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Role of translocator protein (18 kDa) in cell function and metabolism

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Role of translocator protein (18 kDa) in cell function and metabolism

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Content ROLE OF TRANSLOCATOR PROTEIN (18 kDa) IN CELL
FUNCTION AND METABOLISM
By
Priyadarshini Raju Bahadure
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master of Science in
Pharmaceutical Sciences
August 2024
Copyright 2024 Priyadarshini Bahadure

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Acknowledgments
I would like to thank Dr. Vassilios Papadopoulos for being my P.I. and my mentor. I'm
grateful for all the support and guidance he has provided me throughout. I would also like
to thank my labmates for supporting me and helping me with any queries.
I extend my gratitude to my committee members, Dr. Martine Culty and Dr. Daryl L.
Davies, for guiding me through the completion of my thesis.
Additionally, thanks to my friends at USC; with their support and motivation, I was able to
move forward personally and professionally.
Lastly, special thanks to my parents and siblings for constantly encouraging me, believing
in me, and helping me throughout this journey.

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Table of Contents
Acknowledgments…………………………………………………………………………..…ii
List of Tables…………………………………………………………………………………...v
List of Figures………………………………………………………………………………….v
Abbreviations…………………………………………………………………………………..viii
Abstract…………………………………………………………………………………………1
Chapter 1: Introduction……………………………………………………………………….2
Chapter 2: Location and Structure of TSPO……….…………………………………………3
2.1. TSPO Ligands………………………………………………………………………….6
Chapter 3: TSPO Expression and Regulation………………………………………………7
3.1 Expression of TSPO gene
Brain and Neuroinflammatory Marker Translocator Protein (TSPO) Expression in the
Normal Brain……………………………………………………………………………….…..7
Cellular Distribution………………………………………………………………………9
3.2 Regulation of TSPO gene expression………………………………………………..11
Regulation of TSPO expression in cancer cells………………………………………11
Expression of TSPO is regulated by chemicals, hormones, and environmental
factors…………………………………………………………………………………………...13
Chapter 4: Functional Roles of TSPO
4.1 Cholesterol Transport Role……………………………………………………………15
4.2 Impact on steroidogenesis………………………………………………….…………19
Chapter 5: TSPO in Cell Functionality
5.1 Role in Hepatic System………………………………………………………………..21

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5.2 Role in Nervous System………………………………………………………………22
5.3 Role in Endocrine System………………………………………………………….…23
Chapter 6: TSPO and Metabolism
6.1 Role in Energy Metabolism………………………………………………………. …..24
6.2 Association with metabolic diseases…………………………………………………26
Chapter 7: Modulation of TSPO activity
7.1 Pharmacological Agents - Overview of TSPO ligands.…………………………….28
Impact on the Ca++ homeostasis………………………………………………………29
Effects of TSPO ligands on mitochondrial respiration…………….…………………30
7.2 Use of TSPO knockout and transgenic models
Anxiety-related behavior in tspo knockout mice………………………………………31
Chapter 8: Clinical Implications of TSPO Modulation
8.1 Neuropsychiatric Disorders……………………………………………………………33
8.2 Neurodegenerative Diseases………………………………………………………….34
8.3 Inflammatory and Autoimmune Diseases……………………………………………36
Chapter 9: Future Direction and Challenges……………………………………………….37
9.1 Mechanistic Understanding
9.2 Emerging Research Areas
9.3 Challenges in TSPO Research
Chapter 10: Conclusion………………………………………………………………………40
List of References……………………………………………………………………………..42

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List of Tables
Table 1: Outcome of using TSPO ligands in metabolic disorders……………….………27
Table 2: Regarding the study that used TSPO ligand to study Neuroinflammation……35
List of Figures
Fig. 1: TSPO location, structure and function……………………………………………….5
Fig. 2: The two archetypal TSPO ligands, (A) benzodiazepine Ro5-4864 and (B)
isoquinoline carboxamide PK 11195, have different chemical structures…………………6
Fig. 3: Immunofluorescence overview of main brain areas' expression of TSPO
(translocator protein 18 kDa) ............................................................................................8
Fig. 4: Major cell types in the normal mouse brain express Tspo. (A) CD31+ vascular
endothelial cells (green) and Tspo (red) show strong colocalization when punctate
staining is used for double immunofluorescence staining. Larger blood arteries,
capillaries, and smaller arterioles and venules all exhibit this. Localization of the
mitochondria can be determined by punctate staining. (B) On the walls of larger blood
vessels, Tspo expression (red) and PDGFRβ+ pericytes/smooth muscle cells (green)
colocalize. (C) No easily noticeable Tspo expression (red) is seen in CD11b+ microglia
(green)............................................................................................................................10
Fig. 5: Samples from biopsies of both normal and metastasized breast tumors showed
evidence of TSPO gene amplification. Biotin-16-dUTP was used to identify DNA, and it
was then hybridized with interphase nuclei made from human biopsies………………..12

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Fig. 6: Decrease in the efflux of cholesterol in Primary Retinal Pigment Epithelial cells
(WT, wild type) and KO, knockout cells, followed by 24 hours of incubation. APOA1:
Apolipoprotein; HDL: High-Density lipoprotein; HS: Human Serum……………………..16
Fig. 7: Total cholesterol, Triglycerides, and phospholipids in (A) sclera for WT (wild type)
and KO (knockout) for 6, 12, and 18 months……………………………………………….17
Fig. 8: Total cholesterol, Triglycerides, and phospholipids in (B) retina for WT (wild type
and KO - Knockout for 6, 12, and 18 months……………………………………………….17
Fig. 9: Total cholesterol, Triglycerides, and phospholipids in (C) Brain for WT - Wild type
and KO - Knockout for 6, 12, and 18 months……………………………………………….18
Fig. 10: Outcome of binding to TSPO results in steroidogenesis, apoptosis or immune
response………………………………………………………………………………………..19
Fig. 11: Diagram in left: H-score and typical IHC pictures of TSPO from TMA in liver
tissues from normal liver and liver cancer tissues; Right: qRT-PCR was used to assess
the expression of TSPO mRNA in matched HCC tissues and nearby non-tumor
tissue……………………………………………………………………………………………21
Fig. 12: (I) Relative TSPO expression of 6 HCC cell lines; (J) To investigate TSPO
expression in TSPO knockdown; (K) To investigate in TSPO overexpression…………22
Fig. 13: Ro5-4864 and PK 11195 are examples of synthetic ligands that compete with
natural ligands to control downstream processes including the synthesis of (neuro-)
steroids, the metabolism of mitochondrial energy, or an unidentified role………………26
Fig. 14: Effect of LPS or TSPO ligands present in mouse BV-2 cell expression of the
mitochondrial TSPO protein…………………………………………………………………..28
Fig. 15: Changes in Ca++ homeostasis due to FCCP loaded BV- 2 cells………………29

