{"id":{"repo_id":"umkc","oai_identifier":"oai:mospace.umsystem.edu:10355/67997"},"canonical_url":"https://search.dev.ndltd.org/etd/umkc/oai:mospace.umsystem.edu:10355/67997","repository":{"repo_id":"umkc","name":"University of Missouri - Kansas City","base_url":"https://mospace.umsystem.edu/oai/request"},"display":{"title":"Development and Evaluation Of HIV gp120 Responsive Microbicide Formulation for the Prevention of HIV Sexual Transmission","abstract":"Sexual transmission of HIV remains the primary route (75 to 85%) of HIV infection among all new infection cases. Furthermore, women represent the most vulnerable population and are more susceptible to HIV infections than their male counterpart. Thus, there is an urgent need to develop topical (vaginal/rectal) microbicide formulations capable of preventing HIV sexual transmission. The objective of this dissertation is to develop a mannose specific, lectin-based topical microbicide formulation capable of targeting HIV gp120 for the prevention of HIV sexual transmission. In Chapters 1 and 2, the general hypothesis, aims and scope of this work are introduced. Chapter 3 covers the literature review of anti-HIV lectins and current delivery approaches. In Chapter 4, the binding interactions between the mannose specific lectin Concanavalin A (ConA) and glycogen from Oster, as well as mannan from Saccharomyces cerevisiae, were studied using a quartz crystal microbalance (QCM). The equilibrium dissociation constant describing the interaction between Con A and glycogen (KD = 0.25 μM) was 12 fold lower than the equilibrium dissociation constant describing the binding between Con A and mannan (KD = 2.89 μM). That is, Con A was found to have a higher affinity for the glucose-base polysaccharide, than for the mannose-based. This observation was mainly attributed to steric effects, the difference in molecular weight and branching pattern of both polysaccharides. The knowledge gained in Chapter 4 was applied in Chapter 5 for the development of HIV 1 gp120 and mannose responsive particle (MRP) formulations. Thus, core dissolved MRP (𝐶−𝑀𝑅𝑃) and core containing MRP (𝐶+𝑀𝑅𝑃) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with the mannose specific lectin (Con A) and a polysaccharide cross-linker (Glycogen). Particles were characterized and tested in vitro on Lactobacillus crispatus, Human vaginal keratinocytes (VK2/E6E7) and murine macrophage [RAW 264.7 (TIB 71)] cell lines. 𝐶+𝑀𝑅𝑃 average size and ζ-potential were 1130±15.72 nm [PDI = 0.153] and 15.1±0.55 mV, (n=3). Similarly, 𝐶−𝑀𝑅𝑃 average size and ζ-potential were 1089±23.33 nm (n=3) and -14.2±0.25 mV (n=3). Tenofovir (TFV) encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% w/w and 6.0% w/w in 𝐶+𝑀𝑅𝑃 and 𝐶−𝑀𝑅𝑃, respectively. Both 𝐶−𝑀𝑅𝑃 and 𝐶+𝑀𝑅𝑃 were nontoxic to L. crispatus and did not induce any significant pro-inflammatory nitric oxide release in VK2 and RAW 264.7 cell culture. However, 𝐶−𝑀𝑅𝑃 was found to significantly affect VK2 and RAW 264.7 cells viability at concentrations ≥ 100 µg/ml. Similarly, 𝐶−𝑀𝑅𝑃 significantly increased pro-inflammatory cytokines (IL1α, IL1β, IL6, IL7, MKC and TNFα) release at concentrations ≥ 100 µg/ml. Conversely, 𝐶+𝑀𝑅𝑃 did not induce any significant changes in VK2 and RAW 264.7 cells viability nor in pro-inflammatory cytokines’ levels, in the concentration range tested (≤ 1000 µg/ml), for 24 h. 𝐶+𝑀𝑅𝑃 was then selected for further in vitro drug release studies as well as ex vivo vaginal mucoadhesion studies. HIV gp120 triggered TFV release from 𝐶+𝑀𝑅𝑃 in a concentration dependent manner, and following Hixson–Crowell and Hopfenberg kinetic models, consistent with drug release from diminishing surface or matrix eroding drug particles. 