Pyrrolobenzodiazepines (PBDs) are a group of naturally occurring compounds that were discovered in the cultures of Streptomyces in the 1960s. Some natural PBDs discovered in these cultures, such as anthramycin and sibiromycin, were shown to possess a broad spectrum of anti-tumor activity. Since cancer is still a leading cause of death globally, the development of novel anti-proliferative derivatives of PBDs is essential for human welfare worldwide. Further synthesis and structure-activity relationship (SAR) studies of the parent natural products and their tetracyclic analogs will lead to the discovery of drug candidates. In this work, thirteen PBD analogues were synthesized using no more than three to four synthetic steps, beginning with commercially obtainable L-proline and isatoic anhydride. The MTT assay, which is a colorimetric assay that uses 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) to assess cell metabolic activity, was initially implimented to test the in vitro cytotoxicity of the compounds using multiple cell lines, namely: SKBR-3, MCF-7, SKMEL-2, CaCo 2, HCT 116, and Mia Paca. Nearly all of the compounds decreased the cell viability of MCF-7 by roughly 20%. Additionally, the anti-proliferative activity of the PBD products were further evaluated by the NCI-60 Human Tumor Cell Lines Screen, which is a part of the National Cancer Institute’s Development Therapeutics Program - Drug Synthesis and Chemistry Branch.

Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009 / Refer to document / National Research Foundation (NRF) and University of Limpopo

Vectors derived from Human Immunodeficiency Virus type 1 (HIV-1) are being widely developed for gene therapy applications, principally because they are able to transduce both dividing and non-dividing cells and result in stable, long term gene expression. However, these vectors are difficult to produce in high titres and sufficient volumes for large scale experiments and clinical application. Therefore, an investigation into methods to improve the production of HIV-1 derived gene transfer vectors was undertaken. One factor that limits the production of recombinant virus is the amount of viral genomic RNA available for packaging into virions. Therefore, a transfer vector was modified with the aim of increasing the amount of genomic RNA produced. Substitution of the polyadenylation (pA) signal, mutation splice donor sites and removal of unnecessary sequences were all examined. pA signal readthrough was quantified to determine the effect of these modifications on the rate of pA signal readthrough. Insertional mutagenesis and vector mobilisation are recognised risk factors with all integrating vectors. Self inactivating (SIN) vectors, which contain a deletion of U3 sequences in the 3’ LTR, demonstrate a reduced rate of mobilisation. Transduction with these vectors results in a provirus containing no viral promoter elements, with transcription of the transgene being controlled from an internal promoter. However, LTR repair of SIN vectors occurs at an appreciable frequency. Therefore, the extent of this deletion was maximised and the effect on the frequency of the repair examined. The production of lentiviral gene therapy vectors by large-scale transient transfection is both time consuming and technically difficult. Therefore, methods to increase the scale of production without compromising virus titre were developed. This resulted in fewer transfections and less handling of the cells when making virus on a large scale (3-4 L). In order to process the virus on this scale in a single day (i.e. 8 hours), new concentration and purification methods were established. The protocol consisted of low speed centrifugation, 0.45 μm filtration, 750 kDa ultrafiltration, 0.8 μm filtration and ultracentrifugation. However, the use of ultracentrifugation means that this protocol is not amenable to further scale up. Therefore, the replacement of the ultracentrifugation step with anion exchange was investigated. A number of different resins and anion exchange devices were investigated, two of which show promise for large scale purification of HIV-1 derived gene transfer vectors. In an ideal world, HIV-1 derived gene transfer vectors would be produced using stable packaging cell lines engineered to produce the desired virus. However, previous attempts to produce such a cell line with the desired properties have had limited success and have generally used outdated helper systems. Therefore, in an attempt to combine the efficiency advantages of having a single helper plasmid with the safety advantages of expressing each protein separately, a single packaging construct that contained separate transcription units for each of the required proteins was produced. Transcription of cyotoxic proteins was controlled by inducible promoters. Initial results suggest that such a system is technically feasible but that further work is required to optimise the expression of helper functions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309550 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2008