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Fig. 16: Impact of TSPO ligands on the rate of oxygen consumption……………………30
Fig. 17: Behavioral changes observed comparison between wild type and tspo knockout
mice……………………………………………………………………………………………..32
Fig. 18: Impaired neurosteroidogenesis in Tspo-KO mice………………………………...33
Fig. 19: TSPO PET-based in vivo differentiation of persistent T1 lesions……………….36
Fig. 20: Basic Function of TSPO gene………………………………………………………40

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List of Abbreviations
CD31+: Cluster of Differentiation 31 Positive
CNS: Central Nervous System
CRAC: Cholesterol-Recognition Amino Acid Consensus
CYP11A1: Cytochrome P450 Family 11 Subfamily A Member 1
DAA1097 : DAA-1097 Compound
DAA1106 : DAA-1106 Compound
DBI: Diazepam Binding Inhibitor
FCCP: Carbonyl Cyanide-p-Trifluoromethoxyphenylhydrazone
FGIN-1-27 : N,N-Dihexyl-2-(4-fluorophenyl)indole-3-acetamide
HLA+: Human Leukocyte Antigen Positive
HS: Human Serum
OCR: Oxygen Consumption Rate
PD-L1: Programmed Death-Ligand
PPRE: Peroxisome Proliferator Response Element
TMEM119+: Transmembrane Protein 119 Positive
TNF: Tumor Necrosis Factor
TRT: Testosterone Replacement Therapy
TSPO-PET: Translocator Protein Positron Emission Tomography
VDAC: Voltage-Dependent Anion Channel

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Abstract
Translocator protein (TSPO, 18 kDa), formerly known as the peripheral-type
benzodiazepine receptor (PBR), is a highly conserved protein located in the outer
mitochondrial membrane. It plays a pivotal role in various cellular functions and metabolic
processes, including cholesterol transport, steroidogenesis, apoptosis, and immune
response modulation. This review thesis provides an understanding of TSPO's structure,
function, and regulatory mechanisms, highlighting its significance in the peripheral tissues
as well as the central nervous system. The review also explores the modulation of TSPO
activity by different ligands, discussing their effects on cellular processes and potential
therapeutic applications. TSPO ligands have shown promise in treating various diseases,
including neurodegenerative disorders, psychiatric conditions, cancer, and inflammatory
diseases. Additionally, the review addresses the technical and conceptual challenges in
TSPO research, such as the lack of highly specific ligands, the complexity of
mitochondrial biology, and the variability in TSPO expression and function across different
tissues and pathological states. Future research directions are proposed to bridge the
current gaps in understanding TSPO's diverse roles and enhance its therapeutic
potential.
Keywords - TSPO, TSPO Ligands, Outer mitochondrial membrane, Steroidogenesis

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1. Introduction
Translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor
(PBR), is a highly conserved protein located in the outer mitochondrial membrane1. It
plays a pivotal role in various cellular functions and metabolic processes, including
cholesterol transport, steroidogenesis, apoptosis, and immune response modulation1.
The TSPO gene encoding the translocator protein in humans is a member of the
tryptophan-rich sensory protein family2. It is present in the mitochondria of both peripheral
organs and the central nervous system (CNS), expressed in neurons, endothelial cells,
glial cells (astrocytes and microglia), and tanycytes3. The significant expression in glial
cell mitochondria necessitated the reclassification from the "peripheral benzodiazepine
receptor" to TSPO, underscoring its presence in both the CNS and peripheral tissues3.
TSPO comprises five transmembrane domains and 169 amino acid residues². It is highly
conserved across species, with mammalian TSPO showing structural and functional
similarities to the non-photosynthetic eubacterium Pseudomonas fluorescens, including
binding affinity for the TSPO ligand PK111952. In bacteria, Tspo homologues function as
oxygen sensors and photosynthetic regulators, and in mammals, TSPO similarly acts as
an oxygen sensor¹. There are two TSPO genes in plants and animals, Tspo1 and Tspo2,
with Tspo2 resulting from gene duplication before the divergence of mammals and birds3.

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TSPO is crucial for cholesterol translocation across the outer mitochondrial membrane, a
key step in the production of pregnenolone and neuroactive steroids2. It is implicated in
various physiological processes, including steroidogenesis in Leydig cells, which produce
testosterone in response to luteinizing hormone (LH) stimulation². TSPO ligands have
shown promise in treating neurodegenerative disorders, psychiatric conditions, cancer,
and inflammatory diseases by modulating neuroinflammation and microglia activation¹².
In animal models, TSPO ligands like PK11195 and XBD173 have demonstrated
neuroprotective effects reducing amyloid-beta deposition and preserving dopaminergic
neurons4.
TSPO ligands modulate its activity, influencing various cellular processes and showing
potential therapeutic applications3. These ligands, including PK11195, Ro5-4864, and
XBD173, enhance cholesterol transport into mitochondria, promoting steroidogenesis
and increasing testosterone production¹³. Additionally, Tspo ligands have demonstrated
anxiolytic effects in animal models of anxiety and neurodegenerative diseases by
reducing neuroinflammation and enhancing mitochondrial steroidogenesis².
2. Location and structure of TSPO
TSPO is found in various tissues across the body, predominantly located in mitochondrial
membranes¹. The name "translocator protein" reflects TSPO's function in transporting
substances across the outer mitochondrial membrane¹. In the mitochondria, TSPO is
closely associated with the 30-kDa adenine nucleotide translocator (ANT) and the 32-kDa
voltage-dependent anion channel (VDAC), which are key components of the

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mitochondrial permeability transition pore (mPTP)¹. The proportion of TSPO to VDAC and
ANT varies with tissue type¹.
Fig. 1: TSPO location, structure and function1
Research has shown that TSPO is involved in the production of reactive oxygen species
(ROS) within mitochondria, which play a role in cardiovascular function2. Additionally,
TSPO influences the release of cytochrome c from the mitochondrial membrane potential
and initiates the mitochondrial apoptosis cascade. Through interactions with VDAC and
ANT, TSPO regulates the flow of electrolytes across mitochondrial membranes¹. TSPO
is also reported to facilitate the transport of proteins and cholesterol into mitochondria,
contributing to mitochondrial membrane biogenesis, crucial for cell division and growth2.

