På huden lever en sammansättning av olika mikroorganismer såsom bakterier, svampar och virus. Dessa mikroorganismer kallas hudens mikrobiom. Sammansättningen av en individs mikrobiom kan ge mycket information om en individens hälsa. För att undersöka sammansättningen av bakterier på hudytan med exempelvis qPCR, behöver bakterier samlas in och DNA extraheras. Bakteriekoncentrationen på hudens torrare områden som exempelvis armar har normalt en relativt låg bakteriekoncentration vid 102-104 bakterier per cm2. Huden koloniseras till stor del av grampositiva bakterier. Grampositiva bakterier är i regel svårare att lysera än andra bakterier och kräver därför hårdare lysering. En bra extraktionsmetod ska erhålla mycket DNA utan att påverka dess kvalité. I detta arbete utvärderades initialt fem olika extraktionsmetoder på bakteriesuspension med Staphylococcus aureus (S. aureus), både direkt på bakteriesuspension men också från svabb. Utvärdering gjordes på PureLink Microbiome DNA Purification Kit, QlAamp PowerFecal Pro, QlAamp DNA Mini Kit och KOH-EDTA. Metoden med QlAamp DNA Mini Kit testades med två olika protokoll och räknades som två separata metoder. Metoderna som gav bäst resultat vid initial utvärdering var PureLink Microbiome och KOH-EDTA. Därefter utvärderades dessa två metoderna på prov insamlat med svabb från huden på 10 frivilliga deltagare.

Hudens mikrobiom utgör ett skydd mot patogener och domineras framförallt av grampositiva bakterier som Staphylococcus, där S. epidermidis är den mest förekommande arten. På huden är S. epidermidis en kommensal art och har en aktiv roll i att begränsa tillväxten och koloniseringen av patogener som S. aureus. S. epidermidis är en viktig art i att bibehålla stabiliteten av hudens mikrobiom och en reduktion av S. epidermidis innebär ökade risker för dysbios. S. aureus är en vanlig orsak till hudinfektioner och har upptäckts finnas i en hög utsträckning på patienter med atopisk dermatit samt hos diabetiker med risk för att utveckla fotsår. Syftet med denna studie var att optimera qPCR och undersöka om metoden kunde användas för att uppskatta förhållandet mellan S. aureus och S. epidermidis, vilket skulle kunna identifiera patienter med risk för hudsjukdomar. Tre primerpar undersöktes. Två var specifika mot S. aureus och en var specifik mot S. epidermidis. Flera tester genomfördes för att optimera qPCR och specificiteten av primerparen. Den annealingtemperatur och primerkoncentration som gav mest adekvata resultat användes vid den relativa kvantifieringen. Den utvecklade metoden kunde uppskatta förhållandet mellan S. aureus och S. epidermidis vid de lägre koncentrationerna (80–280 kopior/μl) bättre än vid de högre koncentrationerna (80 000–280 000 kopior/μl), men vidare optimering av metoden behövs.

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Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent cause of bacterial sexually transmitted disease. Genital chlamydial infections, marked by chronic, intense inflammation, can lead to genital tissue scarring and infertility and is a contributing factor to development of pelvic inflammatory disease and ectopic pregnancy. Iron is required as a cofactor for numerous highly conserved pathways, and nearly all studied organisms rely on iron for growth. In response to iron restriction, the chlamydial developmental cycle arrests at the intracellular reticulate body stage, resulting in a phenomenon termed persistence. Persistence likely plays a role in chlamydial pathogenesis through the expression of virulence factors and antigens in addition to sustaining chronic infection; however, little is known concerning how chlamydiae respond to iron limitation at the molecular level, and no systems for iron acquisition have been identified in Chlamydia. This dissertation presents an investigation into the chlamydial response to iron restriction. Chlamydial heat shock protein 60 (cHsp60) has been implicated in development of the more severe disease sequelae and has been found to increase in expression following iron restriction; however, three cHsp60 homologues were identified following the sequencing of the chlamydial genome. Here, iron restriction is shown to increase expression of cHsp60-2 but not the two other homologs, cHsp60-1 or -3. Next, in order to investigate an alternate model for restricting iron availability to chlamydiae, a cell line with inducible expression of recombinant ferroportin, a eukaryotic iron efflux protein, was examined. Lastly, 10 chlamydial proteins differentially expressed during growth in iron-restricted host cells were identified by proteomic analysis of radiolabeled proteins followed by mass spectrometry analysis; transcripts encoding 5 iron responsive proteins were examined across a timecourse of infection and revealed increased transcript levels at 18 and/or 24 hours post infection. Together, these studies have examined the molecular response of chlamydiae to reduced iron availability and have underlined the importance for pathways involved in protection against oxidative damage and adaptation to stress.

