The accurate replication of the mammalian genome is a complex and logistically challenging process. The entirety of the genome must undergo a single duplication with as little error as possible. This must occur in a coordinated fashion and over suitably short time scale so as to allow timely cellular division within a cell cycle that is typically around 24 hours in a human cell. A great wealth of knowledge already exists describing various aspects of the S phase, during which this replication of the genome occurs. This data has been gathered over a variety of model systems, ranging from inferences from the replicative mechanics of SV40 through to direct observations of replication in mammalian cells.In order integrate this data and determine the value of inferences from different data sources, quantitative models of the mammalian cell S phase are required. This study documents the development of several such models and the exploration of the influences that experimentally determined parameters and different mechanistic theories can have on the behaviour of a simulated S phase. Of particular exploratory interest were the modes of activating replication of replicon clusters, with the aim of simulating experimentally observed dynamics. Additionally, the study also aimed to investigate the variation of replication fork rates and the density of origins of replication, along with the relationship that occurs between the two during both replicational stress and during a normal S phase. Through an iterative series of models, relevant parameters and key theories are sequentially explored so as to better understand the S phase. Particularly influential parameters were identified and studied in detail, with experimental determination where necessary in order to more accurately inform the model system. Conclusions concerning the behaviour of the system and the potential impact of the results were drawn upon the completion of each level of modelling and experimental work.To conclude the study, a linear model simulating the genome of the MRC5 cell line was used to estimate the modes activation of DNA replication along chromosomes in order to recreate experimentally observed replication dynamics. Experimentally determined profiles of replication fork rates and the density of origin firing were also determined for the MRC5 cell line, and were used to populate the model with accurate and appropriate data. Using the model to simulate S phase through a variety of behavioural parameters, realistic S phase dynamics were found to occur through a combination of de novo activation of replicon clusters and a specific probability of neighbour activation by completed clusters. These derived mechanics, when performed on a system correctly parameterised with suitable data, can simulate experimentally observed phenomena. The development of the model highlighted the requirements of data fit for purpose, and the study also stresses the need for critical consideration of inferences made between different model systems.

<p> Monomeric ribosomes and very few subunits are found in mammalian cells and because subunits are required for initiation of protein biosynthesis in both mammalian and bacterial systems, this implies that the dissociation step in the ribosome cycle does not occur spontaneously. Our attention was drawn to the possibility that the monomeric ribosomes in mammalian cells could complex with a dissociation factor. This factor would perhaps be present in the cell in limited supply and would, therefore have to recycle in the course of initiation, from a completed initiation complex to another free ribosome. An assay was set up whereby the existence of a dissociation factor in a subcellular fraction of rat liver could be determined. The perfecting of the assay system for the dissociation factor yielded much information on the ionic concentration necessary for both ribosome and subunit stability. The factor was found to be present in the fraction containing the "native" subunits. This is identical to the situation which exists in E. coli. The factor is capable of dissociating rat liver monomeric ribosomes into 60S and 40S subunits. The factor was found to act on ribosomes freed of both messenger RNA and nascent protein.</p> <p> Purification of the crude dissociation factor preparation was achieved by obtaining at 4°C the 35-65% ammonium sulphate fraction. Purification was also achieved by means of an incubation of the preparation at 40°C for 30 minutes followed by a centrifugation to remove precipitated protein.</p> <p> The DF was determined to have a molecular weight in excess of 85,000 by column chromatography.</p> / Thesis / Master of Science (MSc)

