report_mmaillard

Morgane Maillard 130590213
Bsc Biomedical Sciences
1
Bioinformatic analyses of the Rab small GTPases diversity in the
human parasite Trichomonas vaginalis
Morgane Maillard 130590213, supervised by Professor Robert Hirt
Institute for Cell and Molecular Biosciences, School of Biomedical Sciences, the Medical School
Framlington Place, University of Newcastle, Newcastle Upon Tyne, NE2 4H, UK
Abstract
The sequencing of the unicellular parasite
Trichomonas vaginalis has demonstrated a
large genome which has undergone many
gene family amplifications, including the Rab
small GTPases family. The Rab proteins are
key regulators involved in membrane
trafficking, and their number in an organism
can be correlated with its complexity. With
292 members, Trichomonas vaginalis’ Rab
family is about 4 times larger than in human,
trigging interrogations about the actual
endomembrane complexity of this parasite.
Using in silico methods, this project aims to
rationalize this diversity by studying the
structure of all these sequences, their
evolution and their transcription.
Some of the sequences were shown to lack in
the known small GTPases functional motifs.
However, transcriptomic analyses indicate
that all the 292 TvRab are expressed in the
parasite in the tested conditions, consistent
with functionalities.
The phylogenetic analysis shows that the
amplification of the sequences has been made
preferentially in the Rab subgroup2 related to
the early endosome in model organisms, with
194 members. Further analyses made on this
group suggest a functional diversity rather
than redundancy.
As phagocytosis and endocytosis are thought
to be important in the biology of Trichomonas
vaginalis, these results might indicate the
implementation of a complex endomembrane
system to mediate these two cellular
processes.
Keywords: Trichomonas vaginalis; Rab;
diversification; evolution; transcription;
endocytosis.
Introduction
Trichomonas vaginalis is a unicellular
eukaryotic sexually transmitted parasite of the
human uro-genital tract. It causes the vaginal
inflammation trichomoniasis, and is associated
with several conditions including pelvic
inflammation (1), cervical and prostate
cancers (2, 3), and pregnancy complications
implicating preterm delivery and low birth
weight (4).
In addition to these effects, the most
important health outcome of this parasite
resides in its role in promoting HIV
transmission (5)
With an incidence of 248 million new cases
per year worldwide (6), it is the more common
non-viral sexually transmitted pathogen.
Although the infection by T. vaginalis is mainly
curable, cases of resistance (7) and allergies to
the traditional treatments have been
determined in some patients. These two
difficulties in the management of the parasite
strengthen its medical importance, and trigger
the interest to develop new drugs.
Therefore, understand the molecular basis of
its pathobiology is an important issue in
research, in order to improve actual
treatments and stop its transmission.
Furthermore, this parasite is also an
evolutionary model to study eukaryotic
phylogeny. Indeed, it lacks in true

2
mitochondrion (8), positioning it at the base of
the eukaryote diversification.
All these features have led to the sequencing
of its genome in 2007 by Carlton et al (9). The
results of this study show a large and highly
repetitive genome of 160 Mb, which encodes
about 60 000 predicted proteins. This
surprising size is the most important found in
protozoa parasite, and is due to numerous
gene duplications.
These duplications have led to the
amplification of many gene families, and this
is particularly true for the Rab small GTPase
family, which reaches to 292 members
(TvRab).
The Rab proteins belong to the Ras small
GTPase family, and are involved in membrane
trafficking (10).
They process and regulate the membrane
trafficking by intervening at numerous levels,
including the formation of the transported
vesicle, the movement of the vesicle from an
organelle to another, and the vesicle docking
and fusion on the target organelle (10).
To perform this, they bind a GTP molecule in
their active form, and switch in an inactive
form by hydrolysing the GTP in GDP due to
their intrinsic GTPase activity.
Their functions are mediated by specific
features of their primary sequences, which are
highly conserved among the eukaryotes (11).
Firstly, they bind and hydrolysed the GTP with
five polypeptide loops called the Gbox, which
are composed by highly conserved amino
acids (11).
Secondly, their anchorage on the membrane is
made through lipids which bind two or one
highly conserved cysteines forming the
prenylation motif at the C-terminal of their
sequences (12).
In addition to these two characteristic
features, they also possess the two important
switch regions which undergo conformational
change upon hydrolysis of the GTP, allowing
the effectors and regulator protein binding
the Rab to sense the state of GTP/ GDP (13).
Figure 1 Schematic diagram of the structure of the Ras small GTPase protein(14).
The 5 Gboxes, labelled G-1 to G-5, are indicated with the arrows. The two switch regions correspond
to the darker sections. The GppCp molecule corresponds to a nonhydrolyzable GTP analog, and is
represented by a ball-and-stick model.

3
Because a specific Rab is associated with a
specific organelle and a specific pathway, their
number in an organism has been correlated
with tissue complexity and cell polarity (10).
Thus, there is usually between 5 and 20 Rab in
unicellular organisms, and a higher number is
associated in multicellular (15).
For example, the unicellular parasites Gardia
lambia and Trypanosoma cruzi possess
respectively 8 and 19 Rab, and the unicellular
free living Monosiga cruzi and Saccharomyces
cerevisiae possess 23 and 11 Rab (16).
In contrast, 66 and 57 are find in human and
in the flowering plant Arabidobsis thaliana
respectively (16).
Therefore, the 292 members of the Rab family
in Trichomonas vaginalis are surprising, and
trigger interrogations about the actual
endomembrane complexity of this parasite.
The functionality of all these sequences has
not been determined yet, and a possibility
could be that only a small subset of the TvRab
is functional. The TvRab can also be mostly
redundant, optimizing a few function rather
than performing a complex membrane
trafficking. In addition to these hypotheses,
this expansion can also have led to new
functions, as it has been shown in the
unicellular Trypanosoma, with the TbRAB23
involved with the nucleus (17).
However, the fact that the gene amplification
has not been made equally among all the
protein families of the parasite suggest a
functional diversity among the TvRab. Indeed,
other small GTPase have not been amplified,
such as the Ran which is composed by only 2
members. This fact could suggest that the
amplification have been made preferentially
for different protein families, suggesting that
they have a functional importance in the
parasite biology.
Since their annotation in 2007 by Carlton et al
(9), this entire set of Rab have not been
precisely analysed.
Thus, this project initiates their
characterization and studies their potential
functionality using three different and
complementary bioinformatic methods
Firstly, the analysis of the primary structure of
the sequences will be made, by assessing the
presence of the functional residues specific to
the Rab in model organism.
Secondly, the diversification of the protein
family will be study through a phylogenetic
analysis, and by comparing the evolution of
the TvRab since the Last Eukaryotic Common
Ancestor (LECA).
Finally, this project will take advantage of two
recent transcriptomic papers from Gould et al
(18, 19).
The analyses of these transcriptomic data will
permit to assess the expression of the TvRab,
and their modulation across a range of
different conditions.
However, as no replicates have been made for
these analyses, the results should be
interprets with care.
Material and Method
Sequences of T. vaginalis
The TvRab sequences have been extracted
from a file containing the small GTPases
sequences of T. vaginalis (9) regarding the
presence of the following Rab characteristics:
the first criterion was the presence of the
double cystein prenylation motif. Then, the
Rab with a unique cystein have been identified
by testing the presence of the 5 Rab specific
motifs IGVDF, KLQIW, RFRSIT, YYRGA, LVYDIT
(12).
The relevance of the original alignment have
been verified with the Rab specific motifs
alignment, using the software Jalview (20).
This have led to manual corrections of the
original alignment with the software Seaview
(21).
The determination of the functional and
specific motifs has been assessed using the
software Jalview.

