Colorectal cancer is the third most common cancer and the leading cause of cancer deaths in Western industrialised countries. Despite recent advances in the screening, diagnosis, and treatment of colorectal cancer, an estimated 608,000 people die every year due to colon cancer. Our current knowledge of colorectal carcinogenesis indicates a multifactorial and multi-step process that involves various genetic alterations and several biological pathways. The identification of molecular markers with early diagnostic and precise clinical outcome in colon cancer is a challenging task because of tumour heterogeneity. This Ph.D.-thesis presents the molecular and cellular mechanisms leading to colorectal cancer. A systematical review of the literature is conducted on Microarray Gene expression profiling, gene ontology enrichment analysis, microRNA and system Biology and various bioinformatics tools. We aimed this study to stratify a colon tumour into molecular distinct subtypes, identification of novel diagnostic targets and prediction of reliable prognostic signatures for clinical practice using microarray expression datasets. We performed an integrated analysis of gene expression data based on genetic, epigenetic and extensive clinical information using unsupervised learning, correlation and functional network analysis. As results, we identified 267-gene and 124-gene signatures that can distinguish normal, primary and metastatic tissues, and also involved in important regulatory functions such as immune-response, lipid metabolism and peroxisome proliferator-activated receptors (PPARs) signalling pathways. For the first time, we also identify miRNAs that can differentiate between primary colon from metastatic and a prognostic signature of grade and stage levels, which can be a major contributor to complex transcriptional phenotypes in a colon tumour.

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<p>The brain serotonergic and dopaminergic systems broadly influence our internal experience and the ways in which we interact with the outside environment, with crucial regulatory roles in mood, sleep, appetite and the control of voluntary movement. Serotonin and dopamine neurons are themselves influenced by a wide variety of internal and external factors, many of which remain poorly understood. The central aim of this thesis was to better characterize several of these modulatory influences via exploratory investigations involving pharmaceutical agents or environmental modification. Specifically, I examined the modulatory effects of selective serotonin reuptake inhibitors (SSRIs), atypical neuroleptics and environmental enrichment with exercise on the regulation of brain serotonin and dopamine neurons.</p> <p>This thesis documents, for the first time, that (1) inhibition of the serotonin transporter (SERT) by SSRIs induces a rapid and region-selective reduction of tryptophan hydroxylase (TPH)-immunoreactive neurons in serotonergic brainstem nuclei that persists over a prolonged treatment course; that (2) selective blockade of SERT by SSRIs can rapidly induce a reduction of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra (SN) and the ventral tegmental area (VTA) that, again, persists over a lengthy treatment course; that (3) environmental enrichment with exercise can potentiate the effect of SERT inhibition on SN dopaminergic neurons, but not the dorsal raphe nucleus (DRN) serotonergic neurons; that (4) that SSRI fluoxetine triggers a significant upregulation of microglia in the SN; that (5) environmental enrichment with exercise can reduce TPH immunoreactivity in the DRN and TH immunoreactivity in the SN and VTA, even in the absence of any pharmacological intervention, and finally, that (6) the atypical neuroleptic risperidone significantly reduces TPH in the DRN of both young and aged animals and reduces DRN Nissl counts in aged animals. Taken together, the body of work included in this thesis suggests that SSRIs, atypical neuroleptics and environmental enrichment with exercise can have profound effects on brain serotonergic and dopaminergic neurons, possibly accounting for some of the side effects and therapeutic benefits associated with these interventions.</p> / Doctor of Philosophy (PhD)

