Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ hybridisation protocols for sugarcane culm tissue, and secondly to use the developed techniques to examine the expression patterns of three invertase isoforms in sugarcane internodes of various developmental stages. Sugarcane invertases have been the focus of intense research for many years, yet almost nothing is known of their tissue-specific distribution. It was thought that by characterising their expression patterns using in situ hybridisation, more knowledge of their functions and involvement in sucrose accumulation would be gained. Although in situ hybridisation is now regularly used to study gene expression in plants, there is to date only a single publication describing its use on immature sugarcane tissue. Therefore this technique needed further development, and this was achieved by comparing different tissue preparation methods, as well as by systematically testing the various parameters pertaining to each method. The in situ hybridization technique was also developed by testing and comparing a number of key parameters. It was found that fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High hybridization temperatures and probe concentrations provided the best signal, and including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to the probe. A less viscous detection buffer reduced both osmotic effects and time required for signal detection. In the second part of this study, the developed method was used to examine the expression patterns of the three invertase isoforms in young, maturing and mature internodes of sugarcane, and the results were complemented with Northern blot analysis. Transcript of all three isoforms was found to be present in the storage parenchyma and in the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with soluble acid invertase declining sharply and dropping below detection in maturing and mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall invertase would appear to play important roles in phloem unloading and turgor control. Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of hexose pools, however the function of this enzyme remains unclear. This study has demonstrated the value of in situ hybridization, yet at the same time has shown its limitations, especially when more traditional biochemical techniques are not employed to complement the results. Although the precise functions of the invertase isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific promoter design and future in situ studies of sugarcane invertases / AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie- protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek. Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer. Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word, en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en sistematies die verskillende parameters wat op elke metode van toepassing is te toets. Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur die viskositeit van die buffer te verminder. In die tweede deel van die studie is die ontwikkelde metode gebruik om die uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel. Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie duidelik nie. Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van suikerrietinvertases.

No description available.

The powertrain design and control strategies for three representative hybrid and plug-in hybrid electric vehicles (HEV/PHEVs), a plug-in hybrid passenger car, a plug-in hybrid race car, and a hybrid electric mining truck, have been investigated through the system modeling, simulation and design optimization. First, the pre-transmission gen-set couple Plug-in Series-Parallel Multi-Regime (SPMR) powertrain architecture was selected for PHEV passenger car. Rule-based load following control schemes based on engine optimal control strategy and Equivalent Consumption Minimization Strategy (ECMS) were used for the operation control of the passenger car PHEV powertrain. Secondly, the rear wheel drive (RWD) post-transmission parallel through road powertrain architecture was selected for race car PHEV. A high level supervisory control system and ECMS control strategy have been developed and implemented through the race car’s on-board embedded controller using dSPACE MicroAutobox II. In addition, longitudinal adaptive traction control has been added to the vehicle controller for improved drivability and acceleration performance. At last, the feasibility and benefits of powertrain hybridization for heavy-duty mining truck have been investigated, and three hybrid powertrain architectures, series, parallel and diesel-electric, with weight adjusting propulsion system have been modeled and studied. The research explored the common and distinct characteristics of hybrid electric propulsion system technology for different vehicular applications, and formed the foundation for further research and development. / Graduate / 0540 / ruicheng@uvic.ca

