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Immune defense of the female lower reproductive tract and the use of monoclonal antibody-based topical microbicide films to protect against HIV infectionCostanzo, Corey 08 April 2016 (has links)
Research on the human-immunodeficiency virus (HIV) and HIV transmission prevention methods is extensive, yet a female-controlled prevention method still does not exist. In many cultures where HIV prevalence is highest there are many social and cultural barriers to the prevention options currently available, specifically around the use of condoms. Topical microbicides could potentially offer a viable solution.
In heterosexually transmitted HIV, the epithelium of the lower female genital tract is the first place of contact with invading pathogens. A topical microbicide that could protect against infection across this barrier, without causing inflammation, would be an ideal product to protect women against infection. In previous studies, several microbicides were shown to cause inflammation and increase the risk of HIV infection; therefore, it is vitally important that any new topical microbicide products developed do not have the same effect. Our laboratory is collaborating with Mapp Biopharmaceuticals in the development of a new vaginal microbicide product based on human monoclonal antibodies produced in plants. Our prototype microbicide, MB66, is a film containing two antibodies: VRC01 (anti-HIV) and HSV-8 (anti HSV-2). We hypothesize that the new MB66 topical microbicide film will provide protection against these sexually transmitted viruses without inducing inflammation in the vaginal epithelium, and will prove to be a viable topical microbicide product that does not cause inflammation and increase the risk of HIV infection.
MatTek vaginal epithelium models were used to test for any potential inflammation by the MB66 film. Active MB66 and placebo films were placed on the MatTek models. Vaginal tissue models grown in the presence of estradiol were also exposed to the active film. After being exposed to the active MB66 film, apical and basal supernatants were collected and analyzed for proinflammatory cytokines; IL-6, IL-8, MCP-1, and TNF-alpha. There were no significant increases in IL-6 or IL-8 expression in vaginal epithelium when exposed to the MB66 film. There was a significant decrease in the expression of TNF-alpha in the apical supernatant of the film-treated cultures at the 24 hour time point, and a very small (<1%) but statistically significant increase in the expression of MCP-1 in the basal supernatant of the film and the placebo after 24 hours. These results indicate that the active MB66 film did not induce a significant amount of inflammation in the vaginal epithelium.
Immunohistochemistry was used to ensure that the MatTek vaginal models are a valid representation of the native vaginal-ectocervical epithelium. Expression of toll-like receptors (TLRs) 2, 3, 4, 5, and 9 was compared in the MatTek models (partial and full thickness), as well as native vaginal and ectocervical tissue. Results show similar expression of all the TLR's in the native tissue and the partial and full thickness MatTek models. There was a greater degree of similarity between the native tissue and the full thickness MatTek models. Namely, there was a lack of expression with TLR 5 and TLR 9, and positive expression of TLRs 2, 4, and 9.
Overall, the TLR expression validated the use of these models in testing current and future topical microbicides in order to find an effective female-controlled prevention method. In addition, cytokine analysis provided evidence that the new Mapp MB66 topical microbicide films do not cause significant inflammation to the vaginal-ectocervical tissue. Our data indicate that there should be further development of the MB66 film as a vaginal topical microbicide.
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Molecular characterization of an HIV-1 drug target and antiviral compound developmentMaselko, Maciej 09 May 2011 (has links)
The Human Immunodeficiency Virus -1 (HIV-1) has been the source of
substantial human misery since it was first discovered in the early 1980s.
Despite remarkable progress that has been made towards understanding HIV-
1, there is no cure, no vaccine and life-prolonging therapies are beyond the
reach of millions in need. Our research sought to both gain insight into
potential therapeutic targets as well as the preclinical testing of drug
candidates.
We demonstrate that a RhoA derived peptide is an effective inhibitor of HIV-1
entry. Although this peptide inhibits HIV-1 due to its poly-anionic nature, it
nevertheless demonstrates that endogenous host proteins may be repurposed
for novel therapeutic uses. We also characterized the mechanism and
effectiveness of Basant, a polyherbal topical microbicide candidate for the
prevention of HIV-1 transmission. Our data demonstrate that it inhibits the
entry of both CCR5 and CXCR4 tropic HIV-1 at non-toxic concentrations.