4
Phylogeny
The organisms chosen to model the Last
Eukaryotic Common Ancestor (LECA) in the
phylogenetic analysis were Monosiga
brevicollis and Trypanosoma cruzi, based on
the similarity of their Rab protein with that of
LECA, according to Klöpper et al results (16).
The Rab sequences of Monosiga brevicollis
and Trypanosoma cruzi have been taken on
the Rab database:
http://bioinformatics.mpibpc.mpg.de/rab/
The alignment of these sequences has been
made by comparing each sequence to the
alignment profile of T vaginalis’ Rab with the
clustal method on the software Seaview.
Only the non-ambiguoussites were
considered, and they were determined using
the heuristic method “automated1” of the
software TrimAl (22). A total of 149 sites have
been conserved for the continuation of the
study.
To avoid bias in the phylogenetic
reconstitution, a total of 4 partial sequences
with less than 60/149 sites were removed
from the phylogenetic analysis
(TVAG_365580, TVAG_172500, TVAG_550870
and MoBr_Rab21).
The evolutionary model fitting the best with
the data have been determined using the
software ProtTest (23), and the option Akaike
information criterion (AIC) have been used to
estimate the adjustment of the models to the
data.
The evolutionary tree has been built by the
maximum likelihood method, on the PhyML
web interface (http://www.atgc-
montpellier.fr/phyml/). According to the
result of ProtTest,The evolutionary model LG
(24) has been used. To accommodate the rate
site variation in the alignment, gamma rate
was fixed to 0.91. Both proportion of
invariables sites and equilibrium frequencies
were optimized with the evolutionary model.
The branch support were estimated with the
aLRT SH-like method (25).
The analysis using the Ran sequences as
outgroup (26) presented the long branch
artefact in this case. Thus, these sequences
have been removed, improving the
phylogenetic result.
The tree has been rooted by the midpoint
rooting method using the software FigTree
(http://tree.bio.ed.ac.uk/software/figtree/).
A Trichomonas vaginalis specific group was
determined when the branch support was
superior to 0.75.
The variation of the sequences of the Rab
subgroup 2 of the TvRab has been assessed
using the software HMMER
(http://hmmer.org/). The group2 profile for
the HMMERsearch has been built with the
HMMERbuild method using the Rab 22, 5, X1
and 21 of M. brevicollis, 5b, 5a, 21 and 21c of
T. cruzi, and the Rab5a-d of T. vaginalis
characterised by Lal et al(26)
Transcriptomic
This analysis take advantage of the two recent
publication from Gould et al (18) and Huang et
al (19)
The data of Kuo-Yang Huang et al were used
for this analysis (19). There consist in the
RPKM values of T. vaginalis ATCC30236
transcriptome for 4 conditions: 12h in glucose
rich (1%) medium, 12, 24 and 36h in glucose
restriction. The YIS medium (27) have been
used for the culture of the parasite.
The second set of data belong to the study of
Gould et al (18), which have analyzed the
effect of oxidative stress and VEC contact on
the strain T016 of the parasite. These data
consist in the number of reads values
calculated for the 11 following conditions:
Anoxic medium; 5, 30 and 120 min after
15%O2 exposure; 5, 30 and 120min after VEC
contact (which drive O2 exposure), and 0,5,30
and 120min after VEC contact for oxygen-
adapted parasite. The TYM medium has been
used for the culture of the parasite.

5
The data for the Rab have been extracted
using the function merge on the software R
(http://www.R-project.org).
In both analyses, the data were normalized,
and importantly did not include neither
biological nor technical replicates. Thus, the
package NOISeq-sim on the software R has
been used to determine the differential of
expression of the Rab between the conditions
(28). In fact, this algorithm allows normalized
data values as input, and permit to compute
differential expression when no replicates are
available. No low count filter have been
applied for the treatment of the data.
The size of the simulated sample was fixed to
20% of the sequencing depth and 10
replicates have been simulated, according to
the advice of the author of NOISeq for an
analysis without replicates.
As a biological coefficient of variation was
fixed to 0.1 in the study of Gould et al (18), the
same value has been chosen for the variation
parameters required in NOISeq-sim. Since this
information wasn’t available in the study of
Kuo-Yang Huang et al (19), the same value has
been used.
A differential of expression for a gene
between 2 conditions was determinated when
the probability of differental expression
calculated by NOISeq_sim algorithm was equal
or superior to 0.9 (limit value advised when no
replicates are available).
The exact Fisher’s test has been used on the
software R to assess the implication of a
specific group in the differential of expression.
A α risk of 0.05 has been considered.
Each TvRab has been classified into 4
categories according to their level of
expression compared to the expression of the
totality of the TvRab. Their distribution have
been made using the K-means clustering
method on the software R, with the Hartigan
Wong algorithm (29), 5 random start, and a
maximum of 10 iterations allowed.
The expression of the gene family Adaptin and
BspA has been assessed on the transcription
level in the references of the two experiments
from Gould et al(18) and Huang et al (19). The
list of locus tag used for these two gene
families has been determined according to the
results of Noël et al (30) and Carlton et al (9).
Results
Primary Structure analyses
To investigate the potential functionality of
the TvRab, the presence of the functional
residues characteristic in model species (11,
13) has been assess trough the analysis of the
amino acid sequences.
189 TvRab present the classical prenylation
motif with two cysteines, in different possible
combinations (CCxxx, CCxx, xCCx, xxCC, xCxC
(12), single letter amino acid code, x
representing any residues),and 3 possess a
unique cysteine (motif CXXX (12)). The
presence of the prenylation motif for all the
TvRab suggests that they are all involved with
the membranes.
The analysis of the primary structure of the
TvRab has determined a total of 107
sequences lacking at least one of the highly
conserved functional residues of the Gbox.
The 5 Gbox are distributed along the Rab
sequence, forming 5 different motifs
composed by one or more highly conserved
amino acid.
The first box, Gbox1, consist in 4 residues
forming the motif GxxxxGK[S/T]. These
residues are involved in the binding of the α
and β phosphate of the GTP/ GDP.
8 TvRab were found to lack all of the 4
residues, and 4 possess a modify version of
this motif by lacking one on the fourth specific
residues.
The second Gbox motif is composed by a
unique highly conserved threonine residue,
which bind a Mg2+
, and coordinate this ion
with the Rab•GTP/GDP complex. 9 TvRab lack
this specific residue (See Table 1).