𝐶+𝑀𝑅𝑃 was further optimized by varying the number of Con A layer in the formulation, and in order to achieve lower HIV gp120 sensitivity (≤ 100 µg/ml). Furthermore, bioadhesion studies, performed ex vivo on porcine vaginal tissue, demonstrated that FITC−𝐶+𝑀𝑅𝑃 adheres to vaginal tissue at levels varying between 10% ± 1 and 20% ± 2, depending on the number of Con A layers in the formulation. In chapter 6, 𝐶+𝑀𝑅𝑃 preclinical safety was evaluated in 8-12 weeks old female C57BL/6 mice model. First, mice were treated with Depo-Provera® to maintain them in a diestrus-like state. Then the microbicide formulation was delivered vaginally at a dose of 100 mg/kg in PBS. Vaginal histology, immunohistochemistry evaluations, as well as pro-inflammatory cytokines release (vaginal lavage and tissue extract) were investigated after 24 h. The vaginal retention of FITC−𝐶+𝑀𝑅𝑃 was also evaluated, up to 24 h. Vaginal and major reproductive organs’ histology did not show major damage of the epithelial layer. This result was also consistent with immunohistochemistry evaluation of CD45+ cells infiltration in the vaginal epithelial layer, unlike the positive control treated groups (BZK and N-9). Furthermore 𝐶+𝑀𝑅𝑃 did not induce any significant changes in pro-inflammatory cytokines IL1α, Ilβ, IL7, IP10 and TNFα. In addition, it was also observed that FITC labeled 𝐶+𝑀𝑅𝑃 does not have a long-term retention in mice vaginal tract. This result suggested that a precoital or multiple vaginal application (i.e., BID) approaches of 𝐶+𝑀𝑅𝑃 should be investigated. Overall, this study demonstrates the feasibility of lectin-based microbicide formulations to target HIV gp120 for the prevention of HIV sexual transmission.","abstract_html":"Sexual transmission of HIV remains the primary route (75 to 85%) of HIV infection among all new infection cases. Furthermore, women represent the most vulnerable population and are more susceptible to HIV infections than their male counterpart. Thus, there is an urgent need to develop topical (vaginal/rectal) microbicide formulations capable of preventing HIV sexual transmission. The objective of this dissertation is to develop a mannose specific, lectin-based topical microbicide formulation capable of targeting HIV gp120 for the prevention of HIV sexual transmission. In Chapters 1 and 2, the general hypothesis, aims and scope of this work are introduced. Chapter 3 covers the literature review of anti-HIV lectins and current delivery approaches. In Chapter 4, the binding interactions between the mannose specific lectin Concanavalin A (ConA) and glycogen from Oster, as well as mannan from Saccharomyces cerevisiae, were studied using a quartz crystal microbalance (QCM). The equilibrium dissociation constant describing the interaction between Con A and glycogen (KD = 0.25 μM) was 12 fold lower than the equilibrium dissociation constant describing the binding between Con A and mannan (KD = 2.89 μM). That is, Con A was found to have a higher affinity for the glucose-base polysaccharide, than for the mannose-based. This observation was mainly attributed to steric effects, the difference in molecular weight and branching pattern of both polysaccharides. The knowledge gained in Chapter 4 was applied in Chapter 5 for the development of HIV 1 gp120 and mannose responsive particle (MRP) formulations. Thus, core dissolved MRP (𝐶−𝑀𝑅𝑃) and core containing MRP (𝐶+𝑀𝑅𝑃) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with the mannose specific lectin (Con A) and a polysaccharide cross-linker (Glycogen). Particles were characterized and tested in vitro on Lactobacillus crispatus, Human vaginal keratinocytes (VK2/E6E7) and murine macrophage [RAW 264.7 (TIB 71)] cell lines. 