Finally, data is presented on the characterization of a novel HIV-1 protein
expressed from an alternative reading frame of the HIV-1 polymerase gene.
We demonstrate that this protein is localized to the nucleolus and is likely
expressed from a novel HIV-1 transcript. This work lays the foundation for
further studies to target this protein for drug development. / Graduation date: 2011 / Access restricted to the OSU Community at author's request from May 9, 2011 - May 9, 2012
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Understanding the impact of participation in a microbicide clinical trial on condom useMzimela, Adelaide Misiwe January 2010 (has links)
Submitted in partial fulfilment of the academic requirements for the degree:
Master of Education - Research Methodology in the Department of Educational Psychology of the Faculty of Education at the University of Zululand, 2010. / There is ongoing debate regarding the public health message of introducing a microbicide that is less efficacious than condoms into the HIV prevention field. One key issue is whether the availability of microbicides would undermine women's bargaining power to negotiate condom use. Most microbicide trials report increased condom use among participants. This analysis attempts to separate the influence of increased safer sex counselling from the direct impact of gel use as a facilitator to condom acceptability.
In-depth interviews were conducted with a random sample of women participating in the MDP 301 Phase III microbicide trial, and a sub-sample of their male partners. Data from 63 female and 5 male interviews were analysed to assess the impact that participating in a clinical trial and using a vaginal microbicide/placebo gel had on condom usage.
Two thirds of women reported not using condoms prior to study participation due to male opposition. Most of the women interviewed were unable to explain their partners' resistance to condoms. Some of the underlying factors were related to men's resistance to using condoms with a main or long term partner (as opposed to casual partners); preference for skin-to-skin contact; or rumours about negative health implications such as condoms containing maggots. A third of women started using condoms, although inconsistently, after joining the trial. Women used participation in the trial as the rationale for discussing the risks of HfV infection and condom use with their partners. Men appeared to be more willing to use condoms in the presence of gel, and this was partially due to the gel counteracting the negative effects of condom use on sexual pleasure.
Joining the trial appeared to create an opportunity for women to discuss sex with their partners, and the introduction of gel into the relationship serves as a bargaining tool for condom use with their partners.
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Innate immune responses of vaginal epithelium and activity of monoclonal antibody-based microbicide in the presence of lactic acid, a lactobacillus metaboliteBayigga, Lois 17 June 2016 (has links)
Sexually transmitted infections (STIs) such as gonorrhea, herpes simplex virus 2 (HSV 2), hepatitis C, human papilloma viruses (HPV) and human immunodeficiency virus type 1 (HIV) are a global health concern affecting millions of lives. Although extensive efforts have been geared towards prevention and treatment of STIs, little progress has been achieved. Recently, efforts to develop microbicides have been focused on the commensal bacterial species that comprise the vaginal microbiome and their role in immunity and disease pathogenesis. The lower FRT which includes the cervix and vagina has endogenous bacterial species that are supported by the mucosal epithelium. Lactobaccilli are the dominant endogenous bacterial species in the vagina of most women; lactobacilli convert glycogen to lactic acid (LA) which maintains a low pH environment in the vagina and serves as a deterrent to infectious organisms. The purpose of this research project was to determine the effects of LA on vaginal integrity and inflammation in a vaginal epithelial cell (VEC) tissue model, and on the ability of the broadly neutralizing anti-HIV antibody, VRCO1, to inactivate HIV in vitro.
Effects of LA exposure on the viability and integrity of vaginal epithelium were determined by histology, MTT assay and measurement of transepithelial electric resistance (TEER). In addition, an enzyme-linked immunosorbent assay (ELISA) was used to measure concentrations of cytokines secreted by the VEC epithelial cells in response to different doses of LA. Using TLR agonists to simulate infection in the VEC model, we tested the hypothesis that LA has anti-inflammatory properties that modulate immune responses of the vaginal epithelium. We assessed the effect of LA on the neutralization activity of the anti-HIV-1 monoclonal antibody VRCO1 in the TZM-bl HIV neutralization assay.
