporter (Gpr30-lacZ) within the Gpr30 locus. The targeting of Gpr30 was verified by Southern blotting and real-time PCR. Gpr30-expressing cell types were identified by colocalization of LacZ along with cell type-specific markers. Further experiments aimed to decipher the phenotype of Gpr30-lacZ mice. To gain information about the signaling complex of human GPR30, yeast two-hybrid screenings were performed with the N- and C-terminal domains as bait. The main LacZ-positive cell populations were (i) endothelial cells in small arterial vessels of various tissues, (ii) smooth muscle cells, pericytes, and neuronal subpopulations in the brain, (iii) gastric chief cells in the stomach, (iv) cells in the intermediate and anterior pituitary, and (v) chromaffin cells in the adrenal glands. Extensive phenotype screening at the German Mouse Clinic revealed reduced numbers of T cells in the peripheral blood of Gpr30-lacZ mice. Especially the proportion of CD62L+ cells was decreased by approx. 50%. Yeast two-hybrid screening led to the identification of Pals1-associated tight junction protein (PATJ) and FUN14 domain-containing 2 (FUNDC2). In conclusion, this study provides a cellular basis for the function of Gpr30 in vivo. Since CD62L+ cells represent the naive T cell compartment, the phenotype of Gpr30-lacZ mice suggests an impaired production of T cells in the thymus. PATJ likely binds the C-terminus of GPR30 with one of its PDZ domains and may represent a scaffolding protein of the GPR30 signaling complex.

In this work, the identification of the promoter for c-cytochromes Cyt c Id-1 in Ideonella dechloratans has been investigated. Additionally, the study examined investigating whether gene expression is affected by the presence and absence of oxygen. This was investigated by amplifying the promoter region of Cyt c Id-1 (389 bp) and then cloning it into a reporter vector lacking a functional promoter for the upstream gene β-galactosidase. The reporter vector was transformed into E. coli RM101 and grown under both aerobic and anaerobic conditions. To examine the gene expression of Cyt c Id-1 the activity of β-galactosidase has been measured in both the aerobic and anaerobic cultures. The result showed that the gene is induced more in an anaerobic environment than in an aerobic environment, by comparing the activity of β-galactosidase under aerobic and anaerobic conditions. / I detta arbete har identifiering av promotorn för c-cytokromer Cyt c Id-1 i Ideonella dechloratans undersökts, samt att undersöka om genuttrycket påverkas av närvaro och frånvaro av syre. Detta är undersökts genom att amplifiera promotorregionen för Cyt c Id-1 (389 bp) för att sedan klona in den in i en reportervektor som saknar en fungerande promotor för uppströms genen β-galaktosidas. Reportervektorn transformerades till E. coli RM101 och odlades under både aeroba och anaeroba förhållanden.  För att undersöka genuttrycket av Cyt c Id-1 har aktiviteten hos β-galaktosidas uppmätts i både de aeroba och anaeroba odlingarna. Resultatet visat att genen induceras mer i anaerob miljö än i aerob miljö, genom att jämföra aktiviteten av β-galaktosidas under både aerob och anaerob förhållande.

Chromosomal replication is a source of spontaneous DNA double-strand breaks (DSBs). In E. coli, DSBs are repaired by homologous recombination using an undamaged sister template. During repair, the RecA protein polymerizes on single-stranded DNA generated at the site of the DSB and catalyses the search for sequence homologies on the undamaged sister template. This study utilized fluorescence microscopy to investigate the spatial and temporal dynamics of the RecA protein at the site of a replication-dependent DSB generated at the lacZ locus of the E. coli chromosome. The DSB was generated by SbcCD-mediated cleavage of a hairpin DNA structure formed on the lagging strand template of the replication fork by a long palindromic sequence. The tandem insertion of a recA-mCherry gene with the endogenous recA gene at the natural chromosomal locus produced no detectable effect on cell viability in the presence of DSB formation. During repair, the fluorescently-labelled RecA protein formed a transient focus, which was inferred to be the RecA nucleoprotein filament at the site of the replication-dependent DSB. The duration of the RecA focus at the site of the DSB was modestly reduced in a ΔdinI mutant and modestly increased in a ΔuvrD or ΔrecX mutant. Most cells underwent a period of extended cohesion of the sister lacZ loci after disappearance of the RecA focus. Segregation of the sister lacZ loci was followed by cell division, with each daughter cell obtaining a copy of the fluorescently-labelled lacZ locus. The RecA focus at the site of the DSB was observed predominantly between the mid-cell and the 1⁄4 position. In the absence of DSB formation, the lacZ locus exhibited dynamic movement between the mid-cell and the 1⁄4 position until the onset of segregation. Formation of the DSB and initiation of repair occurred at the spatial localization for replication of the lacZ locus while the downstream repair events occurred very close to the mid-cell. Genomic analysis of RecA-DNA interactions by ChIP-seq was used to demonstrate that the RecA focus at the lacZ locus was generated by the repair of the palindrome-induced DSB and not the repair of one-ended DSBs emanating from stalled replication forks at the repressor-bound operator arrays. This study has shown that the repair of a replication-dependent DSB occurs exclusively during the period of cohesion of the sister loci and the repair is efficiently completed prior to segregation of the two sister loci.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