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Tspo expression varies across different species, being found in insects, fish, amphibians,
birds, and mammals but not yet discovered in reptiles. The conserved Tspo gene is
present in animals, plants, and prokaryotes, suggesting a fundamental biological role¹
Furthermore, the possibility of free TSPO—that is, TSPO not in complex with VDAC and
ANT—existing in mitochondrial membranes was raised5. It has been shown that TSPO
can also exist as 36-, 54-, and 72-kDa TSPO polymers in addition to its 18-kDa form5.
This assertion further considers the fact that VDAC and ANT are not necessary for the
control of steroidogenesis; rather, they require the formation of a mitochondrial channel
by the mitochondrial TSPO6. The TSPO to VDAC and ANT ratio in these regions may be
connected to this ability6. Other research found in the adult red blood cells, which lack
mitochondria, TSPO is attached to the plasma membrane5.
2.1 TSPO ligands
A variety of molecules that have affinity with TSPO have been found. The porphyrins
(Protoporphyrin IX, Mesoporphyrin IX, Deuteroporphyrin IX, and Haemin) are other
possible endogenous ligands for TSPO7. These proteins are known to interact with other
mitochondrial proteins and alter the enzymatic activity of various enzymes7. Another
natural TSPO ligand is the Diazepam Binding Inhibitor (DBI). As its name implies, DBI
was first demonstrated to prevent [3H] diazepam from attaching to brain membranes and
to induce Cl-channel activation when gamma-aminobutyric acid (GABA) was present7.
These molecules are known as probable endogenous TSPO ligands because of their
affinity for TSPO¹. Presently, the most popular TSPO ligand is PK11195¹. Because of

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their strong affinity for TSPO across all investigated species, isoquinolines, including 01-
(2-chlorophenyl)-N-methyl-N-(1-methyl-prop 1)-3 isoquinoline-carboxamide (PK 11195),
interact with TSPO in a particular way7.
Fig. 2: The two archetypal TSPO ligands, (A) benzodiazepine Ro5-4864 and (B) isoquinoline carboxamide
PK 11195, have different chemical structures2
In binding studies, ligands PK11195 and ligand Ro5-4864 compete with one another,
indicating overlapping but maybe distinct binding sites¹. These results suggest that these
TSPO ligands inhibit the pro-apoptotic properties of TSPO, as they induce responses
comparable to TSPO knockdown, albeit not as strong¹. Ro5-4864 has been shown
through behavioral experiments to have anxiogenic and convulsive qualities; however, it
has been found that PK11195 possesses anxiolytic and anticonvulsant properties¹.
Furthermore, the potential therapeutic implications of TSPO pharmacological ligands for
cancer therapy are assessed based on their capacity to identify tumor cells in vivo, control
apoptotic rates, and control the production of neurosteroid and brain activity. TSPO

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ligands may function as prospective therapeutic agents that could be helpful for the
treatment of a wide range of disorders, according to several reports2.
3. TSPO Expression and Regulation
3.1 Expression of TSPO gene
● Brain and Neuroinflammatory Marker Translocator Protein (TSPO)
Expression in the Normal Brain
In this investigation, the global TSPO deletion mouse model has been utilized to confirm
the TSPO antibody accuracy (#ab109497, Abcam)3. Expression of Tspo in a typical
mouse brain was precisely distributed throughout the major brain areas, and the cell types
exhibiting Tspo expression were identified using immunohistochemistry3. The brain
parenchyma showed a sparse, dispersed, punctate immunohistochemistry stain. The
expression of Tspo across the whole brain was ascertained by obtaining whole-tissue
images of sagittal brain slices3.
As illustrated in Fig. 3 (below), in comparison to gray matter, more TSPO immunostaining
was observed in the brain's white matter areas. Compared to the various parts of the
brain, the cerebellum has a relatively high density of TSPO, as shown in Figs. 3A and
3B3. Fig. 3C shows that the brains of the TSPO knockout mice are devoid of TSPO
immunoreactivity3. TSPO expression was also seen in the olfactory bulb's subependymal
zone (Fig. 3D), the dentate gyrus of the hippocampal region (Fig. 3F), the subventricular

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zone (Fig. E), and the cerebellar cortex's layer (Fig. 3G)3.
Fig. 3: Immunofluorescence overview of main brain areas' expression of TSPO (translocator protein 18 kDa)3
● Cellular Distribution
The diagram shows that low levels of Tspo are found with GFAP-positive cells in the
subependymal zone, which likely indicates TSPO expression in certain neural progenitor
types in that area (Fig. 3B)3. Significant Tspo presence is seen in the glomerular layer,
subependymal zone, rostral migratory stream (RMS), and olfactory nerve layer of the
olfactory bulb, involving a subset of neural progenitors and new neurons (Fig. 3A)3. In Fig.
3C, the olfactory part of the RMS is Tspo-positive and colocalizes with Nestin neural
stem/progenitor cells as it enters the olfactory bulb's subependymal zone3.

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Research indicates that in vascular endothelial cells, Tspo expression results in a distinct
immunohistochemical staining pattern throughout the brain parenchyma, reflecting
mitochondrial location3. This study extends previous research by mapping Tspo
expression across various brain regions and cell types in a normal mouse brain. It
confirms that Tspo levels in the olfactory nerve layers, glomeruli of the olfactory bulb,
choroid plexus, and ependymal layers are comparable to those in peripheral organs, while
lower Tspo levels are observed in cerebellar Purkinje cells².
Fig. 4 - Major cell types in the normal mouse brain express Tspo. (A) CD31+ vascular endothelial cells (green) and
Tspo (red) show strong colocalization when punctate staining is used for double immunofluorescence staining. Larger
blood arteries, capillaries, and smaller arterioles and venules all exhibit this. Localization of the mitochondria can be
determined by punctate staining. (B) On the walls of larger blood vessels, Tspo expression (red) and PDGFRβ+