6
The amino acids constituting the Gbox3 form
the motif DxxG, and bind to the Mg2+
and the
γ phosphate of the GTP. One TvRab lacks the
glycine residue, and 4 lack the entire motif.
The G4 box, [N/T]KxD, is involved in the
affinity of the Rab with the GTP/ GDP by
recognizing the guanine base. 2 sequences
lack this Gbox, and 10 lack one of the three
residues.
The G5box, which strengthen the activity of
G4, is the less conserved motif in the Rab of
the parasite. The consensus of this loop is
[C/S]A[K/L/T], and 92 sequences possess a
modified version of the box. Among these
sequences, 86 have lost one of the three
residues, and 6 have lost the two last residues.
However, the first amino acid is always
conserved among the 292 TvRab.
Only 1 TvRab lack in the switch 1 region, and
three TvRab lack in both switch 1 and switch 2
regions, due the partiality of these 4
sequences.
On the 107 sequences determined in this
analysis, 13 were shown to lack in functional
residues in two or more motifs.
These results are summarized in the Table 1.
Phylogeny
A few set of Rab are broadly shared across the
entire eukaryotic kingdom, and are believed
to have constituted the Rab family of the Last
Eukaryotic Common Ancestor (LECA).
According to the comparative genomic study
of Klöpper et al (16), the ancestral Rab family
appears to be constituted by 20 Rab
distributed in the 6 following phylogenetic
and functional groups: group1- Secretion
(Rab1, 8, 18); group2- Early endosome (Rab5,
21, 22, 24, X1) , group3- Late endosome (Rab
7, 23, 29, 32, 7L1), group4- Recycling from
endosomes to surface (Rab2, 4, 11 and 14),
group5- Recycling from endosomes to the
Golgi (Rab6), and group6- Traffic associated
with cilia/ flagella (Rab28 and L4).
To assess the diversification of the TvRab
within these functional groups, it was
interesting to compare their sequences with
the Rab family of LECA.
With 23 Rabs distributed across the 6
functional groups, the Rab family composition
of the Choanoflagellate Monosiga brevicollis
was the closest to the Last Eukaryotic
Common Ancestor determined by Klöpper et
al (16) .Thus, the Rab sequences of this
organism can be used as a suggorate LECA in
the phylogenetic analysis.
The Kinetoplastida organisms were also found
to have a similar Rab family set to LECA. As
they belong to the phylum excavata, which is
the same as Trichomonas vaginalis, the
sequences of one of their members is also
interesting to use. Thus, the unicellular
Trypanosoma cruzi have been chosen as a
second organism to model LECA in this
analysis.
Consistently with the results of Klöpper et al
(16), the different Rab of these two organisms
form 6 phylogenetic groups corresponding to
the six functional groups.
The phylogenetic analysis shows that a large
part of the TvRab is regrouped within 5 of the
6 groups (Figure 2), permitting to establish
initial hypothesis about their function. In
addition, 6 T. vaginalis specific groups can be
determined.
The group 1, related to the secretion (16), is
composed by 11 TvRab. Although the support
value calculated by the phylogenetic algorithm
is very weak (value = 0), this group were
always found in the analyses, showing its
solidity.
One TvRab cluster with the Rab1B of
Trypanosoma cruzi, and therefore is named
TvRab1.
The 10 other TvRab form a monophyletic
group separately from the Rab1, 8 and 18 of
the two other protists. This preventing their
characterization in Rab1, 8 or 18, they were
renamed TvRab_grp1a-j.

7
The group2 is the more important in size, and
regroups 194 TvRab included the 4 TvRab5
characterized by Lal et al (26). The consistence
of this group is confirmed by a support value
of 0.93. Moreover, this group include also the
sequences of the 5 T. vaginalis specific group
B to F determined by Lal et al (26).Since these
groups were thought to be implied in
endocytosis, this result is consistent with the
previous analysis.
The group3 is composed by 10 TvRab,
including the Rab7a-c characterized by Lal et
al (26).
3 TvRab cluster with the Rab 7 of M.
brevicollis, T. Cruzi and the Rab7a-c of T.
vaginalis, forming a subgroup supported with
a value of 0.803. Thus, these sequences were
renamed TvRab7d-f.
The 4 other TvRab regroup with Rab32 with a
support value of 0.912, allowing their
characterization as TvRab32a-d.
The group4 possesses 6 TvRab. 2 of them
cluster with the Rab 11 of the other protists,
and were already renamed TvRab11a and
TvRab11b by Lal et al (26). The 4 other TvRab
form a monophyletic group. As for the group1,
their characterization as Rab 2, 14, 4 or 11 is
difficult. Nevertheless, as they cluster in the
group4 with a support value of 0.86, they
were considered as “TvRab Group4a-d”.
6 TvRabs are found to cluster in the group5,
which regrouped the Rab6 of M. brevicollis
and T. cruzi. Two of these sequences were
already named as TvRab6a-b (26). Accordingly,
the four other Rab were renamed TvRab6c-f.
Notably, the group6, considered to be
involved in flagella and cilia trafficking (16,
31), is absent in T. vaginalis.
In addition, the analysis determined six T.
vaginalis specific groups, labelled A to F.
The groupA is composed by 35 members,
including the TvRabA1-6 characterized by Lal
et al (26).
This group cluster with the group1 and 4, both
implicated in the exocytic pathway, with a
support value of 0.885. Correlated with the
phylogenetic analysis, the primary sequences
analaysis of these Rab shows the conservation
of several residues known to be conserved
among the exocytic Rab (32), as indicated on
the Figure 3.
Thus, the Rab belonging to this group could
have a role in exocytosis, consistently with the
previous explanation of Lal et al.
The five other group were renamed B to F,
and possess respectively 2, 3, 8, 4 and 6
members.
A total of 4 TvRabs were not grouped into the
groups discussed above, because of z low
support value.

