Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences. / Includes bibliographical references.

Trichomonas vaginalis is the etiologic agent causing human trichomoniasis, a sexually transmitted infection found worldwide. In this study, subtilisin-like serine proteases (subtilases) were examined for their putative role in cell viability. Published data of other eukaryotic protozoan parasites confirm the importance of subtilases for adhesion and invasion. Serine protease inhibitor assays show that 3,4 DIC and TPCK inhibited cell growth from 79.6 to 98.4% respectively. To examine subtilases in more detail, a number of putative subtilases were cloned from T vaginalis. Two proteases were expressed recombinantly for antiserum production. TvSUB-12 (SP12) was localized to the posterior cell surface as using immunofluorescence and a kexin homolog called TvSUB-6 (SP6), revealed perinuclear staining. This study illustrated an essential role for subtilases in trichomonad cell viability and preliminary examination of two specific serine proteases revealing two different locations in the cell. To date, both of these proteases have never been characterized in this important human parasite.

INTRODUCTION: Foods enter the oral cavity and mix with saliva. Some foods are not well tolerated, for instance, gluten proteins in individuals suffering from celiac disease (CD). Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gliadin component of gluten contained in wheat, barley and rye. In previous studies we showed that oral Rothia bacteria can degrade gliadin in vitro. The objective of this study was to gain more insights into the role of Rothia bacteria and subtilisin-A enzyme on gliadin digestion in vivo, with the ultimate goal to find new therapeutic options for CD. MATERIALS AND METHODS: Part I: Rothia bacterial proteins were analyzed for enzyme activity and subjected to LC-ESI-MS/MS. For in vivo, mice chow was prepared with and without R. aeria. Gliadin epitope abolishment was assessed in the mice stomach contents (n=9/group) by ELISA. Part II: Subtilisin-A was dissolved in various solutions, temperatures and incubation time to assess enzyme activity by using enzyme substrate (Suc-AAPF-pNA). Part III: PEGylation of subtilisin-A (Sub-A) was performed by mixing Sub-A with methoxy-polyethylene glycol (mPEG) and further encapsulated by polylactic-glycolic acid (PLGA). The activity of the modified enzyme to detoxify the immunogenic gliadin epitopes was evaluated at pH3.0. RESULTS: Part I: R. aeria gliadin-degrading enzyme was found to be a member of the subtilisin family. In vivo, gliadins immunogenic epitopes were reduced by 32.6%. Part II: Sub-A dissolved at pH1.5 showed a band of 27kDa, while it only showed bands below 10kDa when dissolved at pH7.0, suggesting auto-proteolysis. The enzyme activity was completely lost at temperatures exceeding 60°C and also reduced 4-fold after 6hr incubation at 37°C. Part III: PEGylation protected Sub-A from autolysis. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gliadin immunogenic epitopes were decreased by 60% in the stomach of the mice fed with chow containing Sub-A-mPEG-PLGA. CONCLUSION: The results provide proof for the contribution of oral Rothia bacteria to gliadin digestion and pharmaceutical modification can protect Sub-A from auto-digestion as well as from acidic insults, thus rendering the usefulness of coated subtilisins as a digestive aid for gluten degradation. / 2019-01-13T00:00:00Z

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references.

Oxidative stress is a cellular condition where cells are challenged by elevated levels of reactive oxygen species (ROS) that are produced endogenously or exogenously. ROS can damage vital cellular components, including lipid, protein, DNA and RNA. Oxidative damage to DNA often leads to cell death or mutagenesis, the underlying cause of various human disease states. Previously our laboratory discovered that human PC4 gene can prevent oxidative mutagenesis in the bacterium Escherichia coli and that the yeast homolog SUB1 has a conserved function in oxidation protection. In this thesis I examined the underlying mechanisms of PC4’s oxidation protection function. My initial efforts to examine the predicted role of SUB1 in transcription-coupled DNA repair essentially negated this hypothesis. Instead, results from our experiments suggest that PC4 and yeast SUB1 can directly protect genomic DNA from oxidative damage. While testing SUB1’s role in double strand DNA break (DSB) repair, I found the sub1Δ mutant resects DSB ends rapidly but still ligates chromosomal breaks effectively, suggesting that DSB resection is not inhibitory to nonhomologous end-joining, an important DSB repair pathway. Finally, in the course of studying transcription recovery after UV damage, I found UV induces a longer form of RPB2 mRNA and demonstrated that this is caused by alternative polyadenylation of the RPB2 mRNA and that alternative polyadenylation contributes to UV resistance. Based on results of preliminary experiments, I propose that UV activates an alternative RNA polymerase to transcribe RNA POL II mRNA, a novel mechanism to facilitate recovery from inhibition of transcription resulting from UV damage. The hypothetical polymerase switch may account for the UV-induced alternative polyadenylation of the RPB2 mRNA.