Tissue morphology and integrity were not affected by exposure to LA. Low concentrations of IL-1β and RANTES were expressed by VEC tissues in response to L-LA treatment. VEC tissues expressed significantly elevated concentrations of IL-1RA (p<0.0001), an anti-inflammatory cytokine, in response to LA regardless of incubation time and LA doses. In addition, treatment of VEC tissues with Poly I: C in the presence of LA dampened upregulated expression of IL-1β, TNF-alpha and IL-6 in response to the TLR 3 agonist. LA also elicited significantly higher IL-1RA concentrations when apically added to the TLR agonist-treated VEC tissues. These data suggest that LA elicits an anti-inflammatory response in the vaginal epithelium. LA acidic conditions as low as pH 3.5 did not affect the ability of VRC01, to prevent HIV infection. We found that LA, at concentrations present in vaginal secretions of normal women, inhibited the inflammatory response to a TLR agonist, possibly due to upregulated Il-1RA synthesis. In addition, the functionality of VRCO1 in an acidic milieu was not diminished, providing evidence that antibodies can function in the low pH vaginal environment. This report highlights the potential use of LA in the vagina as a microbicide due to its virucidal activity and anti-inflammatory properties. It also indicates that monoclonal antibody-based vaginal microbicides will not be neutralized by LA. There is still a need to elucidate the exact mechanisms by which LA confers immuno-modulatory properties within the female reproductive tract.
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Dendronized Amphiphilic Copolymers Synthesized from Methacrylate Derivatives as Potential Topical MicrobicidesHardrict, Shauntrece Nicole 10 January 2010 (has links)
The need for a preventative agent to curtail the rampant spread of HIV, other STDs and mucosal pathogens is urgent. Topical microbicides based on amphiphilic compounds have been identified as an attractive means toward this goal. Novel dendritic methacrylate macromonomers––di-tert-butyl 4-(2-tert-butoxycarbonyl-ethyl)-4-[3-(2-methacryoloxyethyl)ureido]heptandioate (MI) and di-tert-butyl 4-(2-tert-butoxycarbonylethyl)-4-[3-methyl-3-(2-methacryoloxyethyl)ureido]heptanedioate (MIII)––were synthesized, characterized, and subsequently polymerized via conventional free radical polymerization in acetonitrile employing AIBN as an initiator.
Methyl 3-mercaptopropionate (MMP), a chain transfer agent, was employed to target low molecular weight (<10,000 g/mol) polymers. Dendronized homopolymers were prepared from MIII with varying MMP concentrations (0–10 mol%). MALDI-TOF MS characterized the homopolymer prepared with 10 mol% MMP (Mn = 2,481 g/mol).
In the presence of MMP, MIII (5 to 25 mol%) was copolymerized with alkyl (methyl, ethyl, and n-butyl) methacrylates. Copolymer composition correlated well with monomer feed; Mns of 3–10K g/mol were observed.
Ionizable polymers were achieved via acidolysis with trifluoroacetic acid; significant increases in Tg were observed. For the copolymers, the Tgs of the acids increased as the copolymer composition increased in MIII. For the homopolymers, Tgs decreased with increasing MMP concentration.
The solubilities of carboxylic acid functional polymers were studied in 5 w/v % aqueous triethanolamine (aq TEA) at a concentration of 12.5 mg/mL. Clear, homogeneous solutions of the deprotected macromonomer, MIII(OH), homopolymer, PMIII(OH), and MIII(OH)/MMA copolymer series were observed; copolymer solubility decreased as the aliphatic chain length of the alkyl methacrylate increased.