8
Figure 2 : Phylogenetic framework for TvRab diversity.
Maximum likelihood tree, rooted by the midpoint rooting method. The branch colored in green
correspond to the sequences belonging to group1, dark pink group3, yellow group4, orange group5
and deep blue group6. The group2 is represented by the collapsed branch in turquoise, and the T.
vaginalis specific groups correspond to the collapsed branch in pink. The numbers following the name
of the different group on the figure correspond to the number of sequences within the group. The
labels MoBr, CRU and TvRab correspond to the Rab sequences belonging to the organisms Monosiga
brevicollis, Trypanosoma cruzi and Trichomonas vaginalis respectively. The TvRab ungrouped in the
tree are represented with a black branch colour and are labelled with their locus tag. The scale bar
represents 40% sequence divergence.

9
Figure 3 Alignment highlighting examples of sequence features for endocytic and exocytic residues
in Rab.
Alignment of the exocytic Rab 1a-b, 11a characterized by Lal et al(26), and the Rab group4-like
determined by the phylogenetic analysis; the endocytic Rab 5a-d and the RabA1-4 characterized by
Lal et al. The amino acid highlighted in yellow are characteristic to the exocytic Rab (32), and those in
blue are characteristic to the endocytic Rab (33). The prenylation motif is highlighted in green. The
Gbox motifs are indicated in bold. The switch 1 and 2 regions are surrounded by the red boxes.
Relation between functional motifs and
phylogeny
As summarized in the Table 1, the
phylogenetic reconstitution has also permit to
determine the repartition of the sequences
lacking in functional residues.
These sequences seem to be distributed
among the different group. However, the
most affected sequences belong to the group
2 and D.

10
Table 1 Sequences lacking in the known functional residues.
Table regrouping the name of the sequences lacking in one or more known functional residues. The
different functional motifs are presented in the same order as found on the Rab sequences from N-
terminal to C-terminal. The name of the functional group or the Rab type is indicated in the last
column, according to the results of the phylogenetic analysis. The term None indicate that the
sequences lack in the entire motif. When the sequences lack in only one residue, the modified motif is
indicated. The term NI abbreviate “Non Included”, and indicate the sequences too partials to be
included in the phylogenetic study (See section Material and Methods)
Gbox1
GxxxxGK[S/T]
Switch1
Gbox2
T
Gbox3
DxxGQ
Switch2
Gbox4
NKxD
Gbox5
[S/C]A[K/T/L]
Group
TVAG_248240
TVAG_491630
None 2
2
TVAG_365580 None None None None None SAA NI
TVAG_172500 None None None None None NI
TVAG_550870 None None None None None SAA NI
TVAG_289630 None None None 2
TVAG_044280 None SAR 2
TVAG_305600 None SAV 2
TVAG_202110 GxxxxDKT 2
TVAG_073560 GxxxxEKT None 2
TVAG_235880 GxxxxGET SAA 2
TVAG_369010 GxxxxGQT SAA 2
TVAG_421510 None None SSK D
TVAG_206050 None SAV 2
TVAG_069950 None D
TVAG_015270 None 2
TVAG_286400 None 2
TVAG_371280 None 2
TVAG_306120 None DxxE MKxD SSQ D
TVAG_202240 None CKxD SSK C
TVAG_202320 AKxD 2
TVAG_020390 CKxD SSK Rab1
TVAG_324060 NKxE D
TVAG_246750 SKxD B
TVAG_361420 SKxD C
TVAG_446610 SKxD A
TVAG_533890 SKxD SKK B
TVAG_339490 SKxD SSK 2
TVAG_037110 SSI 2
TVAG_073350 SVH 2
TVAG_136260 SSI 2
TVAG_280870 SGY 2
TVAG_377960 SSR Rab6
A (10)
F (2)
2 (47)

11
Gbox1
GxxxxGK[S/T]
Switch1
Gbox2
T
Gbox3
DxxGQ
Switch2
Gbox4
NKxD
Gbox5
[S/C]A[K/T/L]
Group
68 sequences SAx Rab4like (1)
Rab11 (2)
Rab3like (3)
Rab6 (2)
6 sequences Sx[K/L] A (1)
2 (5)
Transcriptomic analysis
Two recent transcriptomic studies (18, 19)
have assess the transcription of the entire set
of gene in Trichomonas vaginalis. This provide
analysis of the TvRab transcription in order to
investigate their expression, which is a
prerequise to their functionalities, and their
modulation between different conditions.
The impact of glucose restriction (19),
oxidative stress and binding to female host
tissue (18) have been study, and represent the
different conditions.
The two transcriptomic analyses have used
different growht conditions and the different
strains of T. vaginalis. Thus, these differences
may drive modifications in the gene
expression between the two references.
The comparaison between the transcriptome
in the reference of the two analyses shows
that the expression of the Rab are correlated
with a R2
equal to 0.66 (Figure4). Therefore,
the TvRab are transcribed similarly between
the two strains, and this despite the difference
in growth medium.
This correlation is the strongest when
compared to the entire gene set, and to the
protein familes adaptin and BspA (9, 34).
Moreover, all the TvRab are trancribed,
compared to only 50% of the totality of the
genes. Similarly as the correlation, the
percentage of transcribed is also lower in BspA
family, with 70% of the members being
expressed in the tested conditions.
These result suggest the importance of this
entire set of Rab in the biology of Trichomonas
vaginalis.