Cancer is known for its unregulated and mutagenic characteristics. The topic of targeting cancer by inhibiting the metabolic pathways it uses to thrive has been a focus of modern cancer research. Specifically, in lung cancer, the transformation from non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a focus. This transformation often comes with a grimmer prognosis and reduced survival rate. This is primarily due to SCLC being resistant to epidermal growth factor receptor (EGFR) inhibitors. This frontline treatment for EGFR mutant NSCLC has shown to be quite effective until transformation to SCLC occurs. To further study the metabolic factors responsible for this transformation, a metabolic screening was conducted on SCLC transformed lung tissues and tumor adjacent normal lung tissues. This analysis revealed that the amino acid L-arginine and intermediates in its biosynthetic pathway were severely dysregulated. While L-arginine supplementation has shown to inhibit the growth of breast and colorectal cancers, there is little literature about its effects on lung cancer. Using cell viability and gene expression screening tools, we have identified arginine metabolizing genes ARG2, GATM, and OAT as being upregulated in NSCLC treated with high concentrations of an EGFR inhibitor. These high treatments also correlate with increased expression of neuronal differentiation factor 1 (NEUROD1), which has been shown to drive tumorigenesis, metastasis, and SCLC transformation. These findings show a role for altering arginine metabolism to accomplish drug resistance through SCLC transformation. These findings will hopefully pave the way for later clinical use of arginine converting enzymes and NEUROD1 expression levels as predictive markers of early drug resistance and SCLC transformation.

The global aquaculture industry is expanding to support increased demand in global markets and supplement traditional fisheries. The rapid increase of aquaculture production relies on introducing and using species outside their native range, posing significant regional environmental and socio-economic risks. Non-native species are selected based on transferable large-scale production protocols, fast growth, and existing market demand. Aquaculture is an important sector within Saudi Arabia’s Vision 2030 (anticipated >400% production increase by 2030). Barramundi (Lates calcarifer) was introduced into the Red Sea for open sea-cage farming in 2008, with large-scale production beginning in 2014. Regionally, there are numerous anecdotal reports of escape events of this species, including an incident in 2015 involving the release of ~300,000 fish. Since then, local fishers have caught up to 15 wild individuals per month in Al Lith, central Saudi Arabia. This study presents the first biological information on L. calcarifer aquaculture escapees in the Saudi Arabian Red Sea. Wild-caught L. calcarifer (n=5) were collected from a local fish landing and measured up to 10 kg, eight years of age, were sexually mature, and consumed benthic and coral reef fishes. In contrast, individuals in aquaculture facilities are harvested at < 1 kg and < 400 mm. In their native range, L. calcarifer can reach up to 200 cm and 60 kg. To assist in detecting and monitoring escapees within the Red Sea, I designed and validated a species-specific SYBR-based environmental DNA quantitative PCR assay targeting a 16S mitochondrial region of L. calcarifer rRNA (Barramundi_16S assay). Preliminary results, using DNA metabarcoding and environmental (seawater) samples, detected L. calcarifer near active/historical aquaculture farms and north/south of areas where this species has not been reported previously in the Red Sea (10–250 km from aquaculture facilities). In the future, the Barramundi_16S assay can screen additional eDNA samples (n=250) collected for this study to delineate the geographic range of barramundi in the Red Sea. Cumulatively, this study highlights the need to consider the ecological impacts of aquaculture escapees and provides managers and industry with valuable information and a novel molecular monitoring tool for detecting aquaculture escapees.