<p dir="ltr">To meet the growing demands for pork products, lactating sows are often housed in farrowing crates to reduce piglet crushing. However, the public has raised welfare concerns about farrowing crate systems due to the confinement and barren environment, which can impair social interactions between sow and piglets, change their activity levels, and alter how sows and piglets satisfy their motivated behaviors to chew and explore by oral manipulation of pen and pen mates. Barren and confined environments can result in skin abrasions caused by oral manipulations and fighting, physiological stress, inactivity, and abnormal behaviors, which can have an impact on sows’ and piglets’ welfare, productivity, and behavior. Previous work on environmental enrichments has shown promise to improve average daily gain, activity levels, stress, and behavioral repertoire. However, the majority of studies on pig environmental enrichments take place after weaning and research investigating the impact enrichments have during lactation is limited. In addition, research on maximizing the use of environmental enrichments based on the location inside the farrowing crate systems has not been conducted. To address the knowledge gaps concerning the impact of the location of environmental enrichments on the welfare, behavior, and productivity of sows and piglets in farrowing crate systems and to provide educational material on pig enrichment, this dissertation consists of two parts: Chapters 2 and 3 examine the effects of the location of environmental enrichments in farrowing crate systems on sows’ and piglet’s welfare, productivity, and behavior. Chapter 5 is an extension article on the different types of environmental enrichments and the best strategies for implementing enrichment in swine operations.</p><p dir="ltr">In Chapter 2, sows (n = 37) and focal piglets (n = 148) were assigned to three treatment groups: SPE (both sows and piglets had access to enrichment objects), PE (only piglets had access to enrichment objects), and CON (control group with no enrichment) blocked by sow parity and genetics. Sow posture and piglet behavior during the lactation and nursery phases were observed at various times after birth and weaning. Environmental enrichments significantly influenced the behaviors of suckling piglets, reducing pig-directed and agonistic behaviors. Piglets with enrichments tended to explore the pen less and engage in more social behaviors. The location of enrichments also impacted behaviors, with higher nursing behavior observed during mid-lactation for piglets with access to enrichments (PE) and increased interaction with enrichments when they were accessible to both sows and piglets (SPE). Treatment did not affect sow postural changes or most nursery behaviors, except for walking, which increased in SPE nursery piglets compared to CON piglets. Overall, the study demonstrated positive effects of environmental enrichments on suckling piglets in farrowing crate systems, highlighting the importance of enrichment placement on nursing behaviors and enrichment interaction.</p><p dir="ltr">In Chapter 3, the same sows and piglets were used to investigate the effects of enrichment location on the welfare (skin lesions, pressure sores, salivary cortisol, and tear stains) and performance (average daily gain and piglet crushing) of the sows and piglets. This study used the same animals that were assigned the treatment group, housing, and management practices from Chapter 2. Salivary cortisol samples were collected from sows at four time points: 24 hours after moving into farrowing crates, 24 hours after treatment group assignment, midway between moving into crates and weaning, and on the day of weaning. Pressure sores of sows were scored on days 1, 4, 7, 10, 14, and 17 after farrowing. For suckling piglets, tear stains and skin lesions were assessed on the same days, and their average daily gain (ADG) was analyzed weekly during lactation. In the nursery phase, salivary cortisol was collected from piglets on the day of weaning and days 1, 7, and 14 post-weaning, with tear stains and skin lesions measured on those days as well. Nursery ADG was analyzed from weaning to day 14 post-weaning. Results indicated that control (CON) sows were less likely to have no pressure sores compared to sows with shared (SPE) enrichments, while piglets in the enriched treatment groups (PE and SPE) had smaller tear stain areas than those in the CON group. Treatment influenced skin lesions in suckling piglets, with enriched piglets having fewer lesions in the ear and front body regions. There was no treatment effect on salivary cortisol for both sows and nursery piglets, nor did treatment affect ADG, piglet crushing, or nursery skin lesions. The provision of environmental enrichments in farrowing crates reduced pressure sores of sows, skin lesions of suckling piglets and tear stains of suckling and nursery piglets.