12
Figure 4 Comparison of the genes expression between the references of the two analyses
(A): Correlation of the expression of the Rab family between the reference of Gould et al (18) and
Huang et al(19) (B): Table comparing the correlation of expression between the references of the two
experiments for the entire set of gene, the Adaptin, BspA and Rab protein families of T.vaginalis. The
number of members of each gene family is indicated in the first column. The proportion of genes
expressed for the totality of the geneq has been determined by Huang et al(54%) and Gould et al
.(55%) The proportion expressed for the BspA family has been determined by Gould et al (18)
A) B)
Gene family R2
Proportion
expressed
All genes
(59681)
0.36 54-55%
Rab
(292)
0.66 100%
Rab group2
(194)
0.67 100%
Adaptin
(78)
0.29 100%
BspA
(911)
0.39 79%
Glucose restriction
Huang et al (19) have driven 4 experiments
consisting in 12h in glucose rich condition, and
12h, 24h and 36h in glucose restriction
condition.
Only 1 Rab is found to be over-expressed after
12h in Glucose restriction compared to
Glucose rich condition. This Rab correspond to
the gene labelled TVAG_474310, and belong
to the group A.
With a Log2Fold-change of 4.7 between the
conditions, this gene is about 25 times more
expressed under glucose restriction.
Compared to the result in glucose rich
condition, it is also up-regulated after
exposition during 24h and 36h to glucose
restriction, indicating that the up-regulation
on this gene is maintain by the parasite.
Oxidative stress
The comparison between the transcriptome of
the reference parasite and the transcriptome
of the parasite dealing with oxidative stress
shows that there are no significant
differentially expressed Rab genes.
Host cell binding combined with oxidative
stress
The binding on the Vaginal Epithelial Cell (VEC)
produce oxidative stress in this condition.
When Trichomonas vaginalis is in contact with
VEC in the same time as dealing with oxidative
stress, an up regulation of 16 Rabs occurs
after 5 minutes, and is maintained until the
end.
y = 15.677x + 98.974
R² = 0.6589
0
2000
4000
6000
8000
10000
12000
14000
0 200 400 600 800
DataGouldetal
Data Kuo-Yang Huang et al
Rab Expression

13
This up-regulation involved significantly the
Rab belonging to the group C. Indeed, 2 of the
3 RabC are up-regulated under this condition,
implicating a probability that this result is
randomly produced of 0.02.
VEC contact in oxygen-adapted parasite
The use of oxygen-adapted parasite allows the
assessment of the VEC contact without
oxidative stress.
Surprisingly, the contact of these parasites
with VEC alone during 5 minutes provokes a
down regulation of 168 Rabs. After 5 min, the
level of the differentially expressed genes
stays constant until the end of the
experimentation, indicating that the down-
regulation is maintained.
Unlike the regulation for VEC coupled with
oxidative stress, this gene modulation does
not significantly imply a functional group in
particular.
Absolute expression level
The analyses have shown that the
experiments for 24h and 36h under glucose
restriction (GR) were not significantly different
to 12h under GR, and that the experiments for
30min and 120min under oxidative stress, VEC
contact and oxygen-adapted parasite with VEC
contact were not significantly different to the
same condition after 5min.
Therefore, the rest of the analysis only
consider the 2 references conditions (18, 19),
5min after GR, 5min after Oxidative stress and
VEC contact, and 0min and 5min after oxygen-
adapted parasite exposure to VEC.
In each condition, the TvRab are distributed
into 5 classes of expression level (Figure 5). As
shown in Figure 5, some of the TvRab have an
expression level varying between the different
conditions.
The integration of this feature for the 7
conditions considered by clustering analysis
has permit to classify each TvRab into 4
different categories, according to their
expression profile.
The category 1 correspond to the TvRab genes
which are highly expressed among all the
conditions, and regroups 11 TvRab, distributed
in the groups 1, 2, 3, 4 and A.
The second category regroups the genes
which have a variable expression level among
the different conditions (mainly due to
difference in strain and medium growth
between the two studies), but which are
maintain between a medium and a high level
of expression. 22 TvRab from the group 1, 2, 3,
4, A, D, and from the ungrouped sequences
belong to this category.
The third category is composed by 46 TvRab,
which have an expression varying between a
medium and a low level. Except for the group
B and C, every group possesses members
belonging to this category.
Finally, the fourth category regroups the Rab
which are poorly expressed among all
conditions and studies. With 213 members,
this category contains 73% of the TvRabs and
includes sequences from all the phylogenetic
groups.
These results, summarized in Table 2, permit
to determine 2 different patterns between the
different groups.
The first tendency is observed in the groups 1,
2, 3 and A. These groups possess few member
highly expressed, and a majority of Rab
belonging to the fourth and fifth category.
The second tendency is find in for the group 5,
B, C, D, E and F, which are all medium-low and
poorly expressed (category3 and 4).

14
Figure 5 Level of expression of the TvRab for 3 transcriptomic experiments.
(A): Expression value for the reference condition of Gould et al (18). The Rab are ranked from the
more expressed to the less expressed. 5 classes can be determined: In red are the most expressed, in
yellow the TvRab medially-highly expressed, in green the medially expressed, in purple the medially-
lowly expressed and in blue the poorly expressed. (B): Expression values of the TvRab for the oxygen-
adapted parasite before VEC contact condition. The TvRab are ranked accordingly to the ranking in
Fig5A, and the colours represent the same feature as described above. (C): Expression values of the
TvRab for the reference of Huang et al (glucose rich condition)(19). The colour and the ranking are
similar as described above.
A)
B)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Numberofreads
TvRab
Condition reference Gould et al
0
2000
4000
6000
8000
10000
12000
Numberofreads
TvRab
Oxigen-adapted T. vaginalis before VEC contact

15
C)
Table 2 Distribution of the TvRab among the 4 expression categories.
Table representing the number of gene distribute in the 4 categories of expression level for the
different phylogenetic group. Category 1 corresponds to the gene highly expressed among all the
conditions of the two transcriptomic analysis, Categories 2 and 3 correspond the genes varying
between high-medium and medium-low level of expression respectively and Category 4 corresponds
to the genes poorly expressed among all the conditions.
Functional
Groups :
Grp
1
Grp
2
Grp
3
Grp
4
Grp
5
Grp
A
Grp
B
Grp
C
Grp
D
Grp
E
Grp
F
Ungrouped/
Non
included
Expression
Category
1
2 3 1 1 0 4 0 0 0 0 0 0
Expression
Category
2
2 11 1 2 0 3 0 0 2 0 0 1
Expression
Category
3
4 24 3 2 1 8 0 0 2 1 1 0
Expression
Category
4
3 156 5 1 5 20 2 3 4 3 5 6
Relation between functional motifs and
transcription
All the 107 TvRab lacking in the known
functional residues were found to be
transcribed, and some of these sequences
belong to the highly expressed category.
The sequence TVAG_289630, which lack the
Gboxes 1 and 2 and the switch1, have the
weaker level of expression, as it is not
expressed in the results of Gould et al (18)and
has very small RPKM values in the results of
Huang et al (19) (0.9, 1.3, 2.6 and 3.1 in the
condition reference, 12h, 24h and 36h under
glucose restriction respectively). However, the
absence of replicates prevents to make solid
conclusion about a potential non-
functionality.
0
50
100
150
200
250
300
350
400
450
500
RPKM
TvRab
Reference condition Huang et al