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pericytes/smooth muscle cells (green) colocalize. (C) No easily noticeable Tspo expression (red) is seen in CD11b+
microglia (green)3
As Tspo is distributed separately expressed in endothelial mitochondria across the normal
brain, Tspo molecular imaging studies should result in a low baseline signal3. To ascertain
the cellular origins and regional distribution of Tspo in the normal mouse brain, this work
employed immunohistochemistry3.
3.2 Regulation of TSPO gene expression
TSPO is distributed and regulated in healthy tissues, being present in almost all tissues.
Cardiac and renal tissues exhibit intermediate levels of TSPO, while the liver and brain
show lower levels5. TSPO is particularly abundant in secretory and glandular tissues,
including the pineal gland, salivary glands, olfactory epithelium, ependyma, and gonads5.
In the kidney, TSPO is specifically found in the thick ascending loop of the loop of Henle,
the distal convoluted tubules, and the testis’ Leydig cells4. The cortex has high levels of
TSPO expression. Remarkably, TSPO is also found in adult human erythrocytes, which
lack mitochondria and nuclei8. Additionally, TSPO is localized both perinuclearly and
nuclearly in breast cancer cells8. These findings suggest that differences in transcriptional
regulation may explain some of the tissue-specific variations in TSPO expression8.
Adrenal gland, kidney, spleen, lung, skeletal muscle, heart, and testis have moderate
amounts of steady-state mRNA, whereas the liver and brain have low levels9.
● Regulation of TSPO expression in cancer cells

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TSPO expression was enhanced in aggressive metastatic MDA-MB-231 cells compared
to the MCF-7 human breast cancer cells10. These findings imply that TSPO gene
amplification may play a role in the enhanced expression of TSPO in cancer cells and
may serve as a key marker of the advancement of breast cancer⁵. The enhancement or
proliferation of the gene expression is also used as a marker for the detection of the
disease⁵. The mechanism for regulating the TSPO genes was also studied⁵. The cell lines
were compared, and mammary epithelial cells from humans showed that the TSPO
gene's transcription starts at several locations within an about 40–50 bp window that
maps both inside and next to the anticipated first exon, all of which are driven by a similar
promoter8.
Fig. 5 - Samples from biopsies of both normal and metastasized breast tumors showed evidence of TSPO gene
amplification. Biotin-16-dUTP was used to identify DNA, and it was then hybridized with interphase nuclei made from
human biopsies8
Epigenetic mechanisms are also investigated to understand the TSPO expression⁷. The
paper used 5-Azacytidine to treat different cell lines that were transfected with the TSPO

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promoter in order to look into whether methylation contributes to the different ways that
TSPO is expressed in different tissues⁷. When introduced to cells, this substance
integrates into DNA and suppresses DNA methylation, offering a useful method to
examine the demethylation of particular gene areas and the activation of the
corresponding genes11. One of the most well-studied processes of epigenetic control is
methylation, which involves adding a methyl group to the cytosine pyrimidine ring's fifth
carbon in a CpG island to decrease gene expression12.
● Expression of TSPO is regulated by chemicals, hormones, and environmental
factors
Import of cholesterol is facilitated with the help of TSPO from the outer to the inner
mitochondrial membrane13. Thus, this makes this a rate-determining and hormonesensitive step. Hormones are essential for constitutive TSPO expression because
hypophysectomy tests revealed large reductions in TSPO in the ovary, testis, and adrenal
glands13. Tspo mRNA and protein levels were unaffected by acute administration of
human choriogonadotropin or adrenocorticotropin to Leydig and adrenal cells,
respectively; nevertheless, the protein's structure was altered, and a higher affinity
binding site developed14. Considering that TSPO binding was reduced by acute ANG II
injection in the kidney, heart, and brain cortex, it has been proposed that ANG II functions
as an endogenous regulator of TSPO in several organs during stress⁹. Stress-inducing
conditions such as forced swimming raises the density of TSPO ligand binding in the
kidney and cerebral cortex of rats⁸. There are plenty of environmental factors like these
that disrupt TSPO's normal expression⁹. According to reports, flavonoids cause human

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neuroblastoma and SNU-CF colorectal adenocarcinoma cells to produce more Tspo
mRNA transcription, which raises apoptosis rates and cytotoxicity, respectively⁹.
The commonly used fibrate hypolipidemic medications bezafibrate and clofibrate are
examples of peroxisome proliferators (PPs), a large family of chemical substances used
in industry as corrosion inhibitors, wetting agents, lubricants, surfactants, and phthalate
ester plasticizers. In rats, PPs have been demonstrated to have a substantial impact on
the expression of many genes related to lipid metabolism¹⁰. These effects are attributed
to the activation of certain receptors known as PP-activated receptors (PPARs). Leydig
cells produce these proteins in the form of PPARɑ and PPARβ, and most recently, PPARγ
has been found expressed in MA10 cells¹⁰. Mechanisms that negatively regulate gene
expression, in contrast to those that positively regulate it through interactions between
PPAR-RXR and positive PP response element (PPRE) sequences, remain poorly
understood. These receptors may be essential for the chemical and pharmacological
regulation of gene expression because of their ability to be triggered by industrial
chemicals and pharmaceutical agents¹⁰. Through several mechanisms of action, PPs
control the production of TSPO by lowering the amount of Tspo mRNA and inhibiting
transcriptional or post-transcriptional processes15.
Based on evidence to date, it will be important to increase our understanding of how
TSPO is controlled in both health and sickness for prognostic, therapeutic, diagnostic,
and preventive purposes because it is linked to a variety of physiological activities and
diseases8. TSPO is regarded as a housekeeping gene, although hormones can also

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control it. Different substances, pathological states, environmental agents, stress, and
other factors can influence TSPO levels8.
4. Functional Roles of TSPO
4.1 Cholesterol Transport Role
TSPO is thought to play roles in regulating the mitochondrial transition pore, steroid
synthesis, calcium homeostasis, ROS formation, and energy production from nutrients¹¹.
TSPO is made up of five transmembrane domains with α helices and has a C-terminal
cholesterol-recognition amino acid consensus (CRAC) sequence that bends in the
direction of the lipid membrane16. The 169-amino acid protein is nuclear gene-encoded
with four exons and is located in the outer mitochondrial membrane¹¹. TSPO's primary
function is transporting cholesterol across mitochondrial membranes, where it is
converted to oxysterols in non-steroidogenic cells or steroids in steroid-producing cells¹¹.
Studies have shown that deletion of the Tspo gene reduces cholesterol efflux and leads
to lipid accumulation¹¹. In aging rats, Tspo expression is downregulated, resulting in
decreased cholesterol flow in RPE cells¹¹. Elevated oxidized low-density lipoprotein (LDL)
absorption and accumulation, as well as increased generation of proinflammatory