</p><p dir="ltr">Overall, providing environmental enrichments to suckling piglets reduced aggression, as evidenced by fewer agonistic and pig-directed behaviors, and resulted in fewer skin lesions compared to piglets without enrichments. This suggests potentially reduced stress levels in enriched piglets, indicated by smaller tear stain areas. While most behaviors and skin lesion scores showed no significant differences during the nursery phase, enriched piglets continued to have smaller tear stains. Enrichment location influenced the following: sows had fewer pressure sores, and suckling piglets interacted more with enrichments and exhibited fewer pig-directed behaviors when enrichments were accessible to both sows and piglets compared to the only piglet enriched treatment group. Piglets with access only to enrichments also performed more nursing behaviors during mid-lactation compared to the control group. Overall, the study highlights the benefits of environmental enrichments in farrowing crate systems, particularly the positive impact of enrichment location on the welfare and behavior of sows and piglets.</p><p dir="ltr">Lastly, in Chapter 5, the extension article discusses the definition of environmental enrichment and its impact on pigs’ welfare. The article also delves into the five types of environmental enrichments (nutritional, occupational, physical, sensory, and social), providing examples of each. Additionally, the article offers five practical tips for efficiently and successfully implementing environmental enrichments in swine herds.</p>

The breast cancer susceptibility protein, BRCA1, is a tumor suppressor that helps maintain genomic integrity. Changes in BRCA1 that effect DNA repair processes can fuel cancer induction. The Kelly lab, at the Virginia Tech Carilion Research Institute, has recently developed a new methodology that employs silicon nitride (SiN) microchips to isolate BRCA1 assemblies from the nuclear material of breast cancer cells. These microchips are coated with adaptor proteins that include antibodies against target proteins of interest. The adaptor proteins are added in sequential steps to the coated microchips, followed by an aliquot of sample containing the protein of interest, such as BRCA1. The Kelly lab, partnered with Protochips Inc., developed these devices as a robust, tunable platform to monitor molecular processes, and refer to them as 'Cryo-SiN' in cryo-Electron Microscopy (EM) imaging. We are currently using Cryo-SiN to recruit BRCA1 protein assemblies to the microchip surface under mild conditions, while simultaneously preparing them for cryogenic preservation and EM imaging. This strategy presents a viable alternative to antibody affinity columns that require stringent elution steps to obtain protein complexes from the column. Another advantage of the microchip strategy is that it requires only a 30-minute nuclear extraction, a 60-minute enrichment procedure, and a 5-minute microchip capture step--a total of 95 minutes from initially lysing the cells to plunge-freezing the EM specimens. Therefore, these novel approaches represent a major departure from classical separation procedures that often require days to complete, during which time active protein assemblies can readily dissociate or become inactive. Overall, our use of BRCA1-specific microchips may reveal changes in the BRCA1 architecture during various stages of cancer progression--a major gap in knowledge that persists in cancer research. / M. S. / Modern advances in the imaging technology used for cryogenic electron microscopy (cryo-EM) have offered researchers an extraordinary view into the world of biology at the nanoscale. Supplemental to these technical innovations is the development of tunable substrates based on functional new materials that revolutionize the sequestering of biological components from human cells, such as protein complexes formed in breast cancer cells. New developments of novel viewing substrates, given traditional electron microscopy viewing grids have remained unchanged for decades, is the logical next step into the future of enhanced cryo-EM imaging. Tunable microchip substrates, made using recently enhanced micro-engineering techniques, are currently under development for use in cryo-EM imaging. In this work I have examined these microchip substrates for their capacity to streamline the isolation of biomolecules such as the protein most prominently cited in breast cancer, known as the breast cancer susceptibility protein (BRCA1). Utilizing these novel microchip substrates in the Kelly Lab, I have collected and analyzed data containing BRCA1 proteins, formed in human breast cancer cells, toward the development of 3-dimensional protein structures that allow us to peer into the structure-function relationships of these proteins. New and exciting Cryo-EM data, collected using these newly developed microchips, has the potential to reveal obscure disease mechanisms being propagated at the molecular level in modern clinical practice, such as breast cancer.