Cloning of trees using somatic embryogenesis could have a major impact on tree breeding and commercial plantation forestry. To initiate somatic embryogenesis in Anatolian black pine (Pinus nigra Arnold. subspecies pallasiana), one-year old cones containing immature seeds were collected from eight trees located in METU campus, Ankara. Embryogenic tissues were derived from immature zygotic embryos excised from the seeds. The zygotic embryos at the time of collection were at the precotyledonary stage of development. For this study, Douglas-fir cotyledon revised medium (DCR) supplemented with 13.6&micro / M 2,4-D, 2.2&micro / M BAP, 0.5 g/L casein hydrolysate, 0.25 g/L L-glutamine and 3% sucrose was used. The media was solidified with 0.2% gelrite. Embryogenic tissue initiation was calculated for each genotype and collection date. Overall initiation frequencies were recorded as 0.92% for 2004 and 1.96% for 2005. Highest initiation frequency was calculated for 5-July 2005 sampling time (4.06). ANOVA revealed significant differences between trees and collection date for initiation frequencies. Also, ECL (Established cell lines) recorded after five subcultures. Overall, 0.38% and 0.62% of the initial explants were converted into ECLs for 2004 and 2005 respectively.

The ultimate objective of this research was to develop a low cost, reliable system that would lead to early detection of lung cancer. Tests involved the quantitation of gaseous metabolic emissions from immortalized lung cancer cell lines in order to correlate the chemical markers to be of cancerous origin. The specific aims of the project were the study of gas emissions in selected cancer cell lines and identification of volatile organic compounds (VOCs) in them. Disadvantages of earlier studies were that the measurements were not real time or state specific so that molecular identification was often inconclusive. Furthermore the methods of study used in the past were not quantitative, which limited their practicality for medical applications. We felt the need to prove or disprove these earlier results using a new technique. The method we proposed is different and unique when compared to previous methods because cell lines have not been studied extensively for cancer markers. We have studied cancer cell lines which are adherent, immortalized cultures originating from primary tumors obtained from patients with no prior treatment for lung cancer. We have used an alternative method for the spectrometric analysis and quantitation of the selected chemical markers. The pre-concentration method involved a Purge and Trap unit with a thermal desorber where the vapor concentration was enhanced. The concentrated head space gases were analyzed using a Gas Chromatograph ?? Mass Spectrometer setup. This setup eliminated the bulky apparatus used in earlier studies. It is simpler in design and more comprehensive so that external factors such as patient??s diet, habitat and lifestyle do not contribute to our study of recognition of cancer markers. Based on the results obtained in the above experiments, a more comprehensive, inexpensive study of lung cancer related markers could be made. The first section, after giving an introduction to lung cancer, goes on to explain the background work done by other researchers on cancer. The third section gives a detailed explanation of the experimental setup. This is followed by all the tests conducted with corresponding results. The final section deals with the conclusions drawn from all experiments.