15
cytokines are the results of Tspo deletion17.
Fig. 6 Decrease in the efflux of cholesterol in Primary Retinal Pigment Epithelial cells (WT, wild-type) and KO,
knockout cells, followed by 24 hours of incubation. APOA1: Apolipoprotein ; HDL: High-Density lipoprotein ;
HS: Human Serum17
The Fig. 6 given above shows that the cholesterol efflux malfunctioned due to Tspo
depletion. The experiment was analyzed using a two-way ANOVA and then a Bonferroni
test17. As seen, the percentage of apo-A-I, HDL, and HS was reduced for cholesterol
efflux in Tspo knockout mice RPE cells compared to their wild-type17.
It was also observed that because of Tspo loss was associated to lipid accumulation. This
was shown by measuring different levels of triglycerides, phospholipids, and cholesterol
mass in the RPE, choroid, sclera, retina, and brain at the age of 6, 12, and 18 months in
rodents with Tspo gene knockout, compared to wild type17. The amount of all three
drastically increased compared to their wild-type counterparts as shown in the Fig. 7

16
below17.
Fig. 7 - Total cholesterol Triglycerides and phospholipids in (A) sclera for WT - Wild type and KO - Knockout for 6,12,
and 18 months17
For Tspo KO mice, all three were shown a significant increase compared to their wild
type17.
Fig. 8 - Total cholesterol Triglycerides and phospholipids in (B) retina for WT - Wild type and KO - Knockout for 6,12,
and 18 months17
For cholesterol, there is not much difference for 18 months, but there is a visible increase
for 6 and 12 months compared to the WT mice17. When comparing Tspo KO retinas to
those of WT animals, phospholipid levels only started to rise noticeably at the 6-month

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mark. When compared to WT mice, the retinas of Tspo KO animals showed noticeably
higher amounts of triglycerides at all age periods17.
Fig. 9 - Total cholesterol Triglycerides and phospholipids in (C) Brain for WT - Wild type and KO - Knockout for 6,12,
and 18 months17
For Tspo KO brain tissue, at all ages, the brains of Tspo KO mice had noticeably higher
levels of phospholipids and triglycerides than the brains of WT mice17. In contrast to the
brains of WT animals, the brain tissue of Tspo KO animals showed significantly higher
cholesterol levels at 6 and 18 months, but not at 12 months17.

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4.2 Impact on steroidogenesis
Fig. 10: Outcome of binding to TSPO results in steroidogenesis, apoptosis or immune response18
Building evidence indicates that certain cancer cell types can undergo apoptosis when
exposed to steroids like progesterone19. For example, steroids may cause C6 glioma cells
to undergo programmed cell death, according to multiple studies19. Moreover, it has been
demonstrated that pregnenolone sulfate causes retinal cell apoptosis19. Because TSPO
is well recognized to be involved in the endocrine system's steroid synthesis, it is possible
that it also contributes to the host-defense response5. For instance, systemic steroid
levels rise right away in response to several cytokines that stimulate the release of
corticotropin-releasing hormones following an injury, pain, fever, and hypovolemia. The
amount of stress usually causes these rises in steroid levels, with serum cortisol levels
peaking in patients who are moribund and just before they pass away¹¹. Steroids,

19
because of their anti-inflammatory properties, are considered chemical adjuvants for the
treatment of systemic inflammation20.
The primary regulatory mechanism for the rapid increase in steroid production is the
conversion of cholesterol to pregnenolone, facilitated by the cytochrome P450 enzyme,
which cleaves the C27 side chain of cholesterol18. The movement of the precursor,
cholesterol, from intracellular reserves into mitochondria is the pathway's ratedetermining phase18. The final steroid molecules are produced by enzymatic modification
of pregnenolone in the endoplasmic reticulum after it exits the mitochondrion. TSPO is
crucial for steroid production, as it helps convert cholesterol to pregnenolone, especially
when bound by certain high-affinity ligands. Higher TSPO levels in steroid-producing
tissues like the adrenal glands and ovaries are associated with increased
steroidogenesis18.
Both Ro5-4864 and PK11195 (TSPO ligands) strongly increased ACTH and
corticosterone secretion in vivo in a dose-dependent manner. Placental explants exposed
to modest doses (10-8 M) of Ro5-4864 secreted progesterone and 17-estradiol into the
media at significantly higher rates (2.4 and 1.4 times, respectively)21 . The Kd of TSPO
seemed to be closely correlated with the actions PK11195 and Ro5-4846 on aldosterone,
suggesting a function of TSPO in steroidogenesis18.

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5. TSPO in Cell Functionality
5.1 Role in the Hepatic System
Primary liver cancer, which accounts for the third most common type of cancer, is
hepatocellular carcinoma (HCC)¹⁴. There is limited available therapy for this type of
cancer, and the survival rate is only three months, according to reports¹⁴. Blocking the
PD-L1 pathway is one of the cancer treatment mechanisms, but it works only in a limited
percentage¹⁴. Numerous cancer types have been shown to have elevated Tspo
expression, which is linked to tumor development and a bad prognosis¹⁴.
Fig. 11 - Diagram in left : H-score and typical IHC pictures of TSPO from TMA in liver tissues from normal liver and
liver cancer tissues ; Right : qRT-PCR was used to assess the expression of Tspo mRNA in matched HCC tissues
and nearby non-tumor tissues22
One of the studies that looked at TSPO as a possible therapy or treatment for HCC is
shown in the diagram above (Fig. 11)22. It is shown that in tumors of individuals at risk for