BACKGROUND:Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.METHODS:The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.RESULTS:39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted / while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.CONCLUSIONS:Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Invasive alien plants have had diverse ecological and social impacts on recipient ecosystems and are a major problem for land managers. Successful management demands an understanding of the ecology of invading taxa. The invasive status and impacts are documented for Prosopis populations in South Africa. However, unresolved taxonomic issues, the extent of hybridization, the applicability of morphology as a species identification approach, and the role that some traits plays in the invasion success have not been studied. This creates a gap that hinders implementation of effective management policies. In this thesis I use a phylogenetic approach to determine the taxonomic make-up of invasive Prosopis populations in South Africa (Chapter 2) and compare the results to morphological identification (Chapter 3). I also look at seedling growth rates in the context of variation in genome size and seed size (Chapter 4). Almost all regions invaded by Prosopis are characterized by taxonomic uncertainty exacerbated by the ease of inter-specific hybridization. In Chapter 2 I aim to resolve taxonomic issues of invasive Prosopis populations in South Africa using a phylogenetic approach. In addition, I aim to unravel the extent of hybridization and the species involved in South Africa. Here, I found that Prosopis populations in South Africa comprise both reported and previously unreported species, indicating a need for a reassessment of the identity of invasive taxa. Hybridization is prevalent and all confirmed species are involved. These findings call for a rethink of legislation and management approaches, e.g. the selection of classical biological control agents. Overall the extent of hybridization indicates that Prosopis species in South Africa comprise a freely inter-breeding population typical of a syngameon. Proper morphological identification of invasive species is crucial for ecological studies and management of invasions. In Chapter 3, I use the total evidence approach to assess whether morphological approaches for identification are adequate for identifying Prosopis species in South Africa. I found that Prosopis taxa in South Africa cannot be reliably distinguished using existing morphological keys. This is likely due mainly to the proliferation of hybrids with a diverse morphology. Therefore, molecular tools are crucial for confirming any morphological identities and for determining the presence of any unreported species. Genome size and seed size have been reported to be associated with invasiveness in a number of plant groups, but not often in a system with multiple hybrids like Prosopis. In Chapter 4, I first investigate the relationship between genome size and seed size in invasive populations of Prosopis spp. in South Africa and secondly I investigate how genome and seed sizes influence germination and early growth. Here I found that genome size loses its distinctness, being diluted in hybridizing populations, but can still be used to assess hybridization events themselves. Large seed size seems to be important for invasiveness as it positively influences germination and early growth. This thesis confirms the taxonomic conundrum of Prosopis species in invasive ranges. This coupled with inadequacy of morphological identification calls for a global study involving native and invasive range taxa to clarify the existing confusions. In view of the presence of unreported Prosopis species in South Africa and extensive hybridization, a rethink of the current legislation and control is needed. / AFRIKAANSE OPSOMMING: Uitheemse indringer plante het grootskaalse ekologiese en sosiale impakte op die ekosisteme wat hulle indring en stel 'n groot uitdaging vir bestuurders van natuurlike hulpbronne. Suksesvolle bestuur en bestryding van indringer plante verg deeglike kennis oor hulle ekologie. Die indringer status en impakte van Prosopis populasies in Suid Afrika is reeds voorheen beskryf. Nieteenstaande, die problematiese taksonomie, die omvang van hibridisasie, die waarde van morfologiese identifikasie, en die rol wat sekere eienskappe speel in die sukses van hierdie groep is nog nie bestudeer nie. Daar is dus 'n gaping in kennis wat die effektiewe beheer van die groep in Suid Afrika belemmer. In hierdie tesis pas ek 'n filogenetiese benadering toe om die taksonomiese verwantskappe van Prosopis populasies in Suid Afrika te bepaal (Hoofstuk 2) en vergelyk my resultate met morfologiese identifikasie sleutels (Hoofstuk 3). Ek ondersoek ook saailing groei tempos in die konteks van variasie in genoom en saad groote in die groep (Hoofstuk 4). Bykans alle areas in Suid Afrika waar Prosopis voorkom word gekenmerk deur taksonomiese onsekerheid, verder bemoeilik deur die gemak waarmee spesies vrylik hibridiseer. Ek vind dat beide bekende en voorheen-onbeskryfde Prosopis spesies in Suid Afrika aangetref word en beklemtoon die behoefte om die identiteit van spesies in die land te hersien. Hibridisasie kom algemeen voor tussen alle spesies teenwoordig in Suid Afrika. Hierdie bevindinge beklemtoon dat wetgewing en beheermaatreêls hersiening benodig, byvoorbeeld in die toepassing van biologiese beheer. In samevatting kom dit voor asof hibridisasie gelei het tot 'n vrytelende Prosopis groep in Suid Afrika, tipies van 'n singameon. Ordentlike morfologiese identifikasie van indringer spesies is belangrik in enige ekologiese studie en die implementering van doeltreffende beheermaatreëls. In Hoofstuk 3 gebruik ek ʼn ‘totale bewys’ benadering om vas te stel of morfologiese eienskappe alleenlik genoegsaam is om Prosopis spesies in Suid Afrika korrek te kan identifiseer. Ek vind dat spesies nie geloofwaardig geïdentifiseer kan word nie, heel moontlik as gevolg van wydverspreide hibridisasie tussen alle spesies teenwoordig in die land. Genoom en saad groote is voorheen geassosieer met die indringer aard van verskeie plant groepe. In Hoofstuk 4 ondersoek ek die verwantskap tussen genoom en saad groote. Tweedens bepaal ek die invloed van genoom en saad groote op ontkieming en vroeë groei eienskappe van Prosopis. My bevindinge toon dat, terwyl die kenmerklikheid van genoom groote verloor word as gevolg van hibridisasie, dit steeds hibridisasie gebeurtenisse per se kan identifiseer. Groot sade het ook ʼn positiewe invloed op die ontkieming en vroeë groei eienskappe van Prosopis. Die tesis bevestig die taksonomiese onduidelikheid van indringer Prosopis taksa in Suid Afrika. Tesame met die onakkuraatheid van morfologiese sleutels beklemtoon my bevindinge die behoefte vir ʼn dringende wêreldwye studie op indringer en inheemse populasies van Prosopis om taksonomiese onsekerhede op te klaar. Die identifikasie van nuwe spesies in Suid Afrika beklemtoon ook die behoefte om huidige wetgewing en beheer van die groep in die land te hersien.