16
These 107 sequences cluster in the 4
categories similarly to the total set of TvRab,
with 1.9% in the category 1, 6.5% in the
category 2, 13% in the category 3, and 78.8%
in the category 4.
Moreover, a large part of these sequences
have their expression modulated under the
tested conditions.
Indeed, the contact with VEC for the oxygen-
adapted parasite drives the down-regulation
of 69 of these 107 Rab.
Among these 69 sequences, 9 are also found
to be up-regulated under the contact with VEC
correlated with oxidative stress.
These results strongly suggest that despite
lacking in some of the highly conserved
residues, these sequences are functional and
have a role in the biology of the parasite.
Focus on the group2
As determined in the phylogenetic analysis,
194 TvRab composed the group2 considered
to be involved in early endosome (16), which
have been preferentially amplified.
These TvRab seem to have undergone a rapid
evolution, and therefore present structural
difference. This fact is emphasized by the long
branch length in the phylogeny. The
phylogenetic tree in Figure 6, which regroups
a subset of the group2 of Trichomonas
vaginalis, illustrates this point. This figure
represents only a part of the Group2
phylogeny, as the size of the entire tree was
too large to be represented here. However,
similar results were found in the rest of the
phylogeny.
Moreover, a HMMER search have been
realised on all the TvRab of T. vaginalis against
a Group2-consensus built on the group2
sequences of M. brevicollis and T. cruzi, and
the TvRab5a-d which were already
characterized by Lal et al (26).
The result of this analysis shows that the
structural similarity of these 194 sequences to
the characterized group2 is very variable.
Indeed, there p-value varying from 1.30 E-65
to 3.10E-23, the highest p-value of the entire
set of TvRab being 4.00E-12.
The 6 first sequences constituting the top hit
belong to the group2, but the seventh and
ninth closest sequences are TvRab11a and
TvRab11b, and two TvRab of the group 2 are
found in the five later sequences.
This analysis further highlight the structural
diversification which seems to have occurred
within the TvRab related to the early
endosome.
Consistently with these observations, the
primary structure analysis shows that the two
residues specific to endocytosis have not be
strictly conserved within this group, as only
37% (71 sequences) possess both of these
endocytic features.
The residues specific to the Rab involved in
endocytosis consist in an alanine, glycine or
serine in switch1 and switch2 (33) (See Figure
3).
102 sequences lack one of these residues, and
particularly the second one is often replaced
by an isoleucine or a valine.
Interestingly, the exocytic threonine (32) can
be find in 33 sequences, replacing the second
endocytic residue.
On these 33 sequences, 24 possess both an
endocytic and an exocytic residue, and 9
possess the exocytic residue only.
12 TvRab lack both of these residues
characteristic for the Rab involved in
endocytosis or exocytosis pathways.
These characteristics are represented for the
subset of Rab in the Figure 6, with each
branch colour corresponding to one of the
features expressed above.
These sequences are mainly distributed along
the tree of the group2. However, 7 TvRab
possessing an exocytic motifs cluster together,
forming a phylogenetic subgroup. This
subgroup is represented at the top of the tree
in Figure 6, and regroups the sequences
TVAG_453170; TVAG_019470; TVAG_474500;

17
TVAG_179640; TVAG_456910; TVAG_056480
and TVAG_329350.
All these sequences possess the alanine
characteristic to the first endocytic residue,
and an exocytic threonine instead of the
second endocytic residue. This could suggest
their implication in the same process.
The expression level differs between the
different sequences, emphasizing different
role in the biology of T. vaginalis. As it is true
for the entire set of Rab, a few sequences (4
TvRab) are highly expressed (colour red in the
Figure 6), and 80% are poorly expressed
(colour green), indicating that the main part of
the group2 seems to be required at a basal
level of expression in both growth and tested
conditions.
The expression pattern shown in Figure 6 is
heterogeneous between the difference
sequences.
This variability between the sequences
suggests their difference in term of
expression, and hence potentially in term of
function.
Moreover, not all the gene presents a
significant differential of expression for the
tested conditions, indicating difference in the
modulation within this group.
For the oxygen adapted parasite, the genes
significantly regulated under exposition with
VEC appears to be distributed among all the
subgroup forming the group 2. Similarly, the
gene differentially expressed for the parasite
non-adapted to oxygen are distributed among
the group2. Finally, although no significantly
differential of expression have been
determined in these conditions, a tendency of
up-regulation in response to glucose
restriction can be seen on the heatmap in
Figure 6. However, this is not the case for the
oxidative stress conditions.

18
Figure 6 Integration of the phylogenetic, structural and transcriptomic analysis for a subset of the
group2
Part of the topology of the group2, including the TvRab5a-d characterized by Lal et al(26).The tree is
rooted by the Midpoint rooting method. The heatpmap represented for each transcriptomic
experiment corresponds to the Log2 values. Each gene value has been normalized by the average of
expression of the gene for the considered experiment. A red colour on the heatmap indicates that the
gene has a higher expression than its expression average for the experiments, and a green colour
indicates a lower expression. One experiment corresponds to the reference data coupled with the
three time point for one of the condition (glucose restriction, oxidative stress, VEC contact, VEC
contact + Oxidative stress). The label X15_12h, O2_0, VEC_0 and Anox_0.1 correspond to the
reference of the different experiments. The scale bar represents 10% sequence divergence.