Bcl-XL is a membrane-associated protein that inhibits programmed cell death (apoptosis) in mammalian cells. Very little is known about the membrane topology of Bel-XL or how its association with membranes contributes to its function. It was the aim of this thesis to use fluorescence spectroscopy to investigate the location of a specific amino acid ofBcl-XL relative to the membrane. Bel-XL purified from E. coli could bind both to large unilamellar vesicles and endoplasmic reticulum (ER) microsomes isolated from canine pancreas. A cysteine residue at position 151 in Bcl-XL could be covalently labelled with the environmentally sensitive fluorescent molecule NBD. Emission intensity measurements in the presence and absence of membranes, combined with aqueous and lipophilic quenching experiments, indicate that Cys 151 is inserted into the interior of the membrane bilayer when Bcl-XL is bound to membranes. The methods outlined in this thesis form the basis for an experimental system that can be used to determine the membrane topology ofBclXL under a variety of conditions. / Thesis / Master of Science (MSc)

O DNA está constantemente exposto a danos causados tanto por agentes endógenos quanto exógenos. Estes podem causar diferentes tipos de lesões incluindo modificações de bases e do açúcar, além de quebras de fitas simples ou duplas. As quebras de duplas fitas, quando comparadas às demais, constituem as mais citotóxicas e podem resultar em deleções no DNA e instabilidade genética. Deleções no DNA mitocondrial (mtDNA) causam diversas doenças e estão envolvidas no processo de envelhecimento. No núcleo, as quebras de duplas fitas no DNA podem ser reparadas por recombinação homóloga (HR), ligação de pontas não homólogas (NHEJ) e anelamento de fita simples (SSA). No entanto, em mitocôndrias de células de mamíferos, o reparo de quebras de duplas fitas ainda não foi completamente caracterizado. Experimentos in vitro usando extratos mitocondriais de células de roedores mostraram que estes são capazes de reparar essas quebras, no entanto pouco é sabido sobre quais proteínas são responsáveis por cada etapa de reparo, bem como sua implicação na manutenção da integridade do genoma mitocondrial. Sendo assim, nesse trabalho investigamos a localização e função mitocondrial das proteínas ATM, Rad51, Rad52, Ku70/86 e DNA-PKCs, que são sabidamente envolvidas em reparo de quebras de duplas fitas no núcleo. Para identificar essas proteínas em mitocôndrias de células de mamíferos, mitocôndrias foram isoladas a partir de células da linhagem HEK293T, usando centrifugação diferencial seguida por gradiente de Percoll. Para as proteínas de recombinação homóloga, ATM e Rad51, imunodetectamos isoformas semelhantes em todos os compartimentos celulares. Já para a proteína Rad52 o mesmo anticorpo imunodetectou duas bandas distintas na mitocôndria ao passo que no núcleo foram quatro. Além disso, verificamos que baixos níveis de proteína Rad52, induzidos pela expressão de shRNA (short hairping RNA) específico, resultam em diminuição do número de cópias de mtDNA bem como acúmulo de deleções no genoma mitocondrial. Para as proteínas de NHEJ, DNA-PKCs e a subunidade Ku70, identificamos isoformas semelhantes em todos os compartimentos celulares. Já para a subunidade 86 do heterodímero Ku70/86 o anticorpo detectou, somente em mitocôndrias, uma banda menor de 50 kDa, a qual difere na região N-terminal da subunidade detectada no núcleo (86 KDa). Experimentos de co-imunprecitação de proteínas mostraram que essa isoforma menor compõe o heterodímero mitocondrial juntamente com a subunidade 70 (mtKu70/50) e que esse interage com DNA ligase