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Sensory and consumer sciences aim to understand the influences of product acceptability and purchase decisions. The food industry measures product acceptability through hedonic testing but often does not assess implicit or qualitative response. Incorporation of qualitative research and automated facial expression analysis (AFEA) may supplement hedonic acceptability testing to provide product insights. The purpose of this research was to assess the application of AFEA and qualitative analysis to understand consumer experience and response. In two studies, AFEA was applied to elucidate consumers emotional response to dairy (n=42) and water (n=46) beverages. For dairy, unflavored milk (x=6.6±1.8) and vanilla syrup flavored milk (x=5.9±2.2) (p>0.05) were acceptably rated (1=dislike extremely; 9=like extremely) while salty flavored milk (x=2.3±1.3) was least acceptable (p<0.05). Vanilla syrup flavored milk generated emotions with surprised intermittently present over time (10 sec) (p<0.025) compared to unflavored milk. Salty flavored milk created an intense disgust response among other emotions compared to unflavored milk (p<0.025). Using a bitter solutions model in water, an inverse relationship existed with acceptability as bitter intensity increased (rs=-0.90; p<0.0001). Facial expressions characterized as disgust and happy emotion increased in duration as bitter intensity increased while neutral remained similar across bitter intensities compared to the control (p<0.025). In a mixed methods analysis to enumerate microbial populations, assess water quality, and qualitatively gain consumer insights regarding water fountains and water filling stations, results inferred that water quality differences did not exist between water fountains and water filling stations (metals, pH, chlorine, and microbial) (p>0.05). However, the exterior of water fountains were microbially (8.8 CFU/cm^2) and visually cleaner than filling stations (10.4x10^3 CFU/cm^2) (p<0.05). Qualitative analysis contradicted quantitative findings as participants preferred water filling stations because they felt they were cleaner and delivered higher quality water. Lastly, The Theory of Planned Behavior was able to assist in understanding undergraduates' reusable water bottle behavior and revealed 11 categories (attitudes n=6; subjective norms n=2; perceived behavioral control n=2; intentions n=1). Collectively, the use of AFEA and qualitative analysis provided additional insight to consumer-product interaction and acceptability; however, additional research should include improving the sensitivity of AFEA to consumer product evaluation. / Ph. D.

Yes / Emerging applications in surveillance, the entertainment industry and other human computer interaction applications have motivated the development of real-time facial analysis research covering detection, tracking and recognition. In this paper, the authors present a review of recent facial analysis for real-time applications, by providing an up-to-date review of research efforts in human computing techniques in the visible domain. The main goal is to provide a comprehensive reference source for researchers, regardless of specific research areas, involved in real-time facial analysis. First, the authors undertake a thorough survey and comparison in face detection techniques. In this survey, they discuss some prominent face detection methods presented in the literature. The performance of the techniques is evaluated by using benchmark databases. Subsequently, the authors provide an overview of the state-of-the-art of facial expressions analysis and the importance of psychology inherent in facial expression analysis. During the last decades, facial expressions analysis has slowly evolved into automatic facial expressions analysis due to the popularity of digital media and the maturity of computer vision. Hence, the authors review some existing automatic facial expressions analysis techniques. Finally, the authors provide an exemplar for the development of a facial analysis real-time application and propose a model for facial analysis. This review shows that facial analysis for real-time application involves multi-disciplinary aspects and it is important to take all domains into account when building a reliable system.