19

20
Discussion
Functional and diversity evidence
In this project, the combination of the
analyses at the levels of the primary structure,
the phylogeny and the transcription has
permit to initiate the characterization of the
292 Rab of the parasite Trichomonas vaginalis.
The evidence for the transcription for all these
sequences and the modulation of their
expression for most of them between tested
conditions strongly suggest that they are all
functional.
In addition, the highest correlation found for
the protein family between two different
strains growing in two different medium
emphasize their importance in the biology of
the parasite.
The known functional motifs of the Rab
protein are mostly conserved across the
TvRab.
Indeed, the 292 TvRab possess the prenylation
motif, showing that they are likely to be
implicated with the membranes.
The third amino acid of the Gbox5 is found to
be the less conserved among the TvRab.
However, as the Gbox 5 strengthening the role
of Gbox4 (14), this characteristic may not
impair the Rab activity.
If this motif is not taken into consideration, 28
sequences lack residues in the other Gbox,
and could potentially not act as Rab protein.
This number represents only 10% of the entire
set of TvRab, and could be explained by the
apparition of new functional residues
performing the Rab activity, or by the
apparition of new function than small GTPase.
Among these 28 sequences, the TvRab
TVAG_365580, TVAG_172500, TVAG_550870
and TVAG_289630 lack in the first part of the
typical Rab sequence (Table 1). These
sequences could be incomplete, due to an
artefact of sequencing rather than possess
new functionalities.
These results suggest that at least 90% of the
TvRab possess the known structural features
to work as Rab rather than be implicated in
new function.
In addition, the long length branch in the
phylogenetic analysis and the detailed analysis
made on the group2 suggest a functional
diversity rather than redundancy.
In addition to these result, the amplification of
other protein family implicated in membrane
trafficking have been determined (9)(Table 3),
strengthening the hypothesis that the Rab
family expansion have been made to perform
a complex endomembrane system.

21
Table 3 Comparison of the proportion of
genes implicated in membrane trafficking
between T. vaginalis, yeast and human.
Adapted from Carlton et al (26). This table
compares the number of protein implicated in
the endocytic and phagocytic pathway
between T. vaginalis, S. cerevisiae and H.
sapiens. The Rab values for the yeast and
human have been found on the Rab database
from Klöpper et al (16). The values for the
different group of Rab in T. vaginalis have
been determined in the phylogenetic analysis.
T.
vaginalis
S.
cerevisiae
H.
sapiens
Vesicule formation: coat protein
Adaptin 73 13 24
Retromer 12 3 4
COPI 16 7 9
COPII 12 6 9
Vesicule fusion:
Syntaxin_E 10 3 3
SM_E 16 2 3
Small GTPase:
Rab 292 10 66
Rab
Group 1
11 2 33
Rab
Group 2
194 4 9
Rab
Group 3
10 1 8
Rab
Group 4
6 2 10
Rab
Group 5
6 1 4
Rab
Group 6
6 0 2
ARF 36 7 30
Secondary loss
Interestingly, numerous secondary losses
seem to have occurred in T. vaginalis.
Firstly, no homologous to Rab28 and Rab L4
have been determined in the phylogenetic
analysis, indicating that the functional group6
related to flagella traffic is absent.
As Trypanosoma cruzi possess these Rab and
belong to the excavata, which is the same
phylum as T. vaginalis, these result suggest a
secondary loss of this group.
Since this parasite possess 5 flagella, this
result is surprising.
Rab8 (group1) and Rab23 (group3) have also
been shown to be involved in flagella/ cilia
traffic in mammal (35).As shown in the
phylogenetic analysis, no TvRab cluster
directly with these Rab from the two other
protists. However, the diversification of the
TvRab in the group1 and 3 may have result in
sequences having a role in flagella transport,
which could have replaced both Rab 23 and 8,
explicating this secondary loss.
As it has been noted in the phylogenetic
analysis, the TvRab belonging to the group1
cluster together, implicating that they result
from the same type of Rab
Thus, this result suggests secondary losses of
the Rab 1, 18 or 8. As 11 TvRab_group1
isoforms have been determined, they could
have been replaced the function of the Rab
losses, as suggested for the flagella transport.
Similarly to the group 1, 4 TvRab cluster in the
group4 close to the Rab2, 4 and 14 of the two
other protists. However, their characterization
in one of this type of Rab is difficult to
determine.
As suggest for the group 1, the secondary loss
of two of these Rab could have occur,
replaced by the isoform of the third Rab.
Diversification of a Rab within a known
functional group or within the T. vaginalis
specific could have replaced the other.
These secondary losses, and particularly for
the group6, are not specific to T. vaginalis, and
seem to have occur in a large number of
species (16).
As highlighted by Klöpper et al (16), the
membrane trafficking can be performed by a
few set of 5 Rab distributed in the group1-5.
Thus, T. vaginalis possess all the elements to
perform membrane trafficking, and these
secondary losses are not incompatible with a
complex endomembrane system hypothesis.

22
Therefore, all these elements suggest the
implementation of a highly complex
endomembrane system.
A variable environment
As said previously, the Rab diversity has been
mainly correlated with tissue complexity (10).
However, as T. vaginalis is a unicellular, this
cannot explain the huge Rab diversity found in
this parasite.
The genome amplification which has amplified
this gene family appears to have occurred
after the passage of the parasite from enteric
environment to the uro-genital tract (9). Thus,
this change in environment may have
originated the Rab expansion.
The uro-genital tract is an environment
variable, which triggers metabolic stresses
such as nutrient restriction and oxidative
stress.
Thus, T. vaginalis has to deal with several
source of variability during its life cycle. Firstly,
it switches between a female to a male host,
which are two very different environments.
Within the female host, the menstrual cycle is
also a source of variability, driving oxidative
stress and exposure to blood.
This diversity in environment during its life
cycle could be the driver of the
implementation of a highly complex
The transcriptomic analyses used for this
project (18, 19) have tested the impact of
several of these conditions such as glucose
restriction, oxidative stress and binding to the
female host tissue (VEC).
Both oxidative stress and glucose restriction
don’t seem to involve differentially the Rab
proteins, and contradictory results were found
upon binding with host tissues. However, the
absence of replicates biases the statistical
analyses, and other experiments such as qPCR
should be made to have more precise results.
Moreover, other conditions should be tested,
such as the as the difference between male
and female host cell or the contact with blood.
This will allow to determine if the Rab are
significantly involved when the parasite deals
with these environmental stresses.
Amoebae transformation
When T. vaginalis binds to the host cells, its
morphology changes from a free swimming
trophozoite form to an amoeba form.
The other protists parasite Entamoeba
histolyca and Dictyostelium discoideum also
drive this metamorphosis in their life cycle,
and present an importante Rab diversity, with
105 and 54 Rab respectively (36, 37).
Therefore, this metamorphosis could require a
complex endomembrane system, and thus
explain the Rab number found in T. vaginalis.
However, the transcriptomic analysis of Gould
et al assessing both VEC contact and VEC
contact coupled with oxidative stress gives
contradictory results, making the
interpretation difficult.
The utilization of replicates or qPCR for further
analyses may lead to more conclusive results.
The exocytic pathways
The Rab known to be involved in the exocytic
pathway belong to the group 1 and 4.
The group1 seems to have undergone a
moderate amplification in T. vaginalis. Indeed,
11 TvRab cluster in this group, and this
number is higher than for the two other
protists used for the phylogenetic analysis,
which possess 5 and 3 members (16).
However, the group4, which is related to the
recycling from the Golgi to the surface, is
composed by 6 TvRab, similarly to M.
brevicollis and T. cruzi (4 and 5 members
respectively).
According to its position on the tree and its
structural characteristics, the groupA specific
to T. vaginalis seems to have a role in the
exocytosis pathway. With 35 members, it is
the second largest group of TvRab,
representing a consequent amplification.