III mitocondrial. Nossos resultados também mostraram que a estabilidade proteica de mtKu70/50 é regulada por ATM. Tratamento das células com peróxido de hidrogênio, que induz quebras de duplas fitas, aumentou a associação do heterodímero mtKu70/50 com o mtDNA, de forma independente de aumento da concentração proteica intra-mitocondrial. Já a diminuição dos níveis proteicos de Ku, induzida através de shRNA, resultou em diminuição do número de cópias de mtDNA e acumulo de danos nesse genoma. Extratos mitocondriais de células knockdown para Ku apresentaram menor atividade de reparo NHEJ em um ensaio in vitro, sugerindo que o acúmulo de danos nestas células é provavelmente devido a deficiências na via de NHEJ. Em conjunto, nossos dados sugerem que tanto HR quanto NHEJ operam em mitocôndrias. Além disso, a via de NHEJ mitocondrial utiliza o heterodímero mitocondrial Ku70/50 o qual está envolvido na manutenção do mtDNA. Ademais, nossos resultados mostram uma grande conservação molecular e funcional entre as vias de reparo de NHEJ e HR no núcleo e na mitocôndria, o que reforça sua importância para a manutenção da estabilidade genômica mitocondrial e, provavelmente a função mitocondrial. / DNA is constantly exposed to damaging agents from both endogenous and exogenous sources. These can cause different types of DNA lesions that include base and sugar modifications and single and double strand breaks. DNA doublestrand breaks (DSBs) are among the most cytotoxic DNA lesions, which can result in deletions and genetic instability. Deletions in the mitochondrial DNA (mtDNA) cause numerous human diseases and drive normal aging. DSBs in the nuclear DNA are repaired by non-homologous DNA end joining (NHEJ), homologous recombination (HR) or Single Strand Annealing (SSA). Yet, repair of DSBs in mammalian mitochondria has not been fully characterized. Mitochondrial extracts from rodent cells are proficient in ligating DNA ends in vitro, but little is known about which proteins are responsible for each enzymatic step and its implication in mitochondrial genome maintenance. Thus, we investigated mitochondrial localization and function of DSBR (double strand break repair) proteins ATM, Rad51, Rad52, the Ku70/86 heterodimer and DNA-PKCs.To identify DSBR proteins in mammalian mitochondria, highly purified mitochondria from HEK293T cells were isolated using differential centrifugation followed by Percoll gradient. For HR proteins, we detected similar isoforms for ATM and Rad51 proteins in all cellular compartments. Two mitochondriaspecific isoforms of Rad52 were detected, while the same antibody detected four isoforms in the nucleus. In addition, lower Rad52 protein levels, induced by specific shRNA expression, result in decreased mtDNA copy number and accumulation of deleted mitochondrial genomes. For NHEJ proteins, similar isoforms of DNA-PKcs and the Ku70 subunit were detected in all cellular compartments. On the other hand, antibodies against the Ku86 subunit detected a smaller band in mitochondrial extracts (50 KDa), lacking the N-terminal region of the canonical isoform detected in the nucleus (86 KDa). The mitochondrial Ku70/50 heterodimer interacts with mitochondrial DNA ligase III, suggesting a role in DSBR. Moreover, stability of the mtKu heterodimer is regulated by ATM. Hydrogen peroxide treatment, which induces DSBs, increases mtKu70/50 association with the mtDNA and cells with reduced Ku levels, also induced by shRNA transfection, have lower mtDNA copy number and accumulate mtDNA damage. Moreover, mitochondrial extracts from Ku knockdown cells show lower NHEJ repair activity in an in vitro assay, suggesting that damage accumulation in these cells is likely due to deficiencies in NHEJ. Together, our data suggest that both HR and NHEJ operate in mitochondria. Also, mtNHEJ requires the Ku heterodimer and is involved in mtDNA maintenance. Moreover, our results indicate that there is a significant molecular and functional conservation between NHEJ and HR repair pathways in the nucleus and in mitochondria, which reinforces their importance for maintenance of mitochondrial genomic stability and, likely mitochondrial function.