21
HCC, TSPO mRNA expression increases dramatically. Western blot and Q-PCR were
used to examine Tspo expression22. Using tissue microarrays (TMA) including 85 pairs
of HCC samples, immunohistochemistry was performed22.
Fig. 12 - I : Relative TSPO expression of 6 HCC cell lines; J: To investigate TSPO expression in TSPO knockdown;
K: To investigate TSPO overexpression22
In the diagram above, to prove that TSPO can be used as a potential biomarker for
treatment and prognosis, experiments are performed. For Fig. 12I, out of 6 HCC cell lines,
relatively high TSPO expression in HCCLM3 and MHCC97H cells, mediocre expression
in Li-7 and PLC/PRF/5 cells, and less expression in Huh7 and Hep3B cells. Thus it proves
that TSPO is a potential biomarker22.
5.2 Role in Nervous System
Positron emission tomography (PET) imaging is used to measure TSPO and monitor
inflammation. In multiple sclerosis, the PET signal is commonly interpreted as

22
representing pathogenic microglia; however, pathology investigations have revealed a
more diffuse cellular expression23. The expression of TSPO in scattered HLA+ cells
throughout the central nervous system (CNS) in MS tissues that appear normal is around
20 times greater in active lesions and the margin of persistent active lesions than in
normal-appearing white matter24.
Furthermore, T and B cells in the CNS express TSPO in MS, hence TSPO PET imaging
must take this cellular expression into account during the disease5. In MS lesions, TSPO
is expressed by a portion of TMEM119+ and P2RY12+ cells, which are indicators of
homeostatic microglia, proving that pathogenic microglia are not the exclusive reflection
of TSPO PET, even though the precise source of TSPO overexpression in MS in the
central nervous system is yet unclear5.
Research showed that expression happens in astrocytes as well as microglia, mostly in
long-term active and dormant lesions25. Additionally, the astrocyte signal has a major
impact in both the periphery of chronic active lesions and active lesions25. This work also
shows that TSPO is expressed in intermediate microglia/macrophages and some M1
(pathogenic) and M2 (immune-regulatory) traits, but not all of them26.
5.3 Role in Endocrine System
Aging men frequently suffer from hypogonadism; 20–50% of men over 60 are said to have
serum T levels that are noticeably lower than those of young men27. Primary
hypogonadism causes reduced Leydig cell secretion activity, and secondary
hypogonadism is a condition when luteinizing hormone is reduced28. T replacement

23
therapy aims to increase serum T levels into the eugonadal range, which will lessen
hypogonadism symptoms and enhance the quality of life27.
It was observed that Leydig cell TSPO levels decline with age, affecting androgen
formation29. Subsequent research investigated whether direct pharmacological activation
of TSPO in aged cells could increase testosterone production30.
To do this, the authors investigated the effects of T production by Leydig cells isolated
from elderly Brown Norway rats (21 months of age), as well as the effects of administering
FGIN-1-27 (N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide), a high-affinity Tspo drug
ligand, on these animals in vivo30. The blood T level of old rats increased to that of young
rats when FGIN-1-27 was administered to them in vivo30. The impact of stimulation of
FGIN-1-27 was reduced by the CRAC domain inhibitor of the Tspo cholesterol
recognition/interaction amino acid consensus30. In pre-clinical studies, these ligands have
been used to increase neurosteroids in males displaying signs of neurological and mental
illnesses31.
6. TSPO and Metabolism
6.1 Role in Energy Metabolism
The levels of TSPO protein and the binding of specific radiolabeled ligands are
significantly elevated in microglia throughout their transition from a quiescent to an active,
rapidly growing cell type32. Large amounts of energy are needed for this cellular transition
to occur, both for cell division and the disintegration of macromolecules to create carbon
building blocks for development and membrane repair. The most well-explained

24
mechanism for the dynamic regulation of TSPO is the local microglia's activation in
response to neuronal inflammation or brain injury33.
The ability to understand the requirement of Tspo for metabolic shift was examined by
the generation of Tspo − / − mice34. 0–2 day-old pups' brains were used to separate
primary microglia cells, and the oxygen consumption of these cells was examined to
determine the oxidative phosphorylation rate and the cells' ability to create ATP34. It's
interesting to note that the authors demonstrate that Tspo − / − cells had a markedly lower
basal oxygen consumption34. All things considered, these findings imply that Tspo is not
necessary for microglia activation following brain injury, yet it is not ruled out by the
growing population33.
In a separate study, the mitochondria's oxygen consumption obtained from mice lacking
Tspo in the liver for the entirety of their lives was measured35. There were no discernible
variations in the respiratory rate between control mitochondria and liver-specific Tspo
knockout mitochondria. Subsequent studies will determine whether hepatocytes can
sustain cellular respiration without the help of Tspo36.

25
Fig. 13 - Ro5-4864 and PK 11195 are examples of synthetic ligands that compete with natural ligands, to control
downstream processes including the synthesis of (neuro-) steroids, the metabolism of mitochondrial energy, or an
unidentified role36
TSPO is a member of a protein network that adjusts the flux of cytosolic metabolites to
the function of mitochondria36.
6.2 Association with Metabolic Diseases
Several investigations tested the therapeutic benefits of TSPO ligands in various
disorders; surprisingly, and positive results were reported in a surprising number of these
trials.

26
Table 1 - Outcome of using TSPO ligands in metabolic disorders36
TSPO ligand have been shown as an improved hallmark of the disorders. They have
shown reduced symptoms for the disorders36.

27
7. Modulation of TSPO Activity
7.1 Pharmacological Agents - Overview of TSPO ligands
Fig. 14 - Effect of LPS or TSPO ligands present in mouse BV-2 cell expression of the mitochondrial Tspo protein37
Understanding how TSPO ligands affect mitochondrial expression in microglial cells is
made easier by the preceding figure. The ligands of TSPO were incubated along BV-2
wild-type cells for 24 hours37. The cells were also incubated with prototypical endotoxin
LPS to cause an inflammatory reaction. All three ligands (XBD173, PK11195 and Ro5-
4864) revealed a modest rise in the expression of the protein but did not reach
significance37. But when cells were incubated with LPS, cells reached significance along
with expression increase37.