grainy head (grh) genes encode a family of transcription factors conserved from fly to human. Drosophila grh is the founding member of this gene family and has multiple functions, including tracheal tube size control, epidermal barrier formation and reconstruction after wounding. To understand the underlying molecular mechanism of grh functions, we tried to isolate its direct targets and analyze their function. We identified ten grh targets by combining bioinformatics and genetics. Grh directly controls the expression of stitcher (stit), which encodes a Ret family receptor tyrosine kinase (RTK), during both development and wound healing. Stit promotes actin cable assembly and induces extracellular signal-regulated kinase (ERK) phosphorylation around the wound edges upon injury. Stit also activates barrier repair genes and its own expression at the wound sites in a Grh-dependent manner. This positive feedback loop ensures efficient epidermal wound repair. In addition, Grh regulates the expression of multiple genes involved in chitin biosynthesis or modification. Most of the genes are required for tracheal tube size control. Two of them, verm and serp, encode related putative luminal chitin deacetylases. The functional analysis of verm and serp identifies an important role of luminal chitin matrix modification in limiting tracheal tube elongation. Therefore, it is very likely that Grh controls tracheal tube size through regulating multiple targets involved in the assembly or modification of luminal chitin matrix. Grh also directly activates the epidermal expression of Peptidoglycan recognition protein LC (PGRP-LC) gene that is required for the induction of antimicrobial peptides (AMPs) upon infection. Furthermore, ectopically expressing Grh is sufficient to induce AMP Cecropin A lacZ reporter (CecA-LacZ) in the embryonic epidermis. These results suggest a new function of Grh in the local immune responses in Drosophila barrier epithelia. / At the time of the doctoral defense, the following papers was unpublished and had a status as follows: Paper 1: Manuscript.

Water quality in the Vaalharts region in the Northern Cape Province, South Africa, decreased over the past few years and there was a need for the microbiological and physico-chemical assessment. This problem was identified through discussions with Vaalharts Water (Vaalharts Water User Association) in 2010 when the issue of the impact of deteriorating water quality on drinking water production was raised. It was thus important to investigate concerns of the water users association pertaining to water quality issues. The aim of this study was to assess the physico-chemical and microbiological quality of household water in the Vaalharts irrigation scheme. The main residential areas were Hartswater, Pampierstad, Jan Kempdorp and Warrenton. Faecal coliforms were detected in the raw water of all the drinking water distribution systems during 2011 and 2012. No faecal coliforms were detected in the household water during 2011. This was a very positive result, because not only did the household water comply with the SANS 241 (2011) standard (0 CFU/100ml), but the purification processes were successful by removing all the E. coli’s from the raw water. However, during March 2012 faecal coliforms were detected in the household water of Jan Kempdorp (191CFU/100ml). This could be due to point pollution and possible breakage of faecal coliforms in the distribution system. Low amounts of total coliforms were detected in the raw water of some of the drinking water distribution systems. This could be due to high amounts of other colonies (pink and purple) growing on the m-Endo agar which suppress the growth of the metallic green sheen (total coliform) colonies. The total coliform numbers complied with the SANS 241 (2011) standard of ≤10 CFU/100ml at most of the distribution systems, except for Hartswater during July 2011 (14CFU/100ml) and Warrenton during March 2012 (256 CFU/100ml). Heterotrophic plate count bacteria were very high in the household water of some of the distribution systems during 2011 and 2012 which exceeded the SANS 241 (2011) standard of ≤1000 CFU/ml. A large number of pigmented (yellow, orange, pink) and non-pigmented (white) colonies were isolated on R2A agar. This can be an indication of some failure in treatment processes. Other microbiological parameters that were tested such as faceal streptococci, Clostridia, Pseudomonas aeruginosa and fungi did not indicate any danger, but there were high levels of total anaerobic bacteria in the raw water during 2011 and 2012. A high level of anaerobic bacteria was detected in the household water of Hartswater during July 2011. Clostridia were also present in the household water of some of the distribution systems during 2011 and 2012. Sequencing results of the mdh, lacZ and uidA genes indicated that one of the isolates was identified as Enterobacter cloacae and the other isolates were E. coli. Four of the isolates were identified as Escherichia coli O104:H4. This is a pathogenic strain and raised concern. The physicochemical parameters that were measured complied with the SANS 241 (2011) standards during 2011 and 2012, but some of the parameters increased gradually from 2011 to 2012. Statistical analysis indicated that physico-chemical parameters had an influence on microbiological parameters and that deteriorating raw water may have an impact on drinking water quality. Another concern currently is that there is no SANS 241 (2011) for faecal streptococci, Clostridia, Pseudomonas aeruginosa, fungi and anaerobic bacteria. These are all opportunistic pathogenic bacteria and consuming water with high levels of these bacteria may cause health problems. This study indicated good progress in the treatment processes of the distribution systems over the two years. This may be due to the feedback given to Vaalharts Water during this study regarding the water quality of the residential areas. The physico-chemical and microbiological results of the present study indicated possible biofilm formation in the distribution systems. This may have impacts on the drinking water quality of the distribution systems. It was also evident that deteriorating raw water sources may have an impact on drinking water production. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.