O DNA está constantemente exposto a danos causados tanto por agentes endógenos quanto exógenos. Estes podem causar diferentes tipos de lesões incluindo modificações de bases e do açúcar, além de quebras de fitas simples ou duplas. As quebras de duplas fitas, quando comparadas às demais, constituem as mais citotóxicas e podem resultar em deleções no DNA e instabilidade genética. Deleções no DNA mitocondrial (mtDNA) causam diversas doenças e estão envolvidas no processo de envelhecimento. No núcleo, as quebras de duplas fitas no DNA podem ser reparadas por recombinação homóloga (HR), ligação de pontas não homólogas (NHEJ) e anelamento de fita simples (SSA). No entanto, em mitocôndrias de células de mamíferos, o reparo de quebras de duplas fitas ainda não foi completamente caracterizado. Experimentos in vitro usando extratos mitocondriais de células de roedores mostraram que estes são capazes de reparar essas quebras, no entanto pouco é sabido sobre quais proteínas são responsáveis por cada etapa de reparo, bem como sua implicação na manutenção da integridade do genoma mitocondrial. Sendo assim, nesse trabalho investigamos a localização e função mitocondrial das proteínas ATM, Rad51, Rad52, Ku70/86 e DNA-PKCs, que são sabidamente envolvidas em reparo de quebras de duplas fitas no núcleo. Para identificar essas proteínas em mitocôndrias de células de mamíferos, mitocôndrias foram isoladas a partir de células da linhagem HEK293T, usando centrifugação diferencial seguida por gradiente de Percoll. Para as proteínas de recombinação homóloga, ATM e Rad51, imunodetectamos isoformas semelhantes em todos os compartimentos celulares. Já para a proteína Rad52 o mesmo anticorpo imunodetectou duas bandas distintas na mitocôndria ao passo que no núcleo foram quatro. Além disso, verificamos que baixos níveis de proteína Rad52, induzidos pela expressão de shRNA (short hairping RNA) específico, resultam em diminuição do número de cópias de mtDNA bem como acúmulo de deleções no genoma mitocondrial. Para as proteínas de NHEJ, DNA-PKCs e a subunidade Ku70, identificamos isoformas semelhantes em todos os compartimentos celulares. Já para a subunidade 86 do heterodímero Ku70/86 o anticorpo detectou, somente em mitocôndrias, uma banda menor de 50 kDa, a qual difere na região N-terminal da subunidade detectada no núcleo (86 KDa). Experimentos de co-imunprecitação de proteínas mostraram que essa isoforma menor compõe o heterodímero mitocondrial juntamente com a subunidade 70 (mtKu70/50) e que esse interage com DNA ligase III mitocondrial. Nossos resultados também mostraram que a estabilidade proteica de mtKu70/50 é regulada por ATM. Tratamento das células com peróxido de hidrogênio, que induz quebras de duplas fitas, aumentou a associação do heterodímero mtKu70/50 com o mtDNA, de forma independente de aumento da concentração proteica intra-mitocondrial. Já a diminuição dos níveis proteicos de Ku, induzida através de shRNA, resultou em diminuição do número de cópias de mtDNA e acumulo de danos nesse genoma. Extratos mitocondriais de células knockdown para Ku apresentaram menor atividade de reparo NHEJ em um ensaio in vitro, sugerindo que o acúmulo de danos nestas células é provavelmente devido a deficiências na via de NHEJ. Em conjunto, nossos dados sugerem que tanto HR quanto NHEJ operam em mitocôndrias. Além disso, a via de NHEJ mitocondrial utiliza o heterodímero mitocondrial Ku70/50 o qual está envolvido na manutenção do mtDNA. Ademais, nossos resultados mostram uma grande conservação molecular e funcional entre as vias de reparo de NHEJ e HR no núcleo e na mitocôndria, o que reforça sua importância para a manutenção da estabilidade genômica mitocondrial e, provavelmente a função mitocondrial. / DNA is constantly exposed to damaging agents from both endogenous and exogenous sources. These can cause different types of DNA lesions that include base and sugar modifications and single and double strand breaks. DNA doublestrand breaks (DSBs) are among the most cytotoxic DNA lesions, which can result in deletions and genetic instability. Deletions in the mitochondrial DNA (mtDNA) cause numerous human diseases and drive normal aging. DSBs in the nuclear DNA are repaired by non-homologous DNA end joining (NHEJ), homologous recombination (HR) or Single Strand Annealing (SSA). Yet, repair of DSBs in mammalian mitochondria has not been fully characterized. Mitochondrial extracts from rodent cells are proficient in ligating DNA ends in vitro, but little is known about which proteins are responsible for each enzymatic step and its implication in mitochondrial genome maintenance. Thus, we investigated mitochondrial localization and function of DSBR (double strand