28
● Impact on the Ca++ homeostasis
The amount of calcium ions that mitochondria store or release is known as their Ca2+
buffering capacity38. This capacity has both positive and negative impacts because it
helps maintain Ca2+ homeostasis and causes apoptosis when excitotoxicity-induced Ca2+
overload occurs38. For studying the Ca++ homeostasis and its effects on TSPO ligands,
the dye used was ratiometric Ca2+ sensitive dye Fura-2/AM, on the cells Tspo knockdown
and scr-shRNA-treated BV-2 cells38. As a result, Ca2+ is released from the cellular matrix
of mitochondria when FCCP is administered and quantified as a brief rise in cytosolic
[Ca2+]
38. By subjecting the cells to the uncoupling agent FCCP (10–20 μM), which helps
dissolve the mitochondrial proton gradient and causes the MMP to dissipate, this capacity
can be examined38. Ro5-4864's TSPO-dependent impact on BV-2 cells' Ca2+
homeostasis is visible and has some effect compared to the control38.
Fig. 15 - Changes in Ca++ homeostasis due to FCCP loaded BV- 2 cells37

29
● Effects of TSPO ligands on the mitochondrial respiration
Tspo ligands' impact on mitochondrial respiration in BV-2 cells was examined by
comparing the oxygen consumption rate (OCR) of knockdown of non-permeabilized Tspo
cells to scr-shRNA-treated control cells using the Seahorse Flux Analyzer (Agilent
Technologies)39. This study shows that TSPO expression levels affect mitochondrial
dynamics37. Proton leakage and maximum respiration were impacted by Ro5-486437.
Proton leak and maximum respiration were impacted by Ro5-4864 (100 nM)37. There was
no discernible difference in the respiration of Tspo knock-down and scramble BV-2 cells
when XBD173 was applied to the cells37.
Fig. 16 - Impact of Tspo ligands on the rate of oxygen consumption37

30
Regarding the effect of pharmacological therapy, the study found that, in scramble BV-2
cells, but not in Tspo-deficient cells, XBD173, Ro5-4864, and PK11195 significantly
altered basal respiration, ATP-related respiration, and spare capacity37. This implies that
the related respiratory parameters are impacted by these ligands in a way that is particular
to TSPO37.
7.2 Use of Tspo knockout and transgenic models
● Anxiety-related behavior in Tspo knockout mice
For the study described below examined anxiety-related behavior in Tspo
knockout mice40. A mouse model was generated by deleting Tspo exons 2 and
340. Increased anxiety was observed in the changes of the behavioral pattern in
the mouse40. The difference was shown by Tspo knockout mice with movement
restriction (spent 40% less time exploring the open arm) compared to normal WT
mice40.

31
Fig. 17 - Behavioral changes observed comparison between wild-type and Tspo knockout mice40
However, in the second round of observation in distance traveled, mice with the Tspo
knockout gene traveled 10% more distance. No effect was seen in depression-related
behavior for both models40.
To remove peripheral sources of neurosteroids, mice were gonadectomized and
adrenalectomized, and cortical concentrations of P5, P4, AP, and DHEA were measured.
This was done to comprehend brain synthesis, independent of steroids in the periphery40.
DHEA was reduced by 70% in Tspo knockout mice, whereas P5 and P4 were 1000 times
more in abundance. Additionally, 40% of the Tspo-KO mice had undetectable AP levels,
which were significantly reduced in their cortex40. These findings demonstrated that Tspo

32
loss is linked to a notable impairment in the brain's local steroidogenesis40.
Fig. 18 - Impaired neurosteroidogenesis in Tspo-KO mice40
8. Clinical Implications of TSPO Modulation
8.1 Neuropsychiatric Disorders
By promoting cholesterol transfer from the outer to the inner mitochondrial membrane,
TSPO and its ligands promote neurosteroidogenesis41. Even though numerous decades
have passed since the discovery of TSPO overexpression during neuroinflammation, the
function of TSPO and it signaling pathways in the control of neuroinflammation remains

33
unclear. A study that examined the function of TSPO in neuroinflammation and LPSinduced microglia activation (both in vivo and in vitro)
42. On the contrary, with
overexpression of TSPO, in contrast to control BV2 cells, there was a decrease in the
production of proinflammatory cytokines33. Accordingly, the molecular-level experimental
data presented in this work shows that TSPO functions as a negative regulator of
microglia activation and neuroinflammation43. Concomitant adult separation anxiety or
suicidality was correlated with a decrease in TSPO expression in individuals with bipolar
disorder or depression44. Last, those with aggressive behavior had a decreased platelet
density TSPO and far higher ratings on hostility, anxiety, state anger, and emotional
discomfort in contrast to people with nonaggressive and homicidal schizophrenia as well
as the controls45.
8.2 Neurodegenerative Diseases
When examining cerebral blood flow and the activity of neurons and glial cells in vivo in
several neurological diseases like Alzheimer's, Parkinson's, and Huntington's disorders,
brain PET is the most effective method. The nuclear medicine functional imaging method
known as Positron Emission Tomography (PET) allows for the in vivo observation of a
wide range of biological molecules expressed in various human tissues46. It is thought
that measuring TSPO binding indicates the activation level of microglia47. Compared to
the natural condition of microglia, activated microglia have higher levels of TSPO
expression48. PET radioligands that specifically bind to the translocator protein have been
produced for PET imaging of microglia48. A PET study shows that [11C](R)-PK 11195,
which is a TSPO radioligand, attaches itself to the striatum and globus pallidum, two

34
regions of the brain that are inflammatory48. The findings of the study indicate that
elevated adherence of [11C](R)-PK 11195 in these regions is correlated with decreased
binding of 11C-Raclopride, a chemical that is related to dopamine activity48.
The research additionally discovered a robust correlation between elevated levels of
specific immune system markers in the blood and enhanced binding of [11C](R)-PK 11195
in the postcentral gyrus, a region of the brain48. Finally, the beginning of the disease may
be predicted by strong activation of microglia, a kind of brain cell implicated in
inflammation, in areas of the brain linked to thought and memory49 .
Table 2 : Regarding the study that used TSPO ligand to study Neuroinflammation49
8.3 Inflammatory and Autoimmune Diseases

35
Despite having a high specificity for TSPO, the application of [11C](R)-PK 11195 in clinical
trials has been hindered by its relatively low signal-to-noise ratio50. It is known that
reactive astrocytes overexpress TSPO in response to damage, which likely contributes
to the non-microglial-specific binding. An additional obstacle with MS research and TSPO
ligands is the absence of an anatomically distinct reference location with no particular
binding. Several second-generation TSPO radioligands have been created, and
compared to their precursor [11C](R)-PK 11195, they have greater affinity and
specificity50. The second-generation TSPO ligands, PK11195, have also been
demonstrated to attach to activated astrocytes.
Fig. 19 - TSPO PET-based in vivo differentiation of persistent T1 lesions51