23
Therefore, the Rab related to the exocytic
pathways appears to have been amplified.
To maintain itself in the environment and
install infection, T. vaginalis is known to
secrete a number of virulent factor (38), such
as peptidases, the exosomes, or the
trichopores, 12 pore forming proteins (39).
Therefore, the amplification of the Rab
involved in the secretory pathways could
reflect the importance of these secreted
effectors molecules.
This hypothesis should be strengthened with
an up-regulation of these TvRab upon binding
with the VEC. However, as for the amoebae
transformation hypothesis, the transcriptomic
results are ambiguous, making the
interpretation difficult.
The endocytic pathway
Both phagocytosis and endocytosis are
thought to be important in the biology of T.
vaginalis, and are involved for essential
function such as iron acquisition (40).
Interestingly, the presence of a large number
of 88 Rab have been found in the ciliated
protozoan phagocytor T. thermophyla,
suggesting the acquisition of a complex
endomembrane system to perform
phagocytosis and endocytosis (41).
The group 2, 3 and 5 are related to the
endocytic pathway, and are all find to be
amplified in Trichomonas vaginalis.
The main amplification has been made in the
group2, related to the early endosomes. With
194 members, this group is more than 20
times more important than the group2 in
human (See table 3). However, although the
number of members in this group is
exceptional, the group2 appears to be the
more complex in term of evolution across the
eukaryotic kingdom (16).
Moreover, the focus on this group indicates
that the sequences differ in their structure
and their expression, suggesting a functional
diversity rather than redundancy.
However, the loss of the specific motif related
to endocytosis could suggest that these Rab
can be implicated in other function.
With 10 and 5 members respectively, the
groups related to the late endosomes
(group3) and to the recycling from endosome
to the Golgi (group5) have undergone a
moderate amplification.
Consistently with the amplification of the
endocytic Rab, other endocytic protein family
of T. vaginalis appears to have been amplified
(9) (see table3).
Notably, the syntaxin and SM protein involved
in endocytosis (42), are find to be 3 to 4 time
larger than in human.
The ARF small GTPases also intervene at
different stage of the membrane trafficking,
and are find to have been amplify, reaching a
similar number to the ARF family in human.
More interestingly, the analysis of the TvRab
shows that this amplification seems to have
been concentrated on the early stage of the
endocytosis process. Therefore, it could be
interesting to assess if the amplification of
these proteins have been made preferentially
for the members involved in the early stage of
the endocytosis.
The level of intervention of most of these
different proteins is summarized in the Figure
7.
As it has been suggest in the study of Lal et al
(26), this amplification in the early stage of
endocytosis and phagocytosis could be a
consequence of the high diversity of the
surface protein found in T.vaginalis.
The parasite using phagocytosis and
endocytosis for feeding, the glucose
restriction condition in the transcriptomic
analysis should have up-regulated these
endocytic-related TvRab, confirming there
functional diversity. However, no significant
differential of expression has been
determined.

24
As the experiment is made in vitro, this result
can be due to the condition of study, not
optimal for these cellular processes.
Nevertheless, a tendency to the up-regulation
can be seen (Figure6). As there are no
replicates, the analysis may not be sensitive
enough to detect this modulation.
Figure 7 Schematic representation of the principal pathways and the level of intervention of the
different protein involved in membrane trafficking in T. vaginalis
Modified from Carlton et al (43). The protein surrounding by a red box correspond to the protein
intervening at the early stage of the endocytic and phagocytic pathways. The protein underlined in
red corresponds to those intervening at the late stage of these pathways. The ARF small GTPase
proteins intervene at both levels.
Further analyses
To conclude on the role of the TvRab in the
biology of T. vaginalis, several laboratory
experiments should be made.
Firstly, the transcriptomic analysis should be
made considering a larger set of conditions,
and with replicates.
The modulation of this TvRab between the
tested conditions could also be assessed by
qPCR, to give more precise result.
To confirm the functionality of the TvRab, the
analysis of their expression at the protein level
should be assess, as it has been study for the
surface protein by Miguel et al (44).
Finally, the precise role of the TvRab could be
assessed by fluorescent localization, as it has
already been used for the Rab of T.
thermophila (41).This type of experiments will
also determine if the TvRab are mostly
redundant, if they are imply in new functions,
or if they is a functional diversification of
these sequences confirming a complex

25
Conclusion
The in silico methods used in this project to
analyze the high Rab diversity of Trichomonas
vaginalis have shown that the grand majority
of the sequences are likely to be functional.
More detailed analysis on the group2 suggests
a functional diversity rather than redundancy
between these sequences, and direct
experimental work should be made to confirm
this interpretation.
The preferential amplification of other protein
family implicated in the endocytic and
phagocytic pathway corroborate the
hypothesis that T. vaginalis have developed a
highly complex endomembrane system in
order to orchestrate these phagocytosis and
endocytosis.
This gene family expansion seems to be
concentrated at the early stage of these
pathways, and may be a consequence of the
surface protein diversification.
The importance of the TvRab for the biology
of T. vaginalis and the presence of sequences
specific to this organism could potentially
suggest them as therapeutic target in future
treatment (45).
Acknowledgments
I would like to thanks Professor Robert Hirt for
his time and advice, and the members of the
lab for their help throughout this project.
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