The amphiphilic dendronized polymer series incorporates many features shown beneficial in the pursuit of effective antimicrobial agents: amphiphilicity, multiple anionic functional groups, a polymer backbone, and aqueous solubility. Additionally, biological selectivity (e.g. cytotoxicity) is expected to be tunable through the control of molecular weight, alkyl chain length (n*), copolymer composition, and molecular architecture. / Ph. D.
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Mechanistic Models of Anti-HIV Microbicide Drug DeliveryGao, Yajing January 2016 (has links)
<p>A new modality for preventing HIV transmission is emerging in the form of topical microbicides. Some clinical trials have shown some promising results of these methods of protection while other trials have failed to show efficacy. Due to the relatively novel nature of microbicide drug transport, a rigorous, deterministic analysis of that transport can help improve the design of microbicide vehicles and understand results from clinical trials. This type of analysis can aid microbicide product design by helping understand and organize the determinants of drug transport and the potential efficacies of candidate microbicide products.</p><p>Microbicide drug transport is modeled as a diffusion process with convection and reaction effects in appropriate compartments. This is applied here to vaginal gels and rings and a rectal enema, all delivering the microbicide drug Tenofovir. Although the focus here is on Tenofovir, the methods established in this dissertation can readily be adapted to other drugs, given knowledge of their physical and chemical properties, such as the diffusion coefficient, partition coefficient, and reaction kinetics. Other dosage forms such as tablets and fiber meshes can also be modeled using the perspective and methods developed here.</p><p>The analyses here include convective details of intravaginal flows by both ambient fluid and spreading gels with different rheological properties and applied volumes. These are input to the overall conservation equations for drug mass transport in different compartments. The results are Tenofovir concentration distributions in time and space for a variety of microbicide products and conditions. The Tenofovir concentrations in the vaginal and rectal mucosal stroma are converted, via a coupled reaction equation, to concentrations of Tenofovir diphosphate, which is the active form of the drug that functions as a reverse transcriptase inhibitor against HIV. Key model outputs are related to concentrations measured in experimental pharmacokinetic (PK) studies, e.g. concentrations in biopsies and blood. A new measure of microbicide prophylactic functionality, the Percent Protected, is calculated. This is the time dependent volume of the entire stroma (and thus fraction of host cells therein) in which Tenofovir diphosphate concentrations equal or exceed a target prophylactic value, e.g. an EC50.</p><p>Results show the prophylactic potentials of the studied microbicide vehicles against HIV infections. Key design parameters for each are addressed in application of the models. For a vaginal gel, fast spreading at small volume is more effective than slower spreading at high volume. Vaginal rings are shown to be most effective if inserted and retained as close to the fornix as possible. Because of the long half-life of Tenofovir diphosphate, temporary removal of the vaginal ring (after achieving steady state) for up to 24h does not appreciably diminish Percent Protected. However, full steady state (for the entire stromal volume) is not achieved until several days after ring insertion. Delivery of Tenofovir to the rectal mucosa by an enema is dominated by surface area of coated mucosa and whether the interiors of rectal crypts are filled with the enema fluid. For the enema 100% Percent Protected is achieved much more rapidly than for vaginal products, primarily because of the much thinner epithelial layer of the mucosa. For example, 100% Percent Protected can be achieved with a one minute enema application, and 15 minute wait time.</p><p>Results of these models have good agreement with experimental pharmacokinetic data, in animals and clinical trials. They also improve upon traditional, empirical PK modeling, and this is illustrated here. Our deterministic approach can inform design of sampling in clinical trials by indicating time periods during which significant changes in drug concentrations occur in different compartments. More fundamentally, the work here helps delineate the determinants of microbicide drug delivery. This information can be the key to improved, rational design of microbicide products and their dosage regimens.</p> / Dissertation
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Characterization of Fc receptor family proteins in vaginal and endocervical epitheliaGubbala, Supreetha 22 January 2016 (has links)
In the age of highly active antiretroviral therapy (HAART), patients infected with Human Immunodeficiency Virus Type 1 (HIV-1) are now living significantly healthier and longer lives. However, HIV prevention and cure still remain significant challenges. Globally, women face specific barriers to using and accessing both female and male condoms, the primary method recommended by the World Health Organization (WHO) to prevent sexual transmission of HIV-1. Although HAART treatment as prevention (TasP) of HIV has shown promising preliminary results, poor economic feasibility of the method in resource poor settings has yet to be resolved. Since women carry over 50% of the disease burden, there is a significant need for the development of a female-controlled method of prevention. One such approach is the reformulation of topical vaginal microbicides. Our laboratory is developing HIV-targeted microbicide formulations that utilize highly specific, broadly neutralizing anti-HIV antibodies (bNAbs).