Despite over seven decades of speciation research and 25 years of phylogeographic studies, a comprehensive understanding of mechanisms that generate biological species remains elusive. In temperate zones, the pervasiveness of range fragmentation and subsequent range expansions suggests that secondary contact between diverging lineages may be important in the evolution of species. Thus, such contact zones provide compelling opportunities to investigate evolutionary processes, particularly the roles of geographical isolation in initiating, and indirect selection against hybrids in completing (reinforcement), the evolution of reproductive isolation and speciation. The spring peeper (Pseudacris crucifer) has six well-supported mitochondrial lineages many of which are now in secondary contact. Here I investigate the evolutionary consequences of secondary contact of two such lineages (Eastern and Interior) in Southwestern Ontario using genetic, morphological, acoustical, experimental, and behavioural evidence to show accentuated divergence of the mate recognition system in sympatry. Mitochondrial and microsatellite data distinguish these two lineages but also show ongoing hybridization. Bayesian assignment tests and cline analysis imply asymmetrical introgression of Eastern lineage nuclear markers into Interior populations. Male calls are divergent between Eastern and Interior allopatric populations and show asymmetrical reproductive character displacement in sympatry. Female preference of pure lineage individuals is also exaggerated in sympatry, with hybrids showing intermediate traits and preference. I suggest that these patterns are most consistent with secondary reinforcement. I assessed levels of post-zygotic isolation between the Eastern and Interior lineages using a laboratory hybridization experiment. Hybrid tadpoles showed equal to or greater fitness than their pure lineage counterparts, but this may be countered through competition. More deformities and developmental anomalies in hybrid tadpoles further suggest post-zygotic isolation. Despite evidence for pre-mating isolation between the two lineages, isolation appears incomplete (i.e. hybridization is ongoing). I hypothesize that potentially less attractive hybrids may circumvent female choice by adopting satellite behaviour. Although mating tactics are related to body size, genetic status may play a role. I show that pure Eastern males almost always engage in calling, while hybrids adopt a satellite tactic. An absence of assortative mating, despite evidence of female preference, suggests successful satellite interception possibly facilitating introgression. / Thesis (Ph.D, Biology) -- Queen's University, 2013-03-04 16:01:33.892

An examination of the Desmodium canescens complex (D. canescens; D. tweedyi; D. illinoense) has resulted in the delimitation of a previously unreported alliance between D. canescens and D. tweedyi. The following points support this view: (a) morphological data taken from herbarium and garden specimens indicate that for many characters, the mean values of D. canescens and D. tweedy are not significantly different (b) breeding experiments have shown that artificial interspecific hybridization is possible between D. canescens and D. tweedyi (c) cytological studies have shown that D. canescens and D. tweedyi have a base number of x = 11, while D. illinoense has a base number of x = 10. A new combination is suggested: Desmodium canescens var. tweedyi (Britt.) Williams.