Sensory and consumer sciences seek to comprehend the influences of sensory perception on consumer behaviors such as product liking and purchase. The food industry assesses product liking through hedonic testing but often does not capture affectual response as it pertains to product-generated (PG) and product-associated (PA) emotions. This research sought to assess the application of PA and PG emotion methodology to better understand consumer experiences. A systematic review of the existing literature was performed that focused on the Facial Action Coding System (FACS) and its use to investigate consumer affect and characterize human emotional response to product-based stimuli, which revealed inconsistencies in how FACS is carried out as well as how emotional response is inferred from Action Unit (AU) activation. Automatic Facial Expression Analysis (AFEA), which automates FACS and translates the facial muscular positioning into the basic universal emotions, was then used in a two-part study. In the first study (n=50 participants), AFEA, a Check-All-That-Apply (CATA) emotions questionnaire, and a Single-Target Implicit Association Test (ST-IAT) were used to characterize the relationship between PA as well as PG emotions and consumer behavior (acceptability, purchase intent) towards milk in various types of packaging (k=6). The ST-IAT did not yield significant PA emotions for packaged milk (p>0.05), but correspondence analysis of CATA data produced PA emotion insights including term selection based on arousal and underlying approach/withdrawal motivation related to packaging pigmentation. Time series statistical analysis of AFEA data provided increased insights on significant emotion expression, but the lack of difference (p>0.05) between certain expressed emotions that maintain no related AUs, such as happy and disgust, indicates that AFEA software may not be identifying AUs and determining emotion-based inferences in agreement with FACS. In the second study, AFEA data from the sensory evaluation (n=48 participants) of light-exposed milk stimuli (k=4) stored in packaging with various light-blocking properties) underwent time series statistical analysis to determine if the sensory-engaging nature of control stimuli could impact time series statistical analysis of AFEA data. When compared against the limited sensory engaging (blank screen) control, contempt, happy, and angry were expressed more intensely (p<0.025) and with greater incidence for the light-exposed milk stimuli; neutral was expressed exclusively in the same manner for the blank screen. Comparatively, intense neutral expression (p<0.025) was brief, fragmented, and often accompanied by intense (albeit fleeting) expressions of happy, sad, or contempt for the sensory engaging control (water); emotions such as surprised, scared, and sad were expressed similarly for the light-exposed milk stimuli. As such, it was determined that care should be taken while comparing the control and experimental stimuli in time series analysis as facial activation of muscles/AUs related to sensory perception (e.g., chewing, smelling) can impact the resulting interpretation. Collectively, the use of PA and PG emotion methodology provided additional insights on consumer-product related behaviors. However, it is hard to conclude whether AFEA is yielding emotional interpretations based on true facial expression of emotion or facial actions related to sensory perception for consumer products such as foods and beverages. / Doctor of Philosophy / Sensory and consumer sciences seek to comprehend the influences of sensory perception on consumer behaviors such as product liking and purchase. The food industry assesses product liking through consumer testing but often does not capture consumer response as it pertains to emotions such as those experienced while directly interacting with a product (i.e., product-generated emotions, PG) or those attributed to the product based on external information such as branding, marketing, nutrition, social environment, physical environment, memories, etc.( product-associated emotions, PA). This research investigated the application of PA and PG emotion methodology to better understand consumer experiences. A systematic review of the existing scientific literature was performed that focused on the Facial Action Coding System (FACS), a process used determine facially expressed emotion from facial muscular positioning, and its use to investigate consumer behavior and characterize human emotional response to product-based stimuli; the review revealed inconsistencies in how FACS is carried out as well as how emotional response is determined from facial muscular activation. Automatic Facial Expression Analysis (AFEA), which automates FACS, was then used in a two-part study. In the first study (n=50 participants), AFEA, a Check-All-That-Apply (CATA) emotions questionnaire, and a Single-Target Implicit Association Test (ST-IAT) were used to characterize the relationship between PA as well as PG emotions and consumer behavior (acceptability, purchase intent) towards milk in various types of packaging (k=6). While the ST-IAT did not yield significant results (p>0.05), CATA data produced illustrated term selection based on motivation to approach and/or withdrawal from milk based on packaging color. Additionally, the lack of difference (p>0.05) between emotions that do not produce similar facial muscle activations, such as happy and disgust, indicates that AFEA software may not be determining emotions as outlined in the established FACS procedures. In the second study, AFEA data from the sensory evaluation (n=48 participants) of light-exposed milk stimuli (k=4) stored in packaging with various light blocking properties underwent time series statistical analysis to determine if the nature of the control stimulus itself could impact the analysis of AFEA data. When compared against the limited sensory engaging control (a blank screen), contempt, happy, and angry were expressed more intensely (p<0.025) and consistently for the light-exposed milk stimuli; neutral was expressed exclusively in the same manner for the blank screen. Comparatively, intense neutral expression (p<0.025) was brief, fragmented, and often accompanied by intense (although fleeting) expressions of happy, sad, or contempt for the sensory engaging control (water); emotions such as surprised, scared, and sad were expressed similarly for the light-exposed milk stimuli. As such, it was determined that care should be taken as facial activation of muscles/AUs related to sensory perception (e.g., chewing, smelling) can impact the resulting interpretation. Collectively, the use of PA and PG emotion methodology provided additional insights to consumer-product related behaviors. However, it is hard to conclude whether AFEA is yielding emotional interpretations based on true facial expression of emotion or facial actions related to sensory perception for sensory engaging consumer products such as foods and beverages.

