College of Science

Theses and dissertations submitted to the College of Science

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The phylogeny of the terrestrial snail family Helicarionidae is poorly understood. This study aimed to barcode helicarionids found in the Philippines and perform a phylogenetic analysis to infer relationships of the group. A total of nine species, namely Hemiglypta moussoni (n=2), Lepidotrichia luteofasciata (n=1), Nipponochlamys semisericata (n=1), Ryssota uranus (n=6), R. sagittifera batanica (n=5), R. zeus (n=6), and the Philippine endemic Hemiglypta semperi (n=3), R. otaheitana (n=9), and R. lamarckiana (n=4), were barcoded using the standard marker cytochrome c oxidase I (CO1) gene. A further two genera, Hemiglypta sp. (n=3) and Lepidotrichia sp. (n=3), were also included. Helicarionid individuals were obtained from various sites in the Philippines (Aurora, Batanes, Bicol, Laguna, Leyte, Marinduque, Mindoro, Romblon and the Polillo Group of Islands). Highest sequence divergence was found within the family level (mean K2P distance=18.87%) and least within the species level (mean K2P distance=1.64%). Overlap was noted between interspecific and intraspecific variations. Neighbor-joining tree based on the Kimura-2- parameter model of DNA substitution revealed most species to be monophyletic with the exception of R. otaheitana; the species is therefore tagged for taxonomic re-evaluation. The phylogenetic analysis of Philippine helicarionids was done using the mitochondrial genes, CO1 (548 bp) and 16S rRNA (374 bp), and the nuclear rRNA gene cluster (1585 bp). Trees were constructed for each gene using the model-based methods Neighbor-Joining, Maximum Likelihood, and Bayesian Interference Analysis and the non-model based Maximum Parsimony and were rooted to the urocyclid Sheldonia as the outgroup. All of the trees showed clustering of the Philippine samples into one big clade with equivocal support (<50%). Three major subclades were consistently observed, namely, the Ryssota clade, Hemiglypta clade and the Lepidotrichia clade, with high bootstrap supports. A phylogenetic signal was detected for all the genes used using the g1 test. The use of other characters such as shell morphology, reproductive anatomy and ecology complemented by such molecular studies would be helpful to better understand the phylogeny of this group.

The effects of exogenous salicylic acid (SA) on the antioxidative response of cadmium-exposed Euphorbia hirta L. and on the distribution of Cd within the plant were investigated. Mature E. hirta plants were transferred to hydroponics, sprayed with 500 µM SA on their leaves, and exposed to 5 ppm CdCl2 for 3 days. The total chlorophyll concentrations of SA- and Cd-treated plants were significantly reduced compared with that of the control, but no significant reduction was observed in SA+Cd-treated plants. Lipid peroxidation was highest in Cd-treated plants but was slightly reduced in SA- and SA+Cd- treated plants. The levels of H2O2 significantly increased in plants treated with SA or Cd, but the increase was insignificant when SA was applied prior to Cd exposure. Catalase activity was significantly inhibited in plants treated with either SA or Cd treatment or both, whereas, superoxide dismutase activity was enhanced by either or both treatments. SA or Cd treatment significantly increased the total phenols in the plant’s shoot, which was consistent with the observed enhanced phenylalanine ammonia lyase activity in the treated plants. Endogenous SA level was higher in Cd-treated plants relative to that in the control, but was slightly lower whenever exogenous SA was applied. Cd2+ content was greater in the roots compared to the stems and leaves in both Cd- and SA+Cd-treated plants, and was significantly higher in the leaves of SA+Cd-treated plants than in the leaves of Cd-treated plants. The results suggest that SA treatment alleviated the Cd-induced oxidative damage in E. hirta by promoting the production of phytophenols and modifying the antioxidant enzyme activities.

Immune suppression occurs both in tumor microenvironments and in the female reproductive tract (FRT) during the secretory phase of the menstrual cycle. In tumors, this allows cancer cells to evade antitumoral responses. In FRT, this promotes an immunological milieu that is conducive for conception, but also makes it more vulnerable to microbial infections. Previous reports suggested that a tumor-associated glycoprotein-72 (TAG-72) induces the expression of Th2 cytokines by antigen presenting cells at the fetal-maternal interface. TAG-72 is a 10 kDa glycoprotein with mucin-like biochemical and biophysical properties. It is expressed at elevated levels in metastatic tumors and in the endometrium during the secretory phase of the menstrual cycle. In this study, we found that TAG-72: 1) downregulates perforin expression in Natural Killer (NK) cells, 2) inhibits NK cell proliferation, 3) skews cytokine expression to a Th2 profile, and 4) dampens some immune responses to lipopolysaccharide (LPS) stimulation. These results indicate that TAG-72 induces the suppression of some immune functions in tumor microenvironments and in the FRT. Elucidating the role of TAG-72 in the cyclical nature of immune suppression in the FRT may provide important insights for designing novel cancer immunotherapeutic strategies that can restore immune competence in tumor microenvironments.