break repair) proteins ATM, Rad51, Rad52, the Ku70/86 heterodimer and DNA-PKCs.To identify DSBR proteins in mammalian mitochondria, highly purified mitochondria from HEK293T cells were isolated using differential centrifugation followed by Percoll gradient. For HR proteins, we detected similar isoforms for ATM and Rad51 proteins in all cellular compartments. Two mitochondriaspecific isoforms of Rad52 were detected, while the same antibody detected four isoforms in the nucleus. In addition, lower Rad52 protein levels, induced by specific shRNA expression, result in decreased mtDNA copy number and accumulation of deleted mitochondrial genomes. For NHEJ proteins, similar isoforms of DNA-PKcs and the Ku70 subunit were detected in all cellular compartments. On the other hand, antibodies against the Ku86 subunit detected a smaller band in mitochondrial extracts (50 KDa), lacking the N-terminal region of the canonical isoform detected in the nucleus (86 KDa). The mitochondrial Ku70/50 heterodimer interacts with mitochondrial DNA ligase III, suggesting a role in DSBR. Moreover, stability of the mtKu heterodimer is regulated by ATM. Hydrogen peroxide treatment, which induces DSBs, increases mtKu70/50 association with the mtDNA and cells with reduced Ku levels, also induced by shRNA transfection, have lower mtDNA copy number and accumulate mtDNA damage. Moreover, mitochondrial extracts from Ku knockdown cells show lower NHEJ repair activity in an in vitro assay, suggesting that damage accumulation in these cells is likely due to deficiencies in NHEJ. Together, our data suggest that both HR and NHEJ operate in mitochondria. Also, mtNHEJ requires the Ku heterodimer and is involved in mtDNA maintenance. Moreover, our results indicate that there is a significant molecular and functional conservation between NHEJ and HR repair pathways in the nucleus and in mitochondria, which reinforces their importance for maintenance of mitochondrial genomic stability and, likely mitochondrial function.

Understanding of bio-nano-interfaces of living mammalian cells will benefit the identification of cellular alterations (e.g. nucleic acids, amino acids, biomechanics, etc.) due to external stimuli, the design of biomaterials (e.g. nanoparticles, nanotubes) and the investigation of the interaction between cells and bio-nano-interfaces (e.g. cell differentiation on 3D nanostructured materials). Analytical techniques can be applied to evaluate the chemical, physical, and mechanical properties of mammalian cells when exposed to such bio-nano-interfaces. In this study, non-invasive advanced spectroscopy techniques including atomic force microscopy (AFM) and Raman microscopy (RM), in conjunction with traditional biological methods are utilized to elucidate specific characteristic information for biological samples and how these property changes reflect the interaction with external stimuli. The focus of this dissertation is on the biophysical, biochemical and cytotoxic detection of mammalian cells interacting with bio-nano-interfaces, and this dissertation can be classified into three topics: biomechanics/cellular biopolymers measurement, bio-interfaces and nano-interfaces studies. For the topic of biomechanics/cellular biopolymers measurement, cellular biophysical and biomechanical properties could be used as differentiation markers to classify cellular differentiation. For the bio-interfaces part, it was observed that BRMS1 expression changed cellular biochemical and biomechanical properties, and the expressions of reactive oxidative species (ROS), apoptosis and cell viability of five types of cells displayed similar patterns over doxorubicin (DOX) incubation time. Secondly, A549 cells were treated with diesel exhaust particles (DEP) and resveratrol (RES) to study the effect of RES on the DEP-induced cells, and it was found that RES can alleviate DEP intervention on cellular structure and increase DEP-induced biomechanical and inflammatory changes. For the nano-interfaces topic, first we synthesized a hybrid nanoparticle with the multimodal properties of fluorescence imaging, Surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT) for single living cell analysis of epidermal growth factor receptor (EGFR) and specifically killed cancer cells with high EGFR expression. Additionally, to increase surface area, light-heat conversion efficiency and biocompatibility, we developed a silica coated nanoparticle conjugated with anti-human epidermal growth factor receptor 2 (HER2) antibody. Finally, three-dimensional TiO2 nanotubes with Au nanoparticles coating were synthesized and used to study trophoblast-derived stem-like cells growth on such 3D nanostructures.