Bacterial persistence is considered as one of the primary reason for antibiotic tolerance besides genetically acquired antibiotic resistance. Persisters are the subpopulation of a clonal bacterial population, which can survive environmental extremes and become invulnerable to stresses due to limited metabolic activities and physiological functions. Cognate toxin and antitoxin (TA) pairs, which are transcribed simultaneously from the same or different operons within the bacterial chromosomes or plasmids, play an important role for bacterial survival during stressful growth environments. Pseudomonas aeruginosa PAO1 is one of the most versatile microorganisms in the environment. Despite its ubiquitous presence, no studies have shown the differential expression pattern of its toxin-antitoxins, and persistence related genes. The purpose of the following study is to analyze differential expression of P. aeruginosa PAO1 type II toxin-antitoxins and persistence related genes under different growth conditions and to show how their stoichiometric ratio changes during different growth conditions. Differential expression analysis indicated that the toxins and antitoxin pairs behave differently under different growth conditions. In addition, the genes related to persistence presented relatively consistent differential expression pattern under different growth environment. / Master of Science

<p>Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used.</p><p>This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible.</p><p>The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies.</p>

Automatic analysis of facial actions (AFA) can reveal a person’s emotion, intention, and physical state, and make possible a wide range of applications. To enable reliable, valid, and efficient AFA, this thesis investigates automatic analysis of facial actions through transductive, supervised and unsupervised learning. Supervised learning for AFA is challenging, in part, because of individual differences among persons in face shape and appearance and variation in video acquisition and context. To improve generalizability across persons, we propose a transductive framework, Selective Transfer Machine (STM), which personalizes generic classifiers through joint sample reweighting and classifier learning. By personalizing classifiers, STM offers improved generalization to unknown persons. As an extension, we develop a variant of STM for use when partially labeled data are available. Additional challenges for supervised learning include learning an optimal representation for classification, variation in base rates of action units (AUs), correlation between AUs and temporal consistency. While these challenges could be partly accommodated with an SVM or STM, a more powerful alternative is afforded by an end-to-end supervised framework (i.e., deep learning). We propose a convolutional network with long short-term memory (LSTM) and multi-label sampling strategies. We compared SVM, STM and deep learning approaches with respect to AU occurrence and intensity in and between BP4D+ [282] and GFT [93] databases, which consist of around 0.6 million annotated frames. Annotated video is not always possible or desirable. We introduce an unsupervised Branch-and-Bound framework to discover correlated facial actions in un-annotated video. We term this approach Common Event Discovery (CED). We evaluate CED in video and motion capture data. CED achieved moderate convergence with supervised approaches and enabled discovery of novel patterns occult to supervised approaches.