Doctor of Philosophy / Department of Psychology / Stephen W. Kiefer / Exposing rats to differential rearing conditions, during early post-weaning development, has been shown to produce changes in a number of behaviors displayed during adulthood. The purpose of the current study was to investigate whether rearing alcohol-preferring (P) and non-preferring (NP) rats in an environmental enrichment condition (EC), a social condition (SC), or an impoverished condition (IC) would differentially affect the consumption of and operant responding for 10% ethanol. In Experiment 1 rats were tested for both limited access and free access (two bottle choice between water and ethanol) consumption of 10% ethanol. For, Experiment 2 rats were trained to respond in an operant chamber for ethanol and then provided concurrent access to 10% ethanol (right lever) and water (left lever). After concurrent access, rats were required to respond over a gradually increasing fixed-ratio schedule for 10% ethanol and finally a progressive ratio schedule for 10% ethanol, 15% ethanol, and 10% sucrose. For Experiment 3 rats were trained to respond for 10% sucrose and then assessed for the maintenance of operant responding for 10% sucrose. The data from this series of experiments shows that EC P rats consumed, responded for, and preferred 10% ethanol significantly less than their IC P counterparts. Also, EC P rats did not significantly differ from NP rats during any aspect of testing for all experiments. Experiment 3 failed to reveal a significant effect of rearing although there was a line effect that has been previously observed in the literature. Thus, it would appear from these results that rearing in an EC condition acts to protect alcohol-preferring rats from increased levels of consumption of, preference for, and responding for ethanol compared to rearing in an impoverished environment.

Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity. This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.

Thesis (LLD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This dissertation examines the approach to statutory formalities in South African law. It focuses primarily on formal requirements which result in nullity in the event of non-compliance, and in particular, on those prescribed for alienations of land (section 2(1) of the Alienation of Land Act 68 of 1981) and suretyships (section 6 of the General Law Amendment Act 50 of 1956). To provide context, the study commences with a general historical overview of the development of formal requirements. It also considers the advantages and disadvantages of formalities. The conclusion is reached that an awareness of both is required if a court is to succeed in dealing with the challenges posed by statutory formalities. The dissertation then considers more specific aspects of the topic of formal requirements, including the difference between material and non-material terms. It also reveals that the current interpretation of statutory formalities is quite flexible and tends towards a conclusion of validity if reasonably possible. However, cases involving unnamed or undisclosed principals present particular challenges in this context, and the possibility of greater consistency, without the loss of theoretical soundness, is investigated. A discussion of what should be in writing, and with what exactitude, necessarily involves a consideration of the extent to which extrinsic evidence is admissible. The interaction between formal requirements and the parol evidence rule is therefore investigated. Special attention is paid to incorporation by reference. After an examination of the common-law approach to this topic, the conclusion is reached that room exists for developing this area of South African law, especially where a sufficient reference to another document is concerned. Rectification also enjoys detailed examination, due to the unique approach adopted in South African law. Where formalities are constitutive, a South African court first satisfies itself that a recordal complies with these requirements ex facie the document, before it will consider whether rectification may be appropriate. An analysis of both civilian and common-law judgments suggests that the South African approach is based on a misconception of the purpose of rectification. This leads to the further conclusion that the requirement