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.

A fundamental understanding of bio-interfaces will facilitate improvement in the design and application of biomaterials that can beneficially interact with biological objects such as nucleic acids, molecules, bacteria, and mammalian cells. Currently, there exist analytical instruments to investigate material properties and report information on electrical, chemical, physical, and mechanical natures of biomaterials and biological samples. The overall goal of this research was to utilize advanced spectroscopy techniques coupled with data mining to elucidate specific characteristic properties for biological objects and how these properties imply interaction with environmental biomaterials. My studies of interfacial electron transfer (ET) of DNA-modified gold electrodes aided in understanding that DNA surface density is related to the step-wise order of which a self-assembled monolayer is created on a gold substrate. Further surface modification plays a role in surface conductivity, and I found that electro-oxidation of the DNA involved the oxidation of guanine and adenine nucleotides. Scanning tunneling microscopy (STM) was used to create topography and current images of the SAM surfaces. I also used Raman microspectroscopy (RM) to obtain spectra and spectral maps of DNA-modified gold surfaces. For studies of bacteria, atomic force microscopy (AFM) and scanning electron microscopy (SEM) images showed similar morphological features of Gram-positive and Gram-negative bacteria. Direct classical least squares (DCLS) analysis aided to distinguish co-cultured strains. Fourier transform infrared (FTIR) spectroscopy proved insightful for characteristic bands for Gram-positive bacteria and a combined AFM/RM image revealed a relationship between culture height/density and peak Raman intensity. In our mammalian cell studies we focused on human lung adenocarcinoma epithelial cells (A549), metastatic human breast carcinoma cells MDA-MB-435 (435), and non-metastatic MDA-MB-435/BRMS1 (435/BRMS1). RM revealed similarities between metastatic 435 and non-metastatic 435/BRMS1 cells compared to epithelial A549 cells. AFM showed increases in biomechanical properties for 435/BRMS1 in the areas of cell adhesion, cell spring constant, and Young’s modulus. Fluorescent staining illustrates F-actin rearrangement for 435 and 435/BRMS1. Electric cell-substrate impedance sensing (ECIS) revealed that 435 cells adhere tightly to substrata and migrate rapidly compared with 435/BRMS1. For ECIS, ≤10-fold diesel exhaust particles (DEP) concentration exposure caused clastogenic DNA degradation whereas ≥25-fold DEP exposure caused cytotoxic results. Resveratrol (RES) at 10 μM showed minimal to mild protection against DEP before and after exposure and aided in improving injury recovery.

No description available.

Methodology was developed which allowed the rapid and routine quantitation of subpicomole quantities of diadenosine-5ʹ,5ʹʹʹ-P¹,P⁴-tetraphosphate (Ap₄A) in cultured mammalian cells. This methodology includes the rapid extraction of cellular nucleotides in cold alkali, resolution of Ap₄A from the bulk of cellular materials on a highly specific boronate affinity resin, and quantitation of the dinucleotide in a coupled bioluminescence assay utilizing venom phosphodiesterase and firefly luciferase. The sensitivity and selectivity of this assay is demonstrated and contrasted with previously developed techniques. This assay was used to examine the role of Ap₄A in DNA replication and the cellular stress response.