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Η ακεραιότητα και η λειτουργία της μιτωτικής συσκευής διαδραματίζει ουσιώδη ρόλο για τον ορθό προσανατολισμό και την ολίσθηση των χρωμοσωμάτων στους πόλους της ατράκτου, οδηγώντας στον ισομερή διαχωρισμό των χρωμοσωμάτων κατά τη μιτωτική ή μειωτική διαίρεση. Τροποποιήσεις του δικτύου των μικροσωληνίσκων (α- και β-τουμπουλίνη) και των κέντρων οργάνωσης αυτών (ΜΤΟC, γ-τουμπουλίνη) είναι δυνατόν να προκαλέσουν βλάβες στη μιτωτική συσκευή, διαταράσσοντας το χρωμοσωματικό αποχωρισμό, με αποτέλεσμα το σχηματισμό ανευπλοειδικών κυττάρων. Η διουρητική φαρμακευτική ένωση υδροχλωροθειαζίδιο (HCTZ) χορηγείται κατά της υπέρτασης και είναι γνωστό ότι προκαλεί μη αποχωρισμό σε διπλοειδή στελέχη του μύκητα Aspergillus nidulans. Πρόσφατες μελέτες της ερευνητικής μας ομάδας έχουν δείξει ότι επάγει αυξημένες συχνότητες μικροπυρήνων και διαταράσσει το χρωμοσωματικό αποχωρισμό σε καλλιέργειες ανθρωπίνων λεμφοκυττάρων in vitro. Στην παρούσα εργασία μελετήθηκε η επίδραση του HCTZ στην οργάνωση του δικτύου των μικροσωληνίσκων κατά τη μεσόφαση και τη μίτωση, με συνδυασμένη εφαρμογή μεθόδων διπλής ανοσοσήμανσης των πρωτεϊνών των μικροσωληνίσκων, του κεντροσώματος και του κινητοχώρου. Εφαρμόστηκε επίσης in situ υβριδοποίηση με φθοροχρώματα (FISH), με α-satellite πανκεντρομερικό ανιχνευτή, για τον εντοπισμό μη ενσωματωμένου (lagging) χρωμοσωματικού υλικού. Η μελέτη πραγματοποιήθηκε σε κυτταρικές σειρές μυός C2C12 και ανθρώπου HFFF2. Παρατηρήθηκε ότι το HCTZ προκαλεί μείωση του ρυθμού διαίρεσης, αποδιοργάνωση του δικτύου των μικροσωληνίσκων και αυξημένη συχνότητα ανώμαλων μεταφάσεων με ποικίλο αριθμό σημάτων γ-τουμπουλίνης. Αυξάνει το ποσοστό των μεταφάσεων και μειώνει το ποσοστό των ανα-τελοφάσεων, προκαλώντας συσσώρευση των κυττάρων στο στάδιο της μετάφασης. Επίσης, επάγει τη χρωμοσωματική καθυστέρηση, καθώς αυξάνει τη συχνότητα των μικροπυρήνων που παρουσιάζουν τόσο σήμα κινητοχώρου όσο και σήμα κεντρομέρους. Η γενετική δράση του στα κύτταρα C2C12 δε φαίνεται να επηρεάζεται σημαντικά από τη χρονική διάρκεια έκθεσης των κυττάρων σ’ αυτό. Από τις δυο κυτταρικές σειρές που χρησιμοποιήθηκαν φαίνεται ότι τα κύτταρα C2C12 είναι περισσότερο ευαίσθητα στην απόκριση στο HCTZ. Τα αποτελέσματα μας, υποδεικνύουν ανευπλοειδογόνο δράση της φαρμακευτικής ένωσης, επιβεβαιώνοντας και ενισχύοντας προηγούμενα ευρήματα της ερευνητικής μας ομάδας. / The integrity and function of mitotic apparatus play essential role for the equitable orientation and the slipping of chromosomes to the spindle poles, indicating normal distribution of chromosomes during mitotic or meiotic division. Modifications of the microtubule network (α- and β- tubulin) and microtubule-organizing centers (MTOC, γ- tubulin) may cause severe damage to the mitotic apparatus, disturbing the segregation of chromosomes and resulting to aneuploid cells. The diuretic drug hydrochlorothiazide (HCTZ) is used for the treatment of hypertension and has been found to induce non-disjunction in diploid strains of Aspergillus nidulans. Recent studies of our team have shown that HCTZ produces increased frequencies of micronuclei and disturbs chromosome segregation in human lymphocytes cultures treated in vitro. In the present study was investigated the effect of HCTZ on the organization of the microtubule network during mesophase and mitosis, with combined application of double immunofluorescence staining assay, for the visualization of microtubules, centrosomes and kinetochore proteins. Fluorescence in situ hybridization (FISH), with α-satellite pancentromeric probe, was also applied, for the localization of not integrated (lagging) nuclear material. The study was realized in C2C12 mouse cells and HFFF2 human cells. Our results revealed that HCTZ causes decrease of the cell division rate, disorganization of the microtubule network and increased frequency of abnormal metaphases with various γ-tubulin signals. HCTZ increases the percentage of metaphases and decreases the percentage of ana-telophases, indicating a metaphase arrest. Also, induces chromosome delay, as was shown from the high frequency of micronuclei that presents kinetochore and centromere signal. The genetic activity of HCTZ in C2C12 cells does not appear to be significantly influenced by the duration of the cell’s exposure time to the drug. It appears that C2C12 mouse cells are more sensitive in their response to the HCTZ. These results indicate aneugenic activity of this drug, confirming and enhancing our previous findings.

