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The prenatal environment has been shown to have lasting effects on cardiovascular health. In the present study, pregnant rats were fed a 0.7% NaCl normal salt (NS) diet or an 8% NaCl high salt (HS) diet throughout pregnancy. Adult offspring were fitted with radiotelemetry probes to continuously measure blood pressure and heart rate. Rats were placed in restraining cages to test for a programmed acute stress hyperresponsiveness. Offspring were challenged with HS diet for one week to determine if blood pressure salt sensitivity had been programmed by the prenatal HS diet. Animals were killed following resting and acute stress conditions, after which brains and blood were collected for in situ hybridization for corticotropin releasing hormone (CRH) and radioimmunoassay for corticosterone. In order to determine the contribution of gene expression to differences seen, total brain RNA was analyzed with microarray. Rats were injected with a ganglionic blocker and an adrenergic receptor antagonist before restraint to examine the autonomic component of the stress response. High salt offspring of either sex did not have basal hypertension. Female HS offspring had an increased pressor and tachycardic response to acute stress compared with NS females. There were no differences between male NS and HS offspring during acute stress. Salt sensitivity was not induced during high salt challenge. According to the microarray, 11 genes were upregulated and 10 were downregulated in adult brains, while 17 were upregulated and 17 were downregulated in pup brains. These data indicate that there are long-term changes due to HS diet. CRH levels were higher in the paraventricular nucleus (PVN) of female HS offspring than in female NS offspring during both basal and stressed conditions, though no differences were seen in CRH expression levels of male offspring. Autonomic blockade completely abolished the enhanced tachycardic response seen in female HS offspring. However, a difference in NS and HS blood pressures remained. Thus, female offspring of mothers fed an 8% NaCl diet have alterations in cardiovascular control, indicated by an enhanced tachycardic response as adults, due to changes in the autonomic nervous system, and enhanced pressor response to stress mediated by unknown mechanisms.

Quinoa (Chenopodium quinoa Willd.) is an important food crop for subsistence farmers in the Altiplano (high plains) of Peru, Bolivia, and Argentina. Saponins are part of a diverse family of secondary metabolites that are found in high concentrations in the pericarp of many varieties of quinoa. Due to their bitter taste and anti-nutritive properties, saponins must be removed before the quinoa grain is consumed. There are ‘sweet’ varieties of quinoa that have significantly reduced levels of saponin. Previous research suggests saponin production is controlled by a single locus. The major objective of this research was to elucidate the genetic components in the saponin biosynthesis pathway. Thus, we report the development and annotation of the first large scale expressed sequence tag (EST) collection for quinoa based on Sanger and 454 pyrosequencing of maturing seed tissue expressing saponins. Sanger sequencing produced 18,325 reads with an average read length of 693 nucleotides, while 454 GS-FLX pyrosequencing generated 295,048 reads with an average read length of 202 nucleotides. A hybrid assembly of all sequences generated 39,366 unigenes, consisting of 16,728 contigs and 22,638 singletons. Repeat sequence analysis of the unigene set identified 291 new microsatellite markers. From the unigene set, a custom microarray was developed and used to assay transcriptional changes in developing seeds of saponin-containing and saponin-free quinoa lines. The microarray consisted of 102,834 oligonucleotide probes representing 37,716 sequences of the unigenes set. Three different statistical comparisons, based on comparisons of ‘sweet’ vs. ‘bitter’ seed tissue at two developmental stages, were assayed on the custom array. Using a p-value cutoff threshold of 0.01, we identified a list of 198 significantly differentially expressed candidate genes common to all three comparisons. We also identified a list of candidate genes (p-value ≤ 0.05) that are known to be associated with identified triterpenoid (saponin) biosynthetic pathways that were differentially expressed in all three comparisons. Included in this list are candidate genes that share homology to cytochrome P450s (20), cytochrome P450 monooxygenases (10), and glycosyltransferases (49) suggesting that transcriptional differences in the saponin biosynthesis pathway possibly responsible for the absence or presence of saponin in quinoa are determined after the formation of the β-amyrin skeleton. These candidate genes are suggested for use in future studies in the production of saponin in quinoa.