𝐶+𝑀𝑅𝑃 average size and ζ-potential were 1130±15.72 nm [PDI = 0.153] and 15.1±0.55 mV, (n=3). Similarly, 𝐶−𝑀𝑅𝑃 average size and ζ-potential were 1089±23.33 nm (n=3) and -14.2±0.25 mV (n=3). Tenofovir (TFV) encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% w/w and 6.0% w/w in 𝐶+𝑀𝑅𝑃 and 𝐶−𝑀𝑅𝑃, respectively. Both 𝐶−𝑀𝑅𝑃 and 𝐶+𝑀𝑅𝑃 were nontoxic to L. crispatus and did not induce any significant pro-inflammatory nitric oxide release in VK2 and RAW 264.7 cell culture. However, 𝐶−𝑀𝑅𝑃 was found to significantly affect VK2 and RAW 264.7 cells viability at concentrations ≥ 100 µg/ml. Similarly, 𝐶−𝑀𝑅𝑃 significantly increased pro-inflammatory cytokines (IL1α, IL1β, IL6, IL7, MKC and TNFα) release at concentrations ≥ 100 µg/ml. Conversely, 𝐶+𝑀𝑅𝑃 did not induce any significant changes in VK2 and RAW 264.7 cells viability nor in pro-inflammatory cytokines’ levels, in the concentration range tested (≤ 1000 µg/ml), for 24 h. 𝐶+𝑀𝑅𝑃 was then selected for further in vitro drug release studies as well as ex vivo vaginal mucoadhesion studies. HIV gp120 triggered TFV release from 𝐶+𝑀𝑅𝑃 in a concentration dependent manner, and following Hixson–Crowell and Hopfenberg kinetic models, consistent with drug release from diminishing surface or matrix eroding drug particles. 𝐶+𝑀𝑅𝑃 was further optimized by varying the number of Con A layer in the formulation, and in order to achieve lower HIV gp120 sensitivity (≤ 100 µg/ml). Furthermore, bioadhesion studies, performed ex vivo on porcine vaginal tissue, demonstrated that FITC−𝐶+𝑀𝑅𝑃 adheres to vaginal tissue at levels varying between 10% ± 1 and 20% ± 2, depending on the number of Con A layers in the formulation. In chapter 6, 𝐶+𝑀𝑅𝑃 preclinical safety was evaluated in 8-12 weeks old female C57BL/6 mice model. First, mice were treated with Depo-Provera® to maintain them in a diestrus-like state. Then the microbicide formulation was delivered vaginally at a dose of 100 mg/kg in PBS. Vaginal histology, immunohistochemistry evaluations, as well as pro-inflammatory cytokines release (vaginal lavage and tissue extract) were investigated after 24 h. The vaginal retention of FITC−𝐶+𝑀𝑅𝑃 was also evaluated, up to 24 h. Vaginal and major reproductive organs’ histology did not show major damage of the epithelial layer. This result was also consistent with immunohistochemistry evaluation of CD45+ cells infiltration in the vaginal epithelial layer, unlike the positive control treated groups (BZK and N-9). Furthermore 𝐶+𝑀𝑅𝑃 did not induce any significant changes in pro-inflammatory cytokines IL1α, Ilβ, IL7, IP10 and TNFα. In addition, it was also observed that FITC labeled 𝐶+𝑀𝑅𝑃 does not have a long-term retention in mice vaginal tract. This result suggested that a precoital or multiple vaginal application (i.e., BID) approaches of 𝐶+𝑀𝑅𝑃 should be investigated. Overall, this study demonstrates the feasibility of lectin-based microbicide formulations to target HIV gp120 for the prevention of HIV sexual transmission.","abstract_has_math":false,"creators":["Coulibaly, Fohona S."],"institution":"University of Missouri -- Kansas City","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018","date_published":"2018","updated_at":"2026-07-24T05:17:10Z","subjects":[],"languages":["en_US"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10355/67997","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Coulibaly, Fohona S."