abstract: Researchers in the twentieth and twenty-first centuries identify the study of the intrinsic and external factors that influence human aging as senescence. A commonly held belief in the year 2015 is that at least some kinds of cells can replicate over long periods or even indefinitely, thereby meaning the cell does not undergo senescence (also known as replicative senescence) and is considered immortal. This study aims to provide information to answer the following question: While some scientists claim they can indefinitely culture a stem cell line in vitro, what are the consequences of those culturing practices? An analysis of a cluster of articles from the Embryo Project Encyclopedia provides information to suggest possible solutions to some potential problems in cell culturing, recognition of benefits for existing or historical culturing practices, and identification of gaps in scientific knowledge that warrant further research. Recent research suggests that hESCs, and immortalized cell lines in general, do not escape the effects of senescence. While there exists a constant change in the practices of cell culturing, a large portion of scientists still rely on practices established before modern senescence research: research that seems to suggest that cultured hESCs, among other immortal cell lines, are not truly immortal. / Dissertation/Thesis / Masters Thesis Biology 2015

O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína. / Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.

Un contexte réglementaire de plus en plus strict en évaluation du risque écotoxicologique des milieux aquatiques exige de renforcer les outils d’évaluation. A ce titre, l’étude des biomarqueurs de génotoxicité doit être privilégiée, compte tenu du rôle central de l’ADN dans le fonctionnement du vivant. L’exposition à des agents génotoxiques peut générer des dommages par interaction directe avec l’ADN, mais aussi indirectement, en modulant l’efficacité des mécanismes de réparation de l’ADN, ou la régulation épigénétique de l’expression des gènes. Aujourd’hui, la plupart des biomarqueurs de génotoxicité visent les dommages primaires à l’ADN ou la mutagènicité mais les effets indirects sur sa fonctionnalité sont encore peu étudiés. Dans ce contexte, à l’issue d’une analyse bibliographique comprenant la rédaction d’une revue sur les mécanismes de réparation des dommages à l’ADN chez le poisson, ce travail visait au développement méthodologique de plusieurs biomarqueurs de génotoxicité à l’aide de trois lignées cellulaires pisciaires (RTL-W1, RTGill-W1 et PLHC-1). Pour ce faire, l’essai des comètes en conditions alcalines a été décliné sous plusieurs versions dans l’objectif d’utiliser une technique de base unique permettant la mesure complémentaire de plusieurs biomarqueurs de génotoxicité : les dommages primaires à l’ADN, les activités de réparation et le niveau de méthylation des cytosines du génome. Les résultats soulignent l’intérêt des trois lignées en évaluation de la génotoxicité 1) pour détecter in vitro de manière sensible des atteintes primaires à l’ADN de natures variées à de faibles concentrations grâce à un essai des comètes modifié par une étape de digestion enzymatique avec une glycosylase (Fpg), 2) pour évaluer l’influence des contaminants sur l’activité de réparation par excision de bases (BER) via la mesure de la capacité d’incision d’un ADN substrat porteur de lésions de type 8-oxoGua par des extraits cellulaires (essai BERc). Le niveau de méthylation des cytosines (5-meCyt) des lignées RTgill-W1et RTL-W1 a été mesuré par HPLC-MS-MS, leur valeur élevée permet d’envisager le paramètre méthylation comme biomarqueur potentiel. Ce volet nécessitera cependant des étapes de validations ultérieures car il n’a pas été techniquement possible de mettre au point un essai des comètes modifié pour la mesure du niveau de méthylation de l’ADN. Plusieurs activités de réparation des lignées RTgill-W1 et RTL-W1 ont été caractérisées et révèlent de bonnes aptitudes de réparation de type « Base Excision Repair » (BER) et « Photo Enzymatic Repair » (PER) et une plus faible capacité au « Nucleotide Excision Repair » (NER), soit un profil proche de celui décrit in vivo et sans différence marquée entre les deux lignées. Les biomarqueurs développés sur les lignées cellulaires de poisson au cours de ce travail ont également été appliqués à la mesure des effets génotoxiques d’effluents issus du lessivage de revêtements routiers. / In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazards and risk assessment of pollutants. In this context, genotoxicity endpoints are of a great concern since even when evaluated at a sub-cellular or cellular level, impaired DNA structure, repair and/or functions can have delayed (long term) consequences at higher level of organization such as individual and population. Some genotoxicant can have direct effect on DNA, but they can also interact indirectly, by modulating the repair mechanism efficiency or by acting on epigenetic mechanism such as DNA méthylation level. An unrepaired DNA damage and epigenetic modification can both lead to functional alteration and/or genetic structure at the population level. However, most of the existing genotoxicity test only measure primary DNA damage induces by genotoxicant; thus there is a real need to develop new tools to investigate those different kinds of genotoxicity. For this purpose, this work aims at developing knowledge in DNA repair capacities of to fish cell lines, RTL-W1 and RTG-W1, in order to develop new genotoxicity biomarker, measuring primary DNA damage by means of modified version of the comet assay. The results highlights the interest of in vitro biological models such as fish cell lines for the assessment of environmental genotoxicity, especially using a Fpg-modified comet assay allowing a sufficient increase of the assay sensibility to detect genotoxicity at environmentally relevant concentration. Results also characterize BER and PER capacities as being efficient repair mechanism in those fish cell lines, whereas NER, although also present, seems less efficient. A new biomarker based on the BER incision capacities of cellular extracts has also been developed and used to assess the genotoxicity of environemental effluent.