Milkfish (Chanos chanos Forsskål) is one of the leading fish in Philippine aquaculture. In this study, landmark-based geometric morphometrics (GM) and randomly amplified polymorphic DNA (RAPD) techniques were tested as rapid and inexpensive tools to discriminate milkfish from geographic populations. These markers were used to compare Philippine and Indonesian milkfish populations as well as wild and hatchery populations. This is to assess if there is a genetic advantage for importing fry from Indonesia under the assumption that fry from Indonesia are genetically better than Philippine milkfish. Thirty milkfish specimens were used from each of four hatchery Philippine populations: BFAR- Tiwi, Albay; BFAR-Calape, Bohol; BFAR-Benoni, Camiguin; and Iloilo (Hautea Hatchery) and one from Bali, Indonesia, obtained in Paombong, Bulacan (Valencia Hatchery). Philippine samples from the wild were collected from: BFAR-Antique; Camiguin wild and Sagñay, Camarines Sur while wild Indonesian samples include those from Medan and Sulawesi. In geometric morphometric analysis, one Philippine hatchery population from a private hatchery in Cebu (Oversea) and wild samples from Claveria and Sarangani were included. Results using both GM and RAPD indicate no genetic differentiation between Philippine and Indonesian samples as well as between wild and hatchery populations. Variation was found to occur mostly within the populations, suggesting that these milkfish
populations have high genetic variability. In RAPD analysis, Camiguin wild had the highest genetic variability (Nei’s gene diversity (h)=0.1733, percentage of polymorphic bands (PPB)=62.81%) followed by all Indonesian populations while BFAR-Bohol had the lowest (h=0.1320, PPB=42.98%). Both Camiguin populations also exhibited higher genetic similarity with Indonesian samples than with Philippine populations. Characteristic bands were observed in Medan (≈400 bp) as well as Sulawesi and Bali (≈1,100 bp). The lack of genetic segregation may be indicative of high gene flow and low genetic divergence of milkfish. Both the perception of milkfish growers in the Philippines that milkfish fry from Indonesia is genetically better than Philippine milkfish is not supported by results of the study. There is no need to import Indonesian fry if the only basis is genetic consideration as Philippine and Indonesian milkfish are genetically similar. Perhaps differences in growth from the two sources can be attributed to other factors such as environmental and milkfish culture practices. The lack of genetic differentiation between wild and hatchery milkfish also suggests that any population would be a good seed stock source. The study of genetic diversity and population structure are important in the conservation and management of milkfish.

Antimicrobial drug resistance in Staphylococcus aureus is a serious burden to public health. The occurrence of multidrug-resistant (MDR) strains further complicates the treatment of infections. While antimicrobial resistance in hospitals is monitored by government agencies, studies on the prevalence of MDR S. aureus in other healthcare facilities are limited. This study aimed to determine the prevalence, antimicrobial susceptibility patterns, and risk factors associated with S. aureus, methicillin-resistant S. aureus (MRSA), and MDR S. aureus carriage in the Golden Reception and Action Center for the Elderly and other Special Cases (GRACES). It also aimed to identify the types of staphylococcal cassette chromosome mec (SCCmec) in the MRSA isolates. Sixty-six nasal swabs were collected from asymptomatic elderly residents. Staphylococcus aureus was identified by biochemical tests and PCR amplification of the nuc gene. Antimicrobial susceptibility assays and PCR amplification of mecA gene were performed to determine methicillin resistance. Results show that the prevalence for S. aureus was 37.9% among the residents. Eleven of the 25 S. aureus isolates (44%) were MRSA, corresponding to a prevalence of 16.7% among the residents. SCCmec typing showed that all of the MRSA isolates carried the SCCmec type IV. MDR, which is the resistance of the organism to at least one antimicrobial agent in three or more categories (Magiorakos et al., 2012), was
observed in 14 S. aureus isolates (56%) which correspond to 21.2% prevalence among the residents. Of these, 10 were MDR MRSA and four were MDR methicillin- susceptible S. aureus (MDR MSSA). Carriage of S. aureus (p=0.048; OR=2.86; 95% CI=0.99-8.26) and MRSA (p=0.033; OR=4.67; 95% CI=1.19-18.26) was significantly associated with history of pulmonary tuberculosis, but no association (p>0.05) was observed in MSSA. Significant association between history of pulmonary tuberculosis and carriage of MDR S. aureus (p=0.010; OR=5.40; 95% CI=1.53-19.05) and MDR MRSA (p=0.012; OR=6.38; 95% CI=1.46-27.91) was also observed, but no association (p>0.05) in MDR MSSA. Results show that asymptomatic elderly residents may carry S. aureus and MDR S. aureus including MRSA. Routine surveillance and mitigation controls should, therefore, be implemented to prevent possible outbreaks of S. aureus infections in a residential care facility.