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2019-05-08T16:39:51Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2019-05-08T16:39:51Z"]},{"key":"dc:date.issued","label":"Date","values":["2018"]},{"key":"dc:publisher","label":"Institution","values":["University of Missouri -- Kansas City"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10355/67997"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Sexual transmission of HIV remains the primary route (75 to 85%) of HIV infection among all new infection cases. Furthermore, women represent the most vulnerable population and are more susceptible to HIV infections than their male counterpart. Thus, there is an urgent need to develop topical (vaginal/rectal) microbicide formulations capable of preventing HIV sexual transmission. The objective of this dissertation is to develop a mannose specific, lectin-based topical microbicide formulation capable of targeting HIV gp120 for the prevention of HIV sexual transmission. In Chapters 1 and 2, the general hypothesis, aims and scope of this work are introduced. Chapter 3 covers the literature review of anti-HIV lectins and current delivery approaches. In Chapter 4, the binding interactions between the mannose specific lectin Concanavalin A (ConA) and glycogen from Oster, as well as mannan from Saccharomyces cerevisiae, were studied using a quartz crystal microbalance (QCM). The equilibrium dissociation constant describing the interaction between Con A and glycogen (KD = 0.25 μM) was 12 fold lower than the equilibrium dissociation constant describing the binding between Con A and mannan (KD = 2.89 μM). That is, Con A was found to have a higher affinity for the glucose-base polysaccharide, than for the mannose-based. This observation was mainly attributed to steric effects, the difference in molecular weight and branching pattern of both polysaccharides. The knowledge gained in Chapter 4 was applied in Chapter 5 for the development of HIV 1 gp120 and mannose responsive particle (MRP) formulations. Thus, core dissolved MRP (𝐶−𝑀𝑅𝑃) and core containing MRP (𝐶+𝑀𝑅𝑃) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with the mannose specific lectin (Con A) and a polysaccharide cross-linker (Glycogen). Particles were characterized and tested in vitro on Lactobacillus crispatus, Human vaginal keratinocytes (VK2/E6E7) and murine macrophage [RAW 264.7 (TIB 71)] cell lines. 𝐶+𝑀𝑅𝑃 average size and ζ-potential were 1130±15.72 nm [PDI = 0.153] and 15.1±0.55 mV, (n=3). Similarly, 𝐶−𝑀𝑅𝑃 average size and ζ-potential were 1089±23.33 nm (n=3) and -14.2±0.25 mV (n=3). Tenofovir (TFV) encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% w/w and 6.0% w/w in 𝐶+𝑀𝑅𝑃 and 𝐶−𝑀𝑅𝑃, respectively. Both 𝐶−𝑀𝑅𝑃 and 𝐶+𝑀𝑅𝑃 were nontoxic to L. crispatus and did not induce any significant pro-inflammatory nitric oxide release in VK2 and RAW 264.7 cell culture. However, 𝐶−𝑀𝑅𝑃 was found to significantly affect VK2 and RAW 264.7 cells viability at concentrations ≥ 100 µg/ml. Similarly, 𝐶−𝑀𝑅𝑃 significantly increased pro-inflammatory cytokines (IL1α, IL1β, IL6, IL7, MKC and TNFα) release at concentrations ≥ 100 µg/ml. Conversely, 𝐶+𝑀𝑅𝑃 did not induce any significant changes in VK2 and RAW 264.7 cells viability nor in pro-inflammatory cytokines’ levels, in the concentration range tested (≤ 1000 µg/ml), for 24 h. 𝐶+𝑀𝑅𝑃 was then selected for further in vitro drug release studies as well as ex vivo vaginal mucoadhesion studies. HIV gp120 triggered TFV release from 𝐶+𝑀𝑅𝑃 in a concentration dependent manner, and following Hixson–Crowell and Hopfenberg kinetic models, consistent with drug release from diminishing surface or matrix eroding drug particles. 