The purpose of my research project was to characterize Fc receptor expression in epithelial cell models of the lower female genital tract with a particular focus on the neonatal Fc receptor (FcRn). Fc receptors are a large family of proteins that bind to the Fc region of immunoglobulins (Igs) and function in Ig transport and effector functions. These receptors could function in enhancing the delivery of bNAbs in microbicide formulations or potentially serve as a mechanism of delivering HIV to target cells in tissues via transport of HIV-antibody complexes. Thus, this thesis assesses Fc receptor expression in human vaginal and endocervical organotypic cultures via microarray, quantitative RT-PCR, immunohistology and preliminary functional assays.
Microarray results revealed significant expression of Fc receptor family genes in the epithelial cells of the lower female reproductive tract (FRT). The polymeric immunoglobulin receptor (pIgR), a well-characterized receptor that transports secretory IgA across mucosal epithelia, was abundantly expressed and hormonally regulated in epithelial cells of the vagina and endocervix. FcRn, a receptor originally characterized in the placenta and gut where it confers passive immunity from mother-to-child via bidirectional IgG transcytosis, was expressed in both tissue models. Moreover, several members of the novel FC receptor-like (FCRL) family were detected by microarray in both models.
Immunohistological staining revealed pIgR protein in the endocervical mucosal epithelium, confirming current literature describing its expression in the FRT and role in local production of cervicovaginal secretions. FcRn protein expression was detected in the basal cell layer of the stratified squamous vaginal epithelium and in the columnar cells of the endocervix. Preliminary functional assays did not observe FcRn-specific transcytosis of human IgG across vaginal or endocervical epithelia by ELISA or immunohistology. VRCO1, a monoclonal antibody in development for application in microbicide formulation, crossed the epithelium, but was likely not transcytosed via FcRn because immunohistology revealed the presence of antibody between epithelial cells rather than the expected intracellular localization of IgG utilized in the FcRn mechanism.
These preliminary findings indicate that Fc receptors, pIgR and Fc receptor-like proteins may play an important role in antibody-mediated immune responses in the FRT. Further research is require to determine whether FcRn functions in HIV-antibody complex-mediated HIV transmission or monoclonal antibody transcytosis.