A perda auditiva hereditária é uma característica com grande heterogeneidade genética. Mais de uma centena de genes já foram relacionados com a audição e, com o advento do sequenciamento massivo em paralelo, novas variantes têm sido identificadas como candidatas a causar surdez hereditária. Porém, estudos funcionais para verificação do efeito das mutações candidatas são necessários. Modelos animais permitem estudos funcionais eficientes para confirmação do efeito de mutações em genes candidatos, sendo uma ferramenta poderosa para compreender melhor os efeitos genéticos, moleculares, fisiológicos e comportamentais destas alterações. Previamente, foi identificada em nosso laboratório uma mutação de sentido trocado em um gene que codifica um coativador nuclear como principal candidata a causar surdez hereditária em uma família de São Paulo. A função principal deste gene é regular positivamente a transcrição gênica mediada por receptores nucleares. Contudo, não há dados na literatura sobre a expressão e o papel deste gene no sistema auditivo. Desta forma, tivemos como objetivo principal investigar a expressão do gene candidato em sistemas mecanossensoriais de camundongo e zebrafish. Detectamos, por meio de RT-PCR, a expressão do RNAm na cóclea inteira e no órgão de Corti associado à estria vascular de camundongos com idades P4, P10 e P16. Posteriormente, experimentos de qRT-PCR mostraram maior expressão nos estágios P10 e P16, em relação ao estágio P4, com parcela significativa da expressão gênica concentrada no órgão de Corti associado à estria vascular. Com relação à proteína, foi detectada, por meio de ensaios de imunofluorescência, a sua expressão nos cortes histológicos de cóclea de camundongos P4, P10 e P14, em várias estruturas diferentes da cóclea: membrana basilar, membrana de Reissner, órgão de Corti, estria vascular, limbo espiral e gânglio espiral. Experimentos de hibridação in situ em zebrafish inteiro foram realizados e a expressão do RNAm foi observada na orelha interna de larvas com idade 3 e 5 dias pós-fertilização (dpf) e de juvenis com 5 e 7 semanas pós-fertilização (spf). Nossos experimentos forneceram dados inéditos que sugerem um papel conservado deste gene no desenvolvimento do sistema auditivo de camundongos e no desenvolvimento e fisiologia do sistema auditivo de zebrafish. Para investigarmos se a falta de expressão do gene afeta o sistema auditivo realizamos experimentos visando à edição gênica do gene candidato por meio do sistema CRISPR/Cas9 em zebrafish. Dada a dificuldade para a padronização da técnica, não obtivemos resultados conclusivos durante o período de estudo. Nosso trabalho mostrou pela primeira vez a expressão do RNAm e da proteína no sistema auditivo de modelos animais, o que é fundamental para reforçar o potencial papel da mutação na surdez hereditária identificada na família. Desta maneira, contribuímos para a delineação de futuros experimentos funcionais que elucidem o papel deste gene e da mutação correspondente no desenvolvimento e na fisiologia do sistema auditivo / Hereditary hearing loss is a characteristic with high genetic heterogeneity. More than one hundred genes have been related to hearing and, with the advent of massive parallel sequencing, new variants have been identified as candidates for causing hereditary deafness. However, functional studies to verify the effect of candidate mutations are necessary. Animal models provide efficient functional studies to confirm the effect of mutations and candidate genes, being a powerful tool to understanding the genetic, molecular, physiological and behavioural effects of these genetic variants. Previously, a missense mutation in a gene coding a nuclear receptor coactivator has been identified in our laboratory as the main candidate for causing hereditary hearing loss in a family from São Paulo. The main function of this gene is to positively regulate gene transcription mediated by nuclear receptors. However, there is no data in the literature about its expression or about its function in the hearing system. Thus, we aimed to investigate its expression in mechanosensory systems of mice and zebrafish. Through RT-CPR, we have detected expression of the mRNA in whole cochlea and in organ of Corti associated to stria vascularis of P4, P10 and P16 mice. In addition, qRT-PCR revealed higher expression in P10 and P16 stages, compared to P4 stage, and that a significant fraction of the expression is concentrated in the organ of Corti associated to stria vascularis. Regarding the protein, immunofluorescence assays revealed expression of the coactivator in histological sections of P4, P10 and P14 mice cochlea, with fluorescencent signals in several structures: basilar mebrane, Reissner\'s membrane, organ of Corti, stria vascularis, spiral limbus and spiral ganglion. Whole-mount in situ hybridization assays were conducted in zebrafish, revealing the mRNA expression in the inner ear of 3 and 5 days-post-fertilization (dpf) larvae and of 5 and 7 weeks-post-fertilization (wpf) juveniles. Our experiments provided unprecedented data suggesting a conserved role of this gene in the development of the auditory system of mice and in the development and physiology of the auditory system of zebrafish. In order to investigate if the lack of the gene expression affects the auditory system, we performed experiments aiming genetic edition through CRISPR/Cas9 system in zebrafish. Given the difficulty to standardize the technique, we could not obtain conclusive results during the study period. Our work has shown for the first time the expression of the candidate gene mRNA and protein in the auditory in animal models, which is fundamental to reinforce the potential role of the mutation in the hearing loss identified in the family. Thus, we have contributed to the delineation of future functional experiments that will unveil the role of the gene and its corresponding mutation in the development and physiology of the auditory system

Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related deaths in Australian women. Colorectal cancer is the second most common cancer in both males and females; after prostate and breast cancer, respectively, and excluding non-melanocytic skin cancer. Both breast cancer and colorectal cancer follow a common progressive course of illness; presenting (at least initially) with benign symptoms that can be treated by ablation (or removal) of the affected area. Cancer progression is associated with breakdown of tissue barriers (such as basement membranes), leading to the spread of cancer cells (via the vasculature or lymphatic system), and the establishment of secondary metastatic disease at green-field sites. Secondary tumours presenting in the lungs, ovaries, liver, bone, or brain are associated with chronic-debilitating symptoms that are difficult to treat, and will result in death. In the case of breast and colon cancer, effective early therapeutic intervention does have a significant impact upon patient survival. Tumour progression in breast and colon carcinomas is characterised by invasion of the surrounding stroma, and the acquisition of stromal characteristics, by previously epithelial cells. This progression is associated with the expression of extracellular proteases (ECPs) and increased motility. The process of mesenchymal transformation that tumour cells undergo is also referred to as the epithelial to mesenchymal transition (EMT). In general terms the aim of the study, presented in this thesis, was to investigate gene expression in cancer biology; and to characterise changes in breast cancer and colon cancer, with a focus on those genes, and gene products that may play a role in metastasis, including a family of ECPs, the matrix metalloproteinases (MMPs). In our laboratory, we have applied methods in microdissection, differential display polymerase chain reaction amplification (DD-PCR), and array hybridisation analysis to identify gene expression patterns in late stage archival formalin fixed paraffin embedded (FFPE) breast tumour biopsies that may be indicative of the EMT; or the response to the surrounding stroma/interstitium to the presence of the tumour.' The quality of nucleic acid obtainable from FFPE material presents a considerable challenge for gene expression studies. In order to identify tissue specific gene expression patterns, DD-PCR products, amplified from message obtained following segregation of tumour tissue from surrounding stroma, was hybridised to arrayed cDNA libraries created from stromal tumours, or sarcomas. In this way, 21 known genes, or expressed sequence tags (ESTs), were identified. These included the cytoskeletal element and EMT marker, vimentin, the mammary developmental factor and, signal transducer and activator of transcription (STAT)-3, and the cargo selection protein (TIP47). Seventeen genes showed differential expression in either the tumour, or stromal fractions. When applied to transformed breast cancer cell lines (MDA-MB-435 & T47D) DD-array analysis revealed a further 17 genes that were differentially regulated in invasive cells, compared with those displaying a less invasive phenotype. Six of the ESTs identified by DD-PCR array analysis, had no known (or predicted) function. For example, bcaf-2 was identified as the 3'-end of a putative open reading frame (ORF) localised to chromosome 6, while bcaf-10 showed homology with a known ORF. In order to analyse the expression of these bcafs further, a stromal cell culture model, representative of the original osteosarcoma cDNA libraries from which they were obtained, was used. In this model, CD14' (or adherent) peripheral blood mononuclear cells (PBMCs) treated with macrophage colony stimulating factor (M-CSF), can be allowed to differentiate into macrophage-like (ML) cells; while cells treated with M-CSF, and the receptor activator of NF-KB ligand (RANKL) will differentiate into multinucleate osteoclast-like (OCL) multinucleate giant cells. Uniquely, the stromal EST, bcaf-2 was expressed only by RANKL-treated (or OCL) cells. bcaf-2 and other ESTs, identified by DD-PCR analysis (and recently published) are the subject of on going research in our laboratory. The role of RANKL in mammary gland development and bone metastasis suggested that the identification of a RANKL-regulated stromal factor in breast tissue (bcaf-2) was not an artefact. RANKL is a membrane-bound, member of the tumour necrosis factor (TNF)-a cytokine super family. In order to test the hypothesis that RANKL might act as an inflammatory cytokine to regulate clinically significant stromal gene expression in the breast, we