36
In the Fig. 19, the upper lesion on TSPO-PET (on the right) exhibits microglial activation,
indicating that it is a chronic active lesion; the lower lesion, on the other hand, does not
exhibit radioligand uptake, indicating that it is a chronic inactive lesion51.
57% of the plaques in the brains of a group of individuals with advanced Secondary
Progressive Multiple Sclerosis (SPMS) were of the chronic active type. Higher levels of
TSPO binding indicate that these plaques have continuous inflammation, especially along
their margins. A marker called TSPO is utilized to show brain inflammation52.
9. Future Directions and Challenges
Despite significant advancements in our understanding of TSPO, several key gaps
remain:
9.1 Mechanistic Understanding: Uncertainty surrounds the precise mechanisms by
which TSPO affects mitochondrial activity and cellular stress responses. More
research is required to understand TSPO's function in mitochondrial dynamics and
interactions with other proteins in the mitochondria53. The specificity and selectivity
of various TSPO ligands remain areas of active investigation53. Understanding how
different ligands modulate TSPO activity and their downstream effects is crucial53.
Functional Diversity: Numerous biological processes, such as steroidogenesis,
apoptosis, and immune response modification, have been linked to TSPO. It is necessary
to make clear in which situations TSPO performs a supporting function and in which it
plays a critical role53.

37
Role in Disease: Further comprehensive research is necessary to differentiate between
TSPO's function as a biomarker its involvement in several illnesses and its potential as a
therapeutic target, especially neurodegenerative diseases, cancer, and psychiatric
disorders53.
9.2 Emerging Research Areas
Neuroinflammation and Neuroprotection: TSPO ligands are being explored for their
potential in treating neuroinflammatory and neurodegenerative diseases. Research into
how these ligands can modulate neuroinflammation and promote neuroprotection is
expanding54.
Cancer Therapy: Given TSPO's role in cell proliferation and apoptosis, it is being
investigated as a target for cancer therapy. Understanding how TSPO modulation affects
tumor growth and progression could lead to new therapeutic strategies54.
Psychiatric Disorders: TSPO ligands are being evaluated for their potential to treat mental
illnesses including schizophrenia, depression, and anxiety55. Research focuses on how
TSPO modulation can influence neurochemical pathways involved in these conditions55.
Mitochondrial Dysfunction: TSPO is a potential target for addressing mitochondrial
dysfunctions, which are implicated in a range of diseases55. Research is ongoing to
develop TSPO-targeted therapies that can restore mitochondrial function and improve
cellular health.
9.3 Challenges in TSPO Research

38
Lack of Specificity: One of the main challenges is the lack of highly specific ligands that
can selectively target TSPO without off-target effects. This specificity is critical for
accurately elucidating TSPO's role in various biological processes55.
The complexity of Mitochondrial Biology: The complexity of mitochondrial biology and its
interplay with cellular and systemic physiology complicates the study of TSPO functions.
Advanced techniques and models are required to dissect these intricate interactions55.
Variable Expression and Function: TSPO expression and function can vary significantly
between different tissues and pathological conditions, making it challenging to draw
general conclusions55. Standardizing methods and approaches across studies is
necessary for comparability.
Translational Research: Translating findings from basic research to clinical applications
is a significant hurdle56. Bridging this gap requires robust preclinical models and welldesigned clinical trials to evaluate the efficacy and safety of TSPO-targeted therapies56.
By addressing these unresolved questions and challenges, future research can better
elucidate TSPO's functions and potential as a therapeutic target, paving the way for novel
treatments for a variety of diseases.

39
10. Conclusion
Fig. 20: Basic Function of TSPO gene
This review underscores the critical roles of TSPO in cellular functionality and
metabolism, particularly through its involvement in cholesterol transport and
steroidogenesis. TSPO's widespread expression and regulation by various factors
highlight its fundamental biological importance. The therapeutic potential of TSPO ligands
extends across multiple domains, including neuropsychiatric and neurodegenerative
disorders, cancer, and metabolic diseases, underscoring the broad clinical relevance of
TSPO modulation. However, significant challenges remain in the field. The development
of highly specific TSPO ligands that accurately target its functions without off-target

40
effects is crucial. A deeper mechanistic understanding of TSPO's interactions and roles
within mitochondrial and cellular contexts is essential to fully elucidate its functions.
Furthermore, translating basic research findings into clinical applications poses a
significant hurdle, requiring robust preclinical models and well-designed clinical trials to
evaluate the efficacy and safety of TSPO-targeted therapies. Addressing these
challenges through advanced research methodologies, collaborative efforts, and
innovative approaches will be essential for realizing the full therapeutic potential of TSPO
modulation. This could pave the way for novel treatments across a wide spectrum of
diseases, ultimately improving patient outcomes and advancing the field of molecular
medicine.

41
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Abstract (if available)

Abstract Translocator protein (TSPO, 18 kDa), formerly known as the peripheral-type benzodiazepine receptor (PBR), is a highly conserved protein located in the outer mitochondrial membrane. It plays a pivotal role in various cellular functions and metabolic processes, including cholesterol transport, steroidogenesis, apoptosis, and immune response modulation. This review provides an analysis of TSPO's structure, function, and regulatory mechanisms, highlighting its significance in the peripheral tissues as well as the central nervous system. The review also explores the modulation of TSPO activity by different ligands, discussing their effects on cellular processes and potential therapeutic applications. TSPO ligands have shown promise in treating various diseases, including neurodegenerative disorders, psychiatric conditions, cancer, and inflammatory diseases. Additionally, the review addresses the technical and conceptual challenges in TSPO research, such as the lack of highly specific ligands, the complexity of mitochondrial biology, and the variability in TSPO expression and function across different tissues and pathological states. Future research directions are proposed to bridge the current gaps in understanding TSPO's diverse roles and enhance its therapeutic potential.

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Asset Metadata

Creator Bahadure, Priyadarshini (author)

Core Title Role of translocator protein (18 kDa) in cell function and metabolism

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2024-08

Publication Date 08/05/2024

Defense Date 08/05/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag OAI-PMH Harvest,outer mitochondrial membrane,steroidogenesis,TSPO,TSPO ligands

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Papadopoulos, Vassilios (committee chair), Culty, Martine (committee member), Davies, Daryl (committee member)

Creator Email bahadure@usc.edu,bahadurepriyadarshini@hmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113998T9T

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Format theses (aat)

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