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Transport Phenomena in Anti-HIV Microbicide Delivery VehiclesGeonnotti, III, Anthony Robert 21 April 2008 (has links)
<p>There were 2.5 million people newly infected with HIV in 2007, clearly motivating the need for additional novel prevention methods. In response, topical vaginal antimicrobials, or microbicides, are being developed. These products aim to stop HIV transmission through local, vaginal delivery of antiviral compounds. To succeed, microbicides require a potent active compound within a well-engineered delivery vehicle.</p><p>A well-engineered delivery vehicle provides an antiviral compound with the greatest opportunity to interact with HIV and/or infected cells, thereby increasing overall microbicide effectiveness. The theoretical and experimental investigations within this dissertation are concerned with the study of HIV and active compound transport within microbicide delivery vehicles and with the mechanisms by which these transport processes can be affected to maximize viral neutralization. To initially investigate the factors contributing to microbicide effectiveness, a combined pharmacokinetic and pharmacodynamic model of HIV transport and neutralization within a microbicide product was created. Model results suggested that thin (~100µm) layers of microbicide product may protect against HIV infection. Model results also indicated that a specific and engineerable property of delivery vehicles - the ability to restrict viral transport - may increase the overall effectiveness of a microbicide. Two new experimental assays were developed to test the hypothesis that delivery vehicles can slow viral transport. First, a novel methodology was created to measure particle diffusion over length scales relevant to microbicide delivery (50-500µm). Results showed that current vehicles significantly restrict the transport of small molecules and proteins. The second assay was designed to test HIV transport in a biologically relevant, layered (fluid-microbicide-tissue) configuration of a microbicide product in vivo; infectious HIV was placed above a thin layer of a microbicide delivery vehicle. Assay results showed that HIV transport is significantly slowed by two different placebo gels. This experimental confirmation of viral restriction in hydrogels, combined with the theoretical finding that viral restriction increased microbicide effectiveness, strongly motivates the future development of new delivery vehicles that intentionally slow viral transport. These new experimental methodologies can also be used to screen and compare future delivery vehicles to produce optimal microbicide products.</p><p>Finally, a two-dimensional, computational finite-element vaginal model was created to evaluate the transport of drugs from an intravaginal ring. This model determined that while IVRs may be effective in the delivery of antiviral compound, their performance is influenced by the flow of vaginal fluid. The analysis also warns about the potential for local toxicity. </p><p>Well-engineered delivery vehicles are an essential component to microbicide performance because they maximize the opportunities for active compounds to interact with and neutralize HIV. The studies in this dissertation demonstrate that delivery vehicles have a significant effect on active compound and HIV transport. To create an effective microbicide, vehicle effects on transport processes must be well understood, purposefully engineered, and carefully optimized to ensure maximal interactions between antiviral compounds and virus. Directed engineering of delivery vehicles contribute to the foundation for microbicide success.</p> / Dissertation
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CD4 Aptamer-SiRNA Chimeras (CD4-AsiCs) Knockdown Gene Expression in CD4+ Cells and Inhibit HIV TransmissionWheeler, Lee Adam January 2012 (has links)
The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.
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Novel Biophotonic Imaging Techniques for Assessing Women's Reproductive HealthDrake, Tyler Kaine January 2013 (has links)
<p>Even though women make up over half the population in the United States, medical advancements in areas of women's health have typically lagged behind the rest of the medical field. Specifically, two major threats to women's reproductive health include human immunodeficiency virus (HIV), and cervical cancer with accompanying human papillomavirus (HPV) infection. This dissertation presents the development and application of two novel optical imaging technologies aimed at improving these aspects of women's reproductive health.</p><p>The presented work details the instrumentation development of a probe-based, dual-modality optical imaging instrument, which uses simultaneous imaging of fluorimetry and multiplexed low coherence interferometry (mLCI) to measure in vivo microbicide gel thickness distributions. The study explores the optical performance of the device and provides proof of concept measurements on a calibration socket, tissue phantom, and in vivo human data. Once the instrument is fully characterized, it is applied in a clinical trial in which in vivo human vaginal gel thickness distributions. The gel distribution data obtained by the modalities are compared in order to assess the ability of mLCI making accurate in vivo measurements. Differences between the fluorimetry and mLCI modalities are then exploited in order to show a methodology for calculating the extent of microbicide gel dilution with the dual-modality instrument data.</p><p>Limitations in cervical cancer screening are then addressed as angle-resolved low coherence interferometry (a/LCI) is used in an ex vivo pilot study to assess the feasibility of a/LCI in identifying dysplasia in cervical tissues. The study found that the average nuclear diameter found by a/LCI in the basal layer of ectocervical epithelium showed a statistically significant increase in size in dysplastic tissue. These results indicate that a/LCI is capable of identifying cervical dysplasia in ectocervical epithelium. The results of the work presented in this dissertation show that dual-modality optical imaging with fluorimetry and mLCI, and the a/LCI technique show promise in advancing technologies that are used in the field of women's reproductive health.</p> / Dissertation
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