employed quantitative real time PCR analysis to examine the relative levels of selected members of a group of metal dependent ECPs, the matrix metalloproteinases (MMPs). RNA was extracted from ML cells and OCL cells, as well as RANK positive breast cancer cell lines (T47D, MDA-MB-435 & MCF-7). When the relative levels of protease mRNA were compared we demonstrated a significant (>20- fold) specific increase in collagenase (collagenase 2lMMP-8 and collagenase 3lMMP-13), and the tissue inhibitor of MMP (TIMP)-2 expression in M-CSF and RANKL treated PBMCs cells. When the assay was applied to RANKL treated breast cancer cell lines (MCF-7, T47D & MDAMB- 231), minor (40-fold) but potentially significant alterations in stromal protease gene expression were observed. The changes observed did not however, support the hypothesis that RANKL might act as an inflammatory cytokine to induce significant alterations in ECP expression in breast cancer cells. To investigate the role of RANKL as a driver of EMT in aberrant breast epithelium, total message (mRNNcDNA) from T47D, MCF-7, MDA-MB-231 cells, and message from the same cell lines treated with RANKL were compared by comparative fluorescent cDNA microarray analysis. Of the 1,700 targets available on the arrays, this study identified 160 that were differentially expressed in RANKL treated cells. The results suggest that RANKL may promote rather than suppress a mammary epithelial phenotype in breast cancer. In fact a putative mesenchymal to epithelial transition (MET) was observed following microscopic analysis, and this finding is the subject of on going research in our laboratory. Sporadic structural alterations in certain mitogenic factors represent important early events in cancer progression, while inherited mutations govern familial susceptibility to disease. In colon cancer, a close link exists between Winglessllnt (WNT) signalling, disease pathology, and the expression of MMPs. To examine the relationship between protease expression and structural genetic alterations in this EMT-linked signalling pathway, and others, we applied combined QPCR analysis of MMP expression and PCR-Single Strand Conformation Analysis (SSCA) to 26 colonic tumours, and patient-matched normal colonic mucosa. In this study, significant correlations between the expression of ECPs, and a key mediator of WNT signalling (p-catenin) were identified. While tumours possessing specific functional mutations in K-Ras, were found to group with phenotypic clustering based on protease gene expression. This result may be due to an interruption of normal interactions between RasIRaf signalling and transforming growth factor (TGF) P signalling, via Sma- and Mad- related protein (SMAD) signalling. These results demonstrate that the already identified link between mutations in kinase signalling, and aspects of gross colon tumour morphology (such as dysplasia) may be due to aberrant MMP expression patterning. The final aim of this research was to utilise methods developed in microdissection and specific Q-PCR analysis, to identify whether tumour-stroma differences in MMP gene expression might be used as markers of disease pathology. Total RNA from tumour, and biopsy-matched adjacent stromal tissue were segregated from 35 FFPE archival breast tumour biopsies. Comparison with stroma identified specific associations between TIMP-2 expression in the stroma and lymph node involvement, as well as stromelysin-3 (MMP-I I ) and TIMP-I expression and calcification of the tumour. Furthermore, a significant correlation was identified in the pattern of gelatinase (gelatinase AIMMP-2 & gelatinaseB1MMP-9) expression; while no significant correlation was identified in tumour-stroma MMP gene expression differences, and tumour grade, or hormone receptor status. These results suggest that coordinated changes within the tumour, and proximal stromal tissues (rather than tissue specific changes per se), regulate pathologically significant changes in breast carcinogenesis. In conclusion, this thesis describes the use of novel techniques in specific and global gene expression analysis that permitted examination of stromal gene expression changes in epithelial tumour progression. Microdissection facilitated localisation of expression to particular tissues, while cell culture models provided material with which to optimise and demonstrate the efficacy of techniques used (where tumour material itself was not abundant). Furthermore, we have identified significant and specific correlations between general stromal protease gene expression changes, a putative mammary epithelial differentiation factor (RANKL), alterations in growth factor signalling, and epithelial tumour pathology in the breast and colon. The combination of techniques developed in this study may assist in improvement of categorisation of tumours in clinical pathology. Specifically, the development of novel grading systems that link underlying molecular genetic changes with changes in tumour pathology. These processes may assist to improve diagnosis and provide more effective patient/tumour-specific drug therapies.

Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used. This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible. The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies. / QC 20101006