Bovine Parvovirus (BPV) belongs to the genus Bocavirus, family Parvoviridae. BPV is the leading etiologic agent among the pathogens that cause primary gastroenteritis of cattle. Many of the intracellular events associated with virus replication are unknown. In this research project, we investigated BPV internalization into the host cell and trafficking in the cytosol. Preliminarily, EBTr cells had abundant clathrin, virus attached to purified clathrin, and EM micrographs revealed virus in endocytic vacuoles. Assays detecting virus infectivity (i.e. viral protein synthesis), virus production (completion of the replication cycle), and quantitative PCR (qPCR) to detect viral transcripts were used to evaluate virus uptake and subsequent trafficking events in the presence of selective inhibitors. Cell toxicity mediated by the drugs was evaluated by the MTT test. Virucidal effects of the drugs were assessed. A control virus was used to verify the inhibitor technology. Immunofluoresceinated virus particles were found in clathrin-rich early endosomes. Clathrin-mediated endocytosis (CME) was examined by clathrin polymerization inhibiting agent (chloropromazine), lysosomotropic agents (ammonium chloride and chloroquine), a vacuolar ATPase inhibitor (bafilomycin A1), and a blocker of transition between endosomes (brefeldin A). Caveosome pathway inhibitors included phorbol 12-myristate 13-acetate (a suppressor of caveolae formation), nystatin and methyl-beta-cyclodextrin (lipid raft blockers), and genistein (a tyrosine kinase phosphorylation inhibitor). Trafficking of BPV was investigated using specific inhibitors of proteasomal activity, actin-myosin function, and microtubule-dynein function. The proteasomal protease suppressor (lactacystin), and a proteasomal chymotrypsin inhibitor (epoxomicin) were used. The role of actin was probed by cytochlasin D, latrunculin A, and ML-7. The microtubule inhibitors nocodazole, vanadate, and EHNA were used to probe microtubule function. The inhibitors of CME reduced virus production and reduced infectivity, a result confirmed by qPCR. The blockers of caveolin-mediated entry did not interfere with virus production nor virus infectivity. Proteasome activity blockage did not affect the virus replication. But the virus cycle was affected by actin blockage and by microtubule blockage detected by qPCR. Taken together these data indicate that BPV uptake is mediated by clathrin coated pits and is acid-dependent. Further processing of BPV in the cytosol does not require proteasomal enzymes. Actin-associated vesicular transport appears to be essential to virus replication and trafficking to the nucleus appears to be mediated by microtubules.

Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

Blood vessel mimics (BVMs) are tissue engineered blood vessels that are intended as an intermediate testing environment for intravascular devices, such as stents. Specifically, Cal Poly’s Tissue Engineering Lab hypothesizes that BVMs can be used to test endothelial cell and smooth muscle cell responses to existing and new vascular stents. Characterization techniques are required for BVMs to be accepted as a valid testing model, prior to being employed as an in vitro model to determine the effects of medical treatments. Quantitative real-time polymerase chain reaction (qPCR) is one available option for evaluating gene expression of tissues. qPCR can be performed on DNA synthesized from RNA isolated from cells, and in this application, will provide quantitative information on what proteins where being transcribed within the cells at the time of RNA isolation. qPCR can be used to determine the proteins expressed in BVMs at baseline in order to then characterize changes in protein expression induced by stent deployment within the BVM. The aim of this thesis was to optimize existing qPCR protocols, and implement the optimized protocols to characterize gene expression of stented and unstented blood vessel mimics (BVMs) and cells from a donor with Diabetes grown in Cal Poly’s Tissue Engineering Laboratory. To accomplish this goal, existing qPCR protocols were evaluated and modified to ensure reproducible, valid results were produced. Standard operating procedures were created for RNA isolation, cDNA synthesis, qPCR and qPCR data analysis. Optimized qPCR methods were then applied to BVMs from umbilical and coronary cell sources to compare the models and to study the BVM responses to stent deployment. Additional primers were also identified for potential usage as reference genes and as diabetic markers for diseased BVMs.