Microarray technology provides powerful tools in the field of biomedicalresearch because it can measure molecular interactions in a highly parallelfashion. It has uses in protein, DNA or cell research, in both discovery anddiagnostic applications. Microfluidics, on the other hand, provides thenecessary tools to rapidly transport and mix small volumes of sample to amicro-sensor area. Bridging these two technologies has the potential todevelop a miniaturized, automated and ease-of-use toolbox for biologicalanalysis. However, the integration of microfluidics with microarrays is notstraightforward, as if a robust and leak-tight seal between the microarray andthe microfluidic channels. Current sealing methods are either impractical,such as mechanical clamping, or not compatible with proteins, such as heat orplasma bonding or gluing. Moreover the former methods create a permanentseal that, once applied prevents the microfluidic structure to be removed later.This work focuses on developing a microfluidic add-ons ("Biosticker") that canbe robustly sealed with protein microarrays with maintained biologicalactivity, but at the same time easily removed to allow for multiple stickersapplied in a sequence or scanning of the microarray in a standard reader. Thefeatures of the novel Biostickers are made possible by the use ofOff-stoichiometry thiol-ene-epoxy (OSTEmer) polymers. In this thesis, wedesign and fabricate Biostickers for rapid integration with pre-spottedmicroarrays and experimentally verify how these micropatterned Biostickerscan be used to significantly facilitate multiplexed assays, by avoiding the useof beads. / Microarray-tekniken är ett kraftfullt verktyg inom biomedicinsk forskningeftersom den kan mäta miljontals molekylära interaktioner parallellt. Den haranvändningsområden i protein-, DNA- eller cellforskning, både i forskningoch diagnostik. Mikrofluidik, å andra sidan, ger de nödvändiga verktygen föratt snabbt transportera och blanda små provvolymer till en sensoryta. Genomatt kombinera dessa två teknologier finns potential att utveckla enminiatyriserad, automatiserad och lättanvänd verktygslåda för biologiskanalys. Emellertid är integrationen av mikrofluidik med mikroarrayer inteenkel, då ytorna är känsliga, kanalerna mycket små men tätningen måste varaperfekt. De vanligast förekommande förseglingsmetoder är antingenopraktiska, som mekaniskt tryck eller så är de inte kompatibla med proteiner,som t.ex. värme- eller plasmabondning. Dessutom syftar de flestaförseglingsmetoder mot att skapa en permanent försegling som vidanvändning förhindrar mikrofluidikstrukturer från att tas bort i ett senareskede, tex. vid avläsning i en skanner. Detta arbete fokuserar på att utvecklamikrostrukturerade plastartiklar ("Biosticker") innehållande kanaler ochkaviteter. Dessa Biosticker kan på ett robust och läckfritt sätt kansammanfogas med proteinmikroarrayer utan att påverka den biologiskaaktivitetet men samtidigt kunna avlägsnas för att tillåta flera Biostickersapplicerade i en sekvens eller scanning i en mikroarrayläsare. Dessafunktioner möjliggörs genom av så kallade icke-stökiometrska-tiol-ene-epoxipolymerer (OSTEmer) används som material. I den här avhandlingenutvecklas och tillverkas Biostickers för snabb integrering medproteinmikroarrayer. Det verifieras även experimentellt hur dessa Biostickerskan användas för att underlätta genomförandet av sk. multiplexadeprotinanalyser.

Immunohistokemi (IHK) är en metod som används för att undersöka närvaron av specifika biomarkörer i vävnad. Metoden utnyttjar antikroppars affinitet till antigen och dess närvaro visualiseras. Caldesmon är ett protein associerat med cytoskelettet som förekommer i glatta muskelceller och i icke-muskelceller. Det används inom IHK som en glatt muskelmarkör men även som en markör för myoepiteliet. Syftet med studien var att utveckla ett nytt protokoll för caldesmon och att jämföra den med SMMS-1, även den en markör för glatt muskulatur och myoepiteliet. Jämförelsen utfördes på fibroadenom vävnad, en bröstcancerform som utvecklats ur epitelialvävnad och stroma, då den innehåller mycket myoepitelial celler. För att effektivisera processen användes tissue microarray tekniken, där vävnad från flera olika preparat (klotsar) stansades ut och sammanfördes till en ny klots. Instrumentet Ventana Benchmark Ultra användes för att utveckla ett nytt protokoll för caldesmon. Det nya protokollet användes sedan vid jämförelsen med SMMS-1. Resultatet visade att SMMS-1 gav en tydligare infärgning av det myoepiteliala lagret då det enbart färgade in det, medan caldesmon även färgade in stroma. Då fibroadenom vävnad innehöll mycket stroma var det svårare att urskilja det myoepiteliala lagret från dess omgivning med caldesmon infärgningen. / Immunohistochemistry (IHC), is a method used for examining the presence of specific biomarkers in tissue. It uses antibodies affinity for antigens and their presence can then be visualized. Caldesmon is a protein that is associated with the cytoskeleton in smooth muscle and non-smooth muscle cells. It is used within IHC as a marker for smooth muscle but also as a marker for the myoepithelium. The intent of this study was to develop a new protocol for caldesmon and to compare it with SMMS-1, also a marker for smooth muscle and the myoepithelium. The comparison was performed on tissue from fibroadenoma, a type of breast cancer that evolved from epithelial tissue and stroma, due to its high content of myoepithelial cells. For optimal effectiveness, the tissue microarray technique was used, where tissue from several blocks are extracted and brought together into a single new block. A Ventana Benchmark Ultra was used to develop a new protocol for the new antibody, caldesmon. The new protocol was used in the comparison with SMMS-1 on fibroadenoma tissue. The results were that SMMS-1 had a clearer and sharper staining of the myoepithelial layer, due to the fact that it only stained the layer. While caldesmon also stained the stroma in the tissue. Because fibroadenom tissue contained so much stroma the staining done with caldesmon made it harder to separate the myoepithelial layer from its surroundings.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders. / Saudi government

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