𝐶+𝑀𝑅𝑃 was further optimized by varying the number of Con A layer in the formulation, and in order to achieve lower HIV gp120 sensitivity (≤ 100 µg/ml). Furthermore, bioadhesion studies, performed ex vivo on porcine vaginal tissue, demonstrated that FITC−𝐶+𝑀𝑅𝑃 adheres to vaginal tissue at levels varying between 10% ± 1 and 20% ± 2, depending on the number of Con A layers in the formulation. In chapter 6, 𝐶+𝑀𝑅𝑃 preclinical safety was evaluated in 8-12 weeks old female C57BL/6 mice model. First, mice were treated with Depo-Provera® to maintain them in a diestrus-like state. Then the microbicide formulation was delivered vaginally at a dose of 100 mg/kg in PBS. Vaginal histology, immunohistochemistry evaluations, as well as pro-inflammatory cytokines release (vaginal lavage and tissue extract) were investigated after 24 h. The vaginal retention of FITC−𝐶+𝑀𝑅𝑃 was also evaluated, up to 24 h. Vaginal and major reproductive organs’ histology did not show major damage of the epithelial layer. This result was also consistent with immunohistochemistry evaluation of CD45+ cells infiltration in the vaginal epithelial layer, unlike the positive control treated groups (BZK and N-9). Furthermore 𝐶+𝑀𝑅𝑃 did not induce any significant changes in pro-inflammatory cytokines IL1α, Ilβ, IL7, IP10 and TNFα. In addition, it was also observed that FITC labeled 𝐶+𝑀𝑅𝑃 does not have a long-term retention in mice vaginal tract. This result suggested that a precoital or multiple vaginal application (i.e., BID) approaches of 𝐶+𝑀𝑅𝑃 should be investigated. Overall, this study demonstrates the feasibility of lectin-based microbicide formulations to target HIV gp120 for the prevention of HIV sexual transmission."]},{"key":"dc:title","label":"Title","values":["Development and Evaluation Of HIV gp120 Responsive Microbicide Formulation for the Prevention of HIV Sexual Transmission"]}]}],"canonical_facts":{"dc:creator":["Coulibaly, Fohona S."],"dc:date.accessioned":["2019-05-08T16:39:51Z"],"dc:date.available":["2019-05-08T16:39:51Z"],"dc:date.issued":["2018"],"dc:description.abstract":["Sexual transmission of HIV remains the primary route (75 to 85%) of HIV infection among all new infection cases. Furthermore, women represent the most vulnerable population and are more susceptible to HIV infections than their male counterpart. Thus, there is an urgent need to develop topical (vaginal/rectal) microbicide formulations capable of preventing HIV sexual transmission. The objective of this dissertation is to develop a mannose specific, lectin-based topical microbicide formulation capable of targeting HIV gp120 for the prevention of HIV sexual transmission. In Chapters 1 and 2, the general hypothesis, aims and scope of this work are introduced. Chapter 3 covers the literature review of anti-HIV lectins and current delivery approaches. In Chapter 4, the binding interactions between the mannose specific lectin Concanavalin A (ConA) and glycogen from Oster, as well as mannan from Saccharomyces cerevisiae, were studied using a quartz crystal microbalance (QCM). The equilibrium dissociation constant describing the interaction between Con A and glycogen (KD = 0.25 μM) was 12 fold lower than the equilibrium dissociation constant describing the binding between Con A and mannan (KD = 2.89 μM). That is, Con A was found to have a higher affinity for the glucose-base polysaccharide, than for the mannose-based. This observation was mainly attributed to steric effects, the difference in molecular weight and branching pattern of both polysaccharides. The knowledge gained in Chapter 4 was applied in Chapter 5 for the development of HIV 1 gp120 and mannose responsive particle (MRP) formulations. Thus, core dissolved MRP (𝐶−𝑀𝑅𝑃) and core containing MRP (𝐶+𝑀𝑅𝑃) were prepared through the layer-by-layer coating of calcium carbonate (CaCO3) with the mannose specific lectin (Con A) and a polysaccharide cross-linker (Glycogen). Particles were characterized and tested in vitro on Lactobacillus crispatus, Human vaginal keratinocytes (VK2/E6E7) and murine macrophage [RAW 264.7 (TIB 71)] cell lines. 