of ex facie compliance should be abolished as a separate step and that a court should rather consider whether awarding a claim for rectification would defeat the objects of formalities in general. Finally, the remedies available to a party who performs in terms of an agreement void for formal non-compliance and the effect of full performance in terms of such an agreement, receive attention. An investigation of the remedies available in other legal systems reveals that the South African approach of limiting a party to an enrichment claim is unnecessarily restrictive. It is argued that local courts should reconsider their exclusion of estoppel in this context, particularly in cases where one party’s unconscionable conduct has led the other to rely on the formally defective agreement. In cases of full performance, no remedies are available, but it is argued that a distinction should be drawn between reciprocal and unilateral performances. / AFRIKAANSE OPSOMMING: Hierdie proefskrif ondersoek die benadering tot statutêre formaliteite in die Suid-Afrikaanse reg. Dit fokus hoofsaaklik op die formele vereistes wat lei tot nietigheid in die geval van nie-nakoming, en in die besonder dié wat voorgeskryf word vir die vervreemding van grond (artikel 2 (1) van die Wet op Vervreemding van Grond 68 van 1981) en borgstellings (artikel 6 van die Algemene Regswysigingswet 50 van 1956). Ten einde die nodige konteks te verskaf, begin die studie met ‘n algemene historiese oorsig van die ontwikkeling van formaliteite. Dit oorweeg ook die voor- en nadele van formaliteite. Die gevolgtrekking is dat ‘n bewustheid van beide vereis word indien ‘n hof die uitdagings wat deur statutêre formaliteite gestel word, suksesvol wil hanteer. Die proefskrif oorweeg dan meer spesifieke aspekte van formaliteite, insluitende die verskil tussen wesenlike en nie-wesenlike bedinge. Dit toon ook dat die huidige opvatting van statutêre formaliteite redelik buigsaam is en tot ‘n bevinding van geldigheid lei waar dit redelikerwys moontlik is. Gevalle van onbenoemde of versweë prinsipale bied egter besondere uitdagings in hierdie verband en die moontlikheid word ondersoek om ‘n meer konsekwente, maar tegelyk teoreties-gefundeerde benadering te volg. ‘n Bespreking van wat op skrif moet wees, en met watter mate van sekerheid, behels noodwendig ‘n oorweging van die mate waarin ekstrinsieke getuienis toelaatbaar is. Die interaksie tussen formaliteite en die parol evidence-reël word derhalwe ondersoek. Spesiale aandag word bestee aan inlywing deur verwysing. Na oorweging van die benadering in gemeenregtelike stelsels, word die gevolgtrekking bereik dat ruimte bestaan vir ontwikkeling op hierdie gebied, veral met betrekking tot ‘n voldoende verwysing na ‘n ander dokument. Rektifikasie word ook breedvoerig hanteer, vanweë die eiesoortige benadering in die Suid-Afrikaanse reg. Waar formaliteite konstitutief van aard is, sal ‘n Suid-Afrikaanse hof eers vasstel dat ‘n ooreenkoms ex facie die dokument aan die formaliteite voldoen, voordat dit sal oorweeg of rektifikasie moontlik is. ‘n Ontleding van sivielregtelike en gemeenregtelike beslissings dui daarop dat die Suid-Afrikaanse benadering op ‘n wanbegrip van die doel van rektifikasie gebaseer is. Dit lei tot die verdere gevolgtrekking dat die vereiste van ex facie nakoming as ‘n afsonderlike stap afgeskaf behoort te word en dat ‘n hof eerder moet oorweeg of die toestaan van ‘n eis vir rektifikasie die oogmerke van die formaliteite in die algemeen sou verydel. Laastens word aandag geskenk aan die remedies beskikbaar aan ‘n party wat presteer ingevolge ‘n ooreenkoms wat nietig is weens nie-nakoming van formaliteite, asook die effek van volle prestasie kragtens so ‘n ooreenkoms. In eersgenoemde geval beperk die Suid-Afrikaanse reg daardie party tot ‘n verrykingseis. ‘n Ondersoek van die remedies beskikbaar in ander regstelsels toon dat dit onnodig beperkend is. Dit word aangevoer dat Suid-Afrikaanse howe die uitsluiting van estoppel in hierdie konteks moet heroorweeg, veral in gevalle waar een party se gewetenlose optrede daartoe lei dat die ander party staat maak op die formeel-gebrekkige ooreenkoms. In gevalle van volledige prestasie is daar geen remedies beskikbaar nie, maar dit word aangevoer dat ‘n onderskeid getref moet word tussen wedersydse en eensydige prestasies.

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