𝐶+𝑀𝑅𝑃 average size and ζ-potential were 1130±15.72 nm [PDI = 0.153] and 15.1±0.55 mV, (n=3). Similarly, 𝐶−𝑀𝑅𝑃 average size and ζ-potential were 1089±23.33 nm (n=3) and -14.2±0.25 mV (n=3). Tenofovir (TFV) encapsulation efficiency in CaCO3 was 74.4% with drug loading of 16.3% w/w and 6.0% w/w in 𝐶+𝑀𝑅𝑃 and 𝐶−𝑀𝑅𝑃, respectively. Both 𝐶−𝑀𝑅𝑃 and 𝐶+𝑀𝑅𝑃 were nontoxic to L. crispatus and did not induce any significant pro-inflammatory nitric oxide release in VK2 and RAW 264.7 cell culture. However, 𝐶−𝑀𝑅𝑃 was found to significantly affect VK2 and RAW 264.7 cells viability at concentrations ≥ 100 µg/ml. Similarly, 𝐶−𝑀𝑅𝑃 significantly increased pro-inflammatory cytokines (IL1α, IL1β, IL6, IL7, MKC and TNFα) release at concentrations ≥ 100 µg/ml. Conversely, 𝐶+𝑀𝑅𝑃 did not induce any significant changes in VK2 and RAW 264.7 cells viability nor in pro-inflammatory cytokines’ levels, in the concentration range tested (≤ 1000 µg/ml), for 24 h. 𝐶+𝑀𝑅𝑃 was then selected for further in vitro drug release studies as well as ex vivo vaginal mucoadhesion studies. HIV gp120 triggered TFV release from 𝐶+𝑀𝑅𝑃 in a concentration dependent manner, and following Hixson–Crowell and Hopfenberg kinetic models, consistent with drug release from diminishing surface or matrix eroding drug particles. 𝐶+𝑀𝑅𝑃 was further optimized by varying the number of Con A layer in the formulation, and in order to achieve lower HIV gp120 sensitivity (≤ 100 µg/ml). Furthermore, bioadhesion studies, performed ex vivo on porcine vaginal tissue, demonstrated that FITC−𝐶+𝑀𝑅𝑃 adheres to vaginal tissue at levels varying between 10% ± 1 and 20% ± 2, depending on the number of Con A layers in the formulation. In chapter 6, 𝐶+𝑀𝑅𝑃 preclinical safety was evaluated in 8-12 weeks old female C57BL/6 mice model. First, mice were treated with Depo-Provera® to maintain them in a diestrus-like state. Then the microbicide formulation was delivered vaginally at a dose of 100 mg/kg in PBS. Vaginal histology, immunohistochemistry evaluations, as well as pro-inflammatory cytokines release (vaginal lavage and tissue extract) were investigated after 24 h. The vaginal retention of FITC−𝐶+𝑀𝑅𝑃 was also evaluated, up to 24 h. Vaginal and major reproductive organs’ histology did not show major damage of the epithelial layer. This result was also consistent with immunohistochemistry evaluation of CD45+ cells infiltration in the vaginal epithelial layer, unlike the positive control treated groups (BZK and N-9). Furthermore 𝐶+𝑀𝑅𝑃 did not induce any significant changes in pro-inflammatory cytokines IL1α, Ilβ, IL7, IP10 and TNFα. In addition, it was also observed that FITC labeled 𝐶+𝑀𝑅𝑃 does not have a long-term retention in mice vaginal tract. This result suggested that a precoital or multiple vaginal application (i.e., BID) approaches of 𝐶+𝑀𝑅𝑃 should be investigated. Overall, this study demonstrates the feasibility of lectin-based microbicide formulations to target HIV gp120 for the prevention of HIV sexual transmission."],"dc:identifier.uri":["https://hdl.handle.net/10355/67997"],"dc:language.iso":["en_US"],"dc:publisher":["University of Missouri -- Kansas City"],"dc:title":["Development and Evaluation Of HIV gp120 Responsive Microbicide Formulation for the Prevention of HIV Sexual Transmission"],"dc:type":["Thesis"]},"